From 3a800c96f6227b32171a1ce1d7586a1aec53717a Mon Sep 17 00:00:00 2001
From: Ryan Du <ryandu9221@gmail.com>
Date: Thu, 12 Oct 2023 11:40:08 -0400
Subject: [PATCH] help me dear wiki gods
---
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diff --git a/public/RCAERCA/index.html b/public/RCAERCA/index.html
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+<!DOCTYPE html>
+<link rel="stylesheet" href="/lambert-ga/lambert-ga/_astro/RCAERCA.96298b81.css" /><h1 id="exponential-rolling-circle-amplification-erca">Exponential Rolling Circle Amplification (eRCA)</h1>
+<p>Although the data from testing rolling circle amplification (RCA) with the target microRNA (miRNA) indicates high sensitivity — detecting lower limits of 2 pM — it lacks the ability to clearly differentiate between various concentrations of miRNA. Throughout multiple iterations of testing, Lambert iGEM has consistently found a significant overlap in error bars for the fluorescent output, especially among lower concentrations of miRNA. Despite taking extra steps to decrease potential error (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: Optimization</a>), we still see a large margin of error. After further discussions with Dr. Mark Styczinski from the Georgia Institute of Technology, we concluded that this overlap in error bars is likely caused by the similarity of fluorescent outputs ‒ due to the minute scale of and minimal difference between concentrations ‒ rather than experimental variation. Exponential RCA (eRCA) is an adaptation of RCA that produces exponentially greater fluorescence per unit of miRNA (Liu et al., 2013; Li et al., 2017); therefore, it has the potential to increase the distinction between lower miRNA concentrations and subsequently decrease the margin of error.</p>
+<h2 id="rca-vs-erca">RCA vs. eRCA</h2>
+<p>There are two major differences between eRCA and RCA: the padlock probe design and the amplification process. The RCA padlock probe consists of two parts: the end or “arms†of the padlock probe and the middle sequence (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: 2022 Overview</a>). The arms are the reverse complement of the target miRNA, while the middle sequence corresponds to the reporter mechanism (Jonstrup et al., 2006). The three main steps of RCA are hybridization, ligation, and amplification (Jonstrup et al., 2006) (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: 2022 Overview</a>). The target miRNA first hybridizes to the padlock probe, bringing the two ends of the probe together. Then, the two ends are ligated by SplintR ligase. The bound miRNA serves as a primer for Phi29 DNA polymerase, initiating the amplification of the padlock probe. The result is a long repeating strand of DNA, known as the rolling circle product (RCP), used to quantify the initial concentration of the miRNA (Jonstrup et al., 2006).
+In eRCA, the padlock probes consist of an additional part between the arms and the reporter sequence: the complement of the endonuclease binding site (see Fig. 1) (Li et al., 2017). When the RCP is synthesized during amplification, the nicking endonuclease Nb.BbvCI will recognize this site and cleave the outer strand of DNA, or the RCP, while it is still bound to the padlock probe (Biolabs). This ensures that the padlock probe remains intact while releasing individual strands of the synthesized miRNA and reporter sequence. The produced miRNA can then bind to the padlock probe and initiate the eRCA process again, essentially creating an endless loop of amplification until the reagents are consumed or the reaction is deactivated (see Fig. 2) (Liu et al., 2013; Li et al., 2017). The exponential increase in output can increase the differences in fluorescence emission between miRNA concentrations, subsequently decreasing the margin of error and accuracy of our overall biosensor.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-11.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. Padlock probe design for eRCA</div></div>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/erca-overall-diagram.jpeg" style="max-width:700" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 2. eRCA process utilizing Nb.BbvCI</div></div>
+<p>Lettuce is a fluorescent DNA aptamer that binds with and induces the fluorescences of the DFHBI-1T dye (VarnBuhler et al., 2022). Initially, we considered producing whole Lettuce aptamers through the RCA reaction. However, Dr. Mark Styczsinski and Megan McSweeney from the Georgia Institute of Technology advised us against this as the secondary structures of the aptamers would likely interfere with each other on a single DNA strand. As a result, we chose to pursue other reporter mechanisms with the traditional RCA assay (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: Outputs</a>). Contrary to RCA, eRCA produces multiple isolated strands of the reporter sequence, offering the potential to create whole Lettuce aptamers without the risk of secondary structure interference (Liu et al., 2013; Li et al., 2017).
+The hsa-miR-1-3p eRCA padlock probe (<a href="http://parts.igem.org/Part:BBa_K4683002">BBa_K4683002</a>) consists of the following parts: 3’ arm for miR-1 (<a href="http://parts.igem.org/Part:BBa_K4245100">BBa_K4245100</a>), 5’ arm for miR-1 (<a href="http://parts.igem.org/Part:BBa_K4245107">BBa_K4245107</a>), Lettuce aptamer complement (<a href="http://parts.igem.org/Part:BBa_K4683000">BBa_K4683000</a>), and Nb.BbvCI binding sites (<a href="http://parts.igem.org/Part:BBa_M31961">BBa_M31961</a>). As the concentration of the target miRNA increases, the padlock probe produces more miRNAs and Lettuce aptamers, resulting in an exponential increase in fluorescence output in the presence of DFHBI-1T.</p>
+<h2 id="results">Results</h2>
+<p>We performed eRCA with 40.8 pM of miR-1 (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: eRCA Protocol</a>) then ran the reaction on a gel electrophoresis (see Fig. 3) (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: blueGelâ„¢ with RCP</a>). Since eRCA produces multiple shorter strands of DNA (~25 nucleotides), we do not expect to see any bands on the gel. Therefore, the gel in Figure 3 indicates that eRCA was likely successful.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rcagel.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes); 2% agarose gel ran for 1 hour at 48V</div></div>
+<p>We then characterized and quantified the RCP from the eRCA reaction through the Lettuce reporting mechanism (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: eRCA Readout</a>). Resultant fluoresence was quantified in a plate reader at exciation wavelength of 480 nm and emission intensity at 528 nm. The triplicate of eRCA with 40.8 pM of miR-1 exhibits significantly more fluorescence than that of the negative control (no enzyme), indicating that the eRCA reaction was successful (see Fig. 4).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/erca-graph.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Fluorescent readout of RCP for eRCA with 40.8 pM miR-1 using Lettuce aptamer</div></div>
+<p>In the future, we plan to test eRCA with the entire range of clinically relevant miRNA concentrations for coronary artery disease (CAD), as well as validate the applicability of eRCA with spiked serum samples. If the system proves to be more accurate than our current RCA biosensor, we will communicate with Dr. Charles Searles from the Emory University School of Medicine to test our biosensors in actual patient serum.</p>
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+<!DOCTYPE html>
+<link rel="stylesheet" href="/lambert-ga/lambert-ga/_astro/RCAERCA.96298b81.css" /><h1 id="overview">Overview</h1>
+<p>One of Lambert iGEM’s major goals in 2023 was to increase the inclusivity of our biosensors for all demographics. Through literature review, we found that healthcare settings tend to emphasize responsive care for men, resulting in lower patient retention among women, especially during coronary artery disease (CAD) diagnosis (Johns Hopkins, 2018). We validated this disparity with Dr. Mindy B. Gentry, a cardiologist in Georgia, and Dr. DeLisa Fairweather, a director of research at the Department of Cardiovascular Diseases at Mayo Clinic. To further address this issue, we specifically targeted microRNAs (miRNAs) correlated to CAD in women (see Inclusivity: Estrogen). Discussion with these experts confirmed the use of hsa-miR-20b (<a href="http://parts.igem.org/Part:BBa_K4683005">BBa_K4683005</a>) as a target for our rolling circle amplification (RCA) assay. Moreover, we extended the correlation of miRNAs to CAD through specific racial groups, utilizing CADmir to identify hsa-miR-328-3p (<a href="http://parts.igem.org/Part:BBa_K4683021">BBa_K4683021</a>) and hsa-miR-146a (<a href="http://parts.igem.org/Part:BBa_K4683025">BBa_K4683025</a>). Further collaboration with Dr. Charles Searles, a professor of medicine at Emory Healthcare Hospital, enabled us to utilize hsa-miR-150-5p (<a href="http://parts.igem.org/Part:BBa_K4683009">BBa_K4683009</a>), hsa-miR-122-5p (<a href="http://parts.igem.org/Part:BBa_K4683013">BBa_K4683013</a>), and hsa-miR-30c-5p (<a href="http://parts.igem.org/Part:BBa_K4683017">BBa_K4683017</a>) as they correlate with CAD in African American populations. Therefore, we can incorporate these miRNAs into our research to further accommodate an inclusive approach for understanding and detecting CAD.</p>
+<h2 id="estrogen-mirna">Estrogen miRNA</h2>
+<p>Women after menopause are more likely to develop heart disease due to a decrease in estrogen hormone levels (Brandt, 2013). Studies conducted by Pare et al. in 2022 found that estrogen directly suppresses CAD through the Estrogen Receptor α (ERα) pathway; when estrogen is present in the bloodstream, it activates the ERα pathway, subsequently activating the production of CAD suppressors. Post-menopausal women have lower levels of estrogen within their bloodstream, resulting in the inhibition of CAD suppressors and therefore increasing patient susceptibility to CAD (Brandt, 2013; Pare et al., 2022).
+The concentration of hsa-miR-20b is positively correlated with that of estrogen (Pérez-Cremades et al., 2018). Like the estrogen molecule, miR-20b activates the ERα pathway, regulating the production of CAD suppressors (Pérez-Cremades et al., 2018). Dr. Fairweather validated the use of this miRNA to detect the risk of CAD in post-menopausal women (see Inclusivity: Estrogen).</p>
+<h2 id="other-mirnas">Other miRNAs</h2>
+<p>We conducted a comprehensive literature review using CADmir to efficiently identify relevant miRNAs (see Fig. 1) (see <a href="https://2023.igem.wiki/lambert-ga/software/">Software: CADmir</a>). In just 4 minutes and 12 seconds, we uncovered three miRNAs and a selection of related articles.</p>
+<p>One of these miRNAs, hsa-mir-328-3p, was found to be correlated with cardiovascular disease, specifically atherosclerosis. Therefore, it serves as a valuable predictive tool for early detection of heart disease, with a specific focus on the African American population (Iwańczyk, 2023). Similarly, hsa-mir-146a is a miRNA associated with the regulation of inflammation and CAD, predominantly linked to the Latin American population (Iwańczyk, 2023). These findings are of particular relevance to our team after considering the demographic makeup of Georgia. Notably, the African American and Latin American population have experienced significant growth, as demonstrated by the statistic: “the Black (non-Hispanic) population had the most growth increasing by 518,670 from 2.9 million in 2010 to 3.5 million in 2021†(Georgia Population by Year, County, Race, & More, 2023).</p>
+<p>To further complement our findings, we consulted with Dr. Searles, who recommended three additional miRNAs correlated with CAD in African American populations: hsa-mir-150-5p, hsa-mir-122-5p, and hsa-mir-30c-5p.</p>
+<h1 id="gel-experimentation">Gel Experimentation</h1>
+<p>We performed RCA on miR-20b, miR-328, miR-146a using our respective padlock probe designs (<a href="http://parts.igem.org/Part:BBa_K4683008">BBa_K4683008</a>; <a href="http://parts.igem.org/Part:BBa_K4683024">BBa_K4683024</a>; <a href="http://parts.igem.org/Part:BBa_K4683028">BBa_K4683028</a>) and ran the subsequent rolling circle product (RCP) on a 1% agarose gel (see Fig. 2). The gel exhibited a fluorescent band of DNA very close to the well, indicating that a long strand of DNA greater than 1 kB - our RCP - was produced. Therefore, we can validate that our reaction was successful. In the future, we plan to expand our collection of biosensors to detect more miRNAs related to CAD and other demographics.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/igem-estrogen-label.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 13. RCP from RCA reaction with miR-20b, miR-328,miR-146; 2% agarose gel ran for 1 hour at 48V </div></div>
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+<link rel="stylesheet" href="/lambert-ga/lambert-ga/_astro/RCAERCA.96298b81.css" /><h1 id="specificity">Specificity</h1>
+<p>While Lambert iGEM has been utilizing rolling circle amplification (RCA) to detect a single isolated microRNA (miRNA), human blood serum contains a total of 204 detectable miRNAs (Wang et al., 2012). Research conducted by Jonstrup et al. in 2006 found that the padlock probe ligates on a perfectly matching RNA template, distinguishing between differences in the target and other sequences. To test whether padlock probes would be able to detect specific miRNA, and therefore be applicable for serum testing, we ran RCA using the hsa-miR-1-3p padlock (<a href="http://parts.igem.org/Part:BBa_K4245200">BBa_K4245200</a>) in the presence of four different miRNA sequences (see Fig. 1). The first is the original miR-1 sequence (<a href="http://parts.igem.org/Part:BBa_K4245006">BBa_K4245006</a>), which is expected to hybridize to the padlock and result in the greatest fluorescence decrease. Two sequences with differing single nucleotide variants (SNVs) found from the National Library of Medicine microRNA 1-1 database were utilized to determine the specificity of RCA: one with a single SNV (<a href="http://parts.igem.org/Part:BBa_K4683003">BBa_K4683003</a>) and one with three SNVs (<a href="http://parts.igem.org/Part:BBa_K4683004">BBa_K4683004</a>). hsa-miR-133a-3p (<a href="http://parts.igem.org/Part:BBa_K4245009">BBa_K4245009</a>) was also included to ensure the padlock would not ligate to any miRNA.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/frame-1.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. Comparison of sequences used to test specificity of hsa-miR-1-3p RCA padlock: 1 bp SNV in the seed region, 3 SNVs outside of seed, and miR-133a-3p</div></div>
+<p>We ran the reactions and control on a gel electrophoresis; only the well with 40.8 pM of miR-1 showed visible bands of DNA near the top of the wells, which is likely our RCP (see Fig. 2) (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: blueGelâ„¢ with RCP</a>).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/specificity-gel.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 2. Gel results: RCA with A: miR-1, B: 1 SNV, C: 3 SNVs, D: miR-133a; 2% agarose gel ran for 1 hour at 48V</div></div>
+<p>We then tested the RCP with linear DNA probes and quantified the resultant fluorescence in a plate reader at an emission wavelength of 480 nm and an excitation wavelength of 528 nm (see Fig. 3) (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Linear DNA Probes with RCP</a>. The RCA reaction utilizing the miR-1 padlock probe with miR-1 exhibited significantly less fluorescence than the other miRNAs. Since linear DNA probes produce a negative correlation between fluorescence and miRNA concentration, this result, along with the gel, indicates that RCA is specific to single nucleotide differences.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/specificty-graph.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. Comparison of RCA with miR-1, 1SNV, 3SNVs, and 133a fluorescence output using linear DNA probes.</div></div>
+<h1 id="emory-testing">Emory Testing</h1>
+<p>Dr. Charles Searles is a cardiologist specialist working with biomarkers regarding coronary artery disease (CAD) and an associate professor of medicine at Emory Healthcare Hospital. Last year, he provided us with hsa-miR-451a (<a href="http://parts.igem.org/Part:BBa_K4245012">BBa_K4245012</a>) - which is unrelated to the pathophysiology of CAD - as a control for our proof of concept experimentation and also offered to test our biosensor in his lab. This year, we continued to communicate with him in order to test whether our biosensor could be practical and applicable as a diagnostic tool. This was done with the help of Kimberly Ann Rooney, a lab technician at the Searles Cardiovascular Lab led by Dr. Charles Searles, who ran the RCA reactions utilizing our protocol and miR-1 padlock probe (<a href="http://parts.igem.org/Part:BBa_K4245200">BBa_K4245200</a>), and their enzymes (see Fig. 4) (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: SYBRâ„¢ Safe with RCP</a>). They ran RCA on 40.8 pM of miR-1 (<a href="http://parts.igem.org/Part:BBa_K4245006">BBa_K4245006</a>) with SYBRâ„¢ Safe dye, which fluoresces when bound to ssDNA, as the output. Resultant fluoresence was quantified in a plate reader at exciation wavelength of 480 nm and emission intensity at 528 nm. As shown in Figure 4, there was a significant increase in fluorescence in the RCA reaction as compared to that of the controls, therefore validating the application of our biosensor in other labs.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/emory.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Triplicate of RCA with SYBRâ„¢ Safe output done by independent hands</div></div>
+<p>After running our assay twice, Ms. Rooney gave us feedback regarding our protocol such as reducing the number of variables as controls and performing serial dilutions for more precise concentrations of miRNA. Ultimately, she told us that our biosensor lacked practicality and applicability for point-of-care testing due to its long reaction time, which is approximately 15-16 hours‒ more than double the time needed for quantification through qRT-PCR. Subsequently, the next step we took to optimize RCA was to reduce the time required to run the reaction.</p>
+<h1 id="time-optimization">Time Optimization</h1>
+<h2 id="ligation-time">Ligation Time</h2>
+<p>Lambert iGEM’s 2022 RCA protocol (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: RCA 2022 protocol</a>) requires samples to be incubated in the thermocycler at 37°C for two hours. However, the properties and usage of SplintR Ligase show that the reaction is successful with a 15-minute ligation time (Avantor Staff). Therefore, we ran RCA utilizing the miR-1 padlock probe (<a href="http://parts.igem.org/Part:BBa_K4245200">BBa_K4245200</a>) with four different ligation times with miR-1 (<a href="http://parts.igem.org/Part:BBa_K4245006">BBa_K4245006</a>): 15 minutes, 30 minutes, one hour, and two hours. After amplification, the reactions and controls were run on a gel; the bright bands near the top of the well showed that DNA product was produced for all reactions except for 15 minutes (see Fig. 5). Moving forward, we implemented a 30-minute ligation time (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Optimized RCA protocol</a>).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/screenshot-2023-10-11-at-4-32-50-pm.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 5. 1: 15-minute ligation, 2: 30-minute ligation, 3: 1-hour ligation, 4: 2-hour ligation, A: control (no enzymes); 2% agarose gel ran for 1 hour at 48V</div></div>
+<h2 id="amplification-time">Amplification Time</h2>
+<p>Lambert iGEM’s 2022 RCA protocol (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: RCA 2022 protocol</a>) requires samples to be incubated in the thermocycler at 37°C for eight hours for amplification. To reduce this time, we ran RCA with two different concentrations of miR-1. After hybridization and ligation, we incubated the reactions in the plate reader at 37°C with 4uL SYBR™ Safe (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: SYBR™ Safe with RCP</a>). The reactions were run overnight and the subsequent fluorescence was quantified in a plate reader (excitation wavelength 480 nm; emission wavelength: 528 nm): 30-minute intervals (see Fig. 6). Over time, the two RCA reactions increased in fluorescence, with no SEM overlap observed between the starting time and 5-hour mark. This suggests that RCP can be produced optimally starting at 5 hours. The significant increase in fluorescence between the RCA reactions and controls shows that SYBR™ Safe can determine the presence of RCP; however, the lack of difference between the 40.8 pM and .41 pM of miR-1 fluorescence indicates that the dye is not sensitive enough to differentiate between miRNA concentrations. Therefore, we did not continue to utilize SYBR™ Safe as a reporter.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/specificty-graph.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 6. RCA amplification optimization reaction with 40.8 pM and 0.41 pM of miRNA. No SEM overlap between starting point and 5-hour incubation time. SEM overlaps between 40. pM and .41 pM show that miRNA concentrations cannot be differentiated by SYBRâ„¢ Safe.</div></div>
+<h2 id="phi29-xt">Phi29-XT</h2>
+<p>Lambert iGEM’s RCA protocol (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Optimized RCA protocol</a>) utilizes Phi29 DNA polymerase to perform amplification, resulting in an 5 hour amplification time. Phi29- XT DNA polymerase is an optimized enzyme with improved thermostability and sensitivity, which could shorten this time down to 2 hours (Biolabs). We ran RCA following the protocol for Phi29-XT on the New England Biolabs website (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Amplification with Phi29- XT</a>). The reactions and controls were run on a gel; no visible bands could be seen on the gel, indicating that RCP was not produced and therefore the reaction was not successful. As a result, we did not pursue utilizing Phi29-XT for further RCA reactions (see Fig. 7).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/phi-xt.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 7. A: control (no enzymes), X: RCA with phi29-XT DNA polymerase; 2% agarose gel ran for 1 hour at 48V</div></div>
+<p>Overall, we were able to reduce the RCA workflow from around 15 hours to 7 (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Optimized RCA Protocol</a>).</p>
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+<link rel="stylesheet" href="/lambert-ga/lambert-ga/_astro/RCAERCA.96298b81.css" /><h1 id="linear-dna-probes-vs-lettuce">Linear DNA Probes vs Lettuce</h1>
+<p>Lettuce DNA aptamer and linear DNA probes were both tested as reporters for rolling circle amplification (RCA) with the hsa-miR-1-3p padlock probe (<a href="http://parts.igem.org/Part:BBa_K4245200">BBa_K4245200</a>); however, only linear probes were used for the successful characterization of miR-1 (<a href="http://parts.igem.org/Part:BBa_K4245006">BBa_K4245006</a>) in the 2022 season (see <a href="https://2022.igem.wiki/lambert-ga/rca">Lambert iGEM Wiki RCA, 2022</a>). Dr. Adam Silverman from Northwestern University and Dr. Mark Styzynski from the Georgia Institute of Technology both suggested utilizing an on-state reporter to make results more accurate and comprehensible; measuring gain of signal reduces fluorescent noise caused by the reaction and has less limitations compared to repressible systems. Linear DNA probes are an off-state reporter, therefore they produce an indirect relationship between microRNA (miRNA) concentration and fluorescence output. Utilizing an on-state reporter such as Lettuce would result in a direct relationship, in which a higher concentration of fluorescence will correspond to a higher concentration of miRNA.</p>
+<h2 id="linear-dna-probes">Linear DNA Probes</h2>
+<p>Fluorophore and quencher-tagged linear DNA probes were used to quantify the presence and concentration of target miRNA in samples. Each probe contains part of the complement to the middle sequence (<a href="http://parts.igem.org/Part:BBa_K4245131">BBa_K4245131</a>) of the rolling circle product (RCP): One is tagged with the fluorophore dye FAM (<a href="http://parts.igem.org/Part:BBa_K4245130">BBa_K4245130</a>), and the other is tagged with the quencher molecule BHQ1 (<a href="http://parts.igem.org/Part:BBa_K4245132">BBa_K4245132</a>). The fluorescent signal from the fluorophore is shut off by the quencher as a result of a fluorescence resonance energy transfer (FRET) reaction (Zhou et al., 2015). The reaction is distance-dependent; when the quencher and fluorophore-tagged linear probes bind to the RCP, they are in close proximity (see Fig. 1), allowing non-radiative energy to be transferred from the excited fluorophore to the quencher (Sekar & Periasamy, 2003). The decrease in fluorescence in the solution can be correlated with a specific concentration of miRNA through characterization.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/frame-2.jpeg" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. Linear DNA probes binding to RCP, leading to diminished fluorescence</div></div>
+<p>Similar to last year, we characterized and quantified RCP through the linear probes reporting mechanism (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Linear DNA Probes with RCP</a>). There is a negative logarithmic correlation between the miRNA concentrations and the relative fluorescence units (RFU) (see Fig. 2). Changes in fluorescence were measured using a plate reader in experimentation: the emission spectrum of FAM is 480 nm in wavelength, while the excitation spectrum is 528 nm in wavelength (Zhou et al., 2015).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/linearprobes.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 2. Characterization curve showing a negative logarithmic relationship between miR-1 concentrations and RFU from linear DNA probes </div></div>
+<h2 id="lettuce">Lettuce</h2>
+<p>Lettuce is a fluorescent DNA aptamer that binds with the dye DFHBI-1T within its secondary structure, thus causing the dye to fluoresce (see Fig. 3) (VarnBuhler et al., 2022). The split Lettuce design includes two halves of the Lettuce aptamer and their flanking sequences. After the RCA reaction, we add the split Lettuce sequences and the DFHBI-1T dye to the RCP. The left flanking sequence (<a href="http://parts.igem.org/Part:BBa_K4245134">BBa_K4245134</a>) will bind to the first half of the middle sequence of the RCP, and the right flanking sequence (<a href="http://parts.igem.org/Part:BBa_K4245135">BBa_K4245135</a>) will bind to the second half of the middle sequence. Once together, the dye is able to bind and produce fluorescence, therefore an increase in miRNA concentration should correlate with an increase in fluorescence.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-9.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. Split Lettuce aptamer with DFHBI-1T binding to RCP</div></div>
+<p>We characterized and quantified RCP through the Lettuce reporting mechanism (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Lettuce with RCP</a>). There is a positive logarithmic correlation between the miRNA concentrations and the relative fluorescence units (RFU) (see Fig. 4). Changes in fluorescence were measured using a plate reader in experimentation: the emission spectrum of DFHBI-1T is 480 nm in wavelength, while the excitation spectrum is 528 nm in wavelength (VarnBuhler et al., 2022).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/lettuce.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Characterization curve showing a positive logarithmic relationship between miR-1 concentrations and RFU from split Lettuce aptamer</div></div>
+<h2 id="comparison">Comparison</h2>
+<p>Both reporter mechanisms resulted in significant SEM overlap between the lower miRNA concentrations, making accurate differentiation of miRNAs difficult. However, there was no indication that Lettuce outperformed linear DNA probes. Therefore, we continued to conduct further experiments with linear probes. In the future, we hope to find another on-state reporter that would make reading RCA results more comprehensible and accurate. Such reporters include molecular beacons and other DNA aptamers.</p>
+<h1 id="capillary">Capillary</h1>
+<p>To create a more accessible miRNA detection system, Lambert iGEM also adopted another output approach: capillary-rolling circle amplification (cpRCA). This method offers faster reaction times and eliminates the need for expensive and specialized equipment like plate readers and fluorometers, making it suitable for rapid point-of-care testing of miRNAs (Hixson & Ward, 2021). Unlike our current reporter mechanisms, cpRCA amplifies miRNA to produce RCP within a glass capillary tube. The small diameter of the capillary tube (0.1mm) enables diffusion of small polymers (such as miRNA), while restricting diffusion of larger polymers (such as the RCP). Due to this principle (see <a href="https://2023.igem.wiki/lambert-ga/model/">Modeling</a>), the miRNA molecules are relatively spaced apart within the tube (see Fig. 5a). As the RCP is being synthesized from the miRNA molecules, the large DNA strands are unable to diffuse, creating spaced apart, isolated regions of ssDNA (see Fig. 5b). When the DNA fluorescent dye SYBR™ Safe is present in the reaction solution, it binds to the synthesized RCP to create regions of high fluorescence, or fluorescent “dots†(see Fig. 5c), a phenomenon that has been explored with PCR in capillary tubes (Choi et al., 2018). We can directly quantify the number of miRNA molecules by counting the number of dots, which can be done through use of a phone camera and counting algorithm, such as Open Computer Vision Library (OpenCV) (Abid et al, 2021).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-12-1.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 5. (a) miRNA and padlock probe diffusion in capillary tube. (b) Isolated regions of RCP within the capillary tube. (c) SYBRâ„¢ Safe fluorescence with RCP
+</div></div>
+<p>After discussion with Dr. Charles Searles from the Emory University School of Medicine, we determined that the upper limit of clinically relevant miRNA concentrations in patients with CAD is around 40 pM. We initially ran cpRCA with the miR-1 padlock probe (<a href="http://parts.igem.org/Part:BBa_K4245200">BBa_K4245200</a>) and 40.8 pM of miR-1 (<a href="http://parts.igem.org/Part:BBa_K4245006">BBa_K4245006</a>) but were not able to visualize any dots of miRNA within the capillary tube (see Fig. 6) (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Capillary RCA</a>). This is likely because the higher concentration of miRNA resulted in a significant overlap of fluorescent regions within the capillary tube, leading to inaccurate quantification through cpRCA.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/screenshot-2023-10-11-at-3-22-41-am.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 6. Visualization of cpRCA with 40.8 pM of miRNA
+</div></div>
+<p>To ensure the amplified products were countable, we diluted the sample down to 1.66 fM, or approximately 50 molecules of miRNA. To validate that cpRCA would still be successful at this lower concentration, we ran a gel electrophoresis on the RCP from the cpRCA reaction after 4 hours of amplification (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: blueGelâ„¢ with RCP</a>) using both 40pM and 1.66fM of miRNA to observe the difference. By analyzing the results on the gel, we concluded that RCP was likely produced with both concentrations as the gel exhibited fluorescent bands of DNA very close to the wells, while no band was expressed in the control (see Fig. 7). As a result, we can infer that cpRCA was successful after 4 hours of amplification and at only 50 molecules of miRNA, which is more efficient than the 8 hour amplification of traditional RCA, and increases the sensitivity of the assay.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/screenshot-2023-10-11-at-3-21-24-am.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 7. Lane 1: ladder, Lanes 2-3: cpRCA with 1.66 fM miR-1, Lanes 4-5: cpRCA with 40.8 pM miR-1, Lanes 6-7: control; 2% agarose gel ran for 1 hour at 48V</div></div>
+<p>In the future, we plan to conduct further experimentation with lower miRNA concentrations to better visualize the fluorescent regions in the capillary tube, as well as characterize the full range of relevant miRNA concentrations for CAD.</p>
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+<link rel="stylesheet" href="/lambert-ga/lambert-ga/_astro/RCAOverview2022.5440303d.css" /><h1 id="overview">Overview</h1>
+<h2 id="problem">Problem</h2>
+<p>Heart disease has been one of the most prevalent diseases in the United States, with 1 in 4 deaths caused by cardiovascular disease (Centers for Disease Control, 2022). According to the Centers for Disease Control and Prevention, heart disease has contributed to over 25% of all deaths in Georgia, Lambert iGEM’s home region, in 2018 - that is 5% greater than the overwhelmingly large national statistic. Historically, the southern region has had issues concerning obesity, high blood pressure, smoking, and diabetes, all of which are exacerbated by a family history of coronary artery disease (CAD). These problems only increase with the “coincidental†ethnic and racial minorities present within these low-income populations (Hames and Greenlund). However, with a process that considers both affordability and accuracy, we can proliferate the means of diagnosing CAD. This starts with inclusive initiatives targeting these overlooked demographics.</p>
+<h2 id="methodology">Methodology</h2>
+<p>Last year, Lambert iGEM created CADlock, an accessible point-of-care diagnostic tool to both detect and quantify microRNA (miRNA) by utilizing rolling circle amplification (RCA). The rolling circle product (RCP) is a continuous transcription of the team’s designed padlock probe, which consists of many interspaced repeats of the reporting mechanism and target miRNA’s complementary sequence (Shi et al., 2012; Song et al., 2010; Ouyang et al., 2019; Ye et al., 2019; Zheng et al., 2016). We identified two miRNAs found to be upregulated to CAD, hsa-miR-1-3p (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245006">BBa_K4245006</a>) and hsa-miR-133a-3p (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245009">BBa_K4245009</a>), to serve as an early indication of the disease (Kaur et al., 2020).</p>
+<p>Initially, we considered Reverse Transcription Quantification PCR (RT-qPCR) which was appealing due to its regularity with current screening, especially with the COVID-19 tests. However, through conversations with experts and further analysis of protocols, we identified the dangers of this method due to the high rate of false positive mismatches and determined that RCA held the most advantages. Not only does RCA eliminate the use of primers–which RT-qPCR requires–but it has no data concerning any mismatch or inconsistent results from our literature studies (Shi et al., 2012; Song et al., 2010; Ouyang et al., 2019; Ye et al., 2019; Zheng et al., 2016).</p>
+<p>Moreover, diagnostic tools such as echocardiograms and angiograms are expensive – costing upwards of $1000 – and not readily available at most primary care facilities, preventing a large portion of the population from receiving effective screening (Hansing C. E.). However, our RCA assay costs approximately $9, which is less than 1% of most common diagnostic procedures.</p>
+<h2 id="padlock-design">Padlock Design</h2>
+<p>A padlock probe is a single-stranded DNA sequence (30-150 nucleotides in length) designed to hybridize to a specific RNA/DNA target– in this case, our target miRNA (Jonstrup et al., 2006). The ends or “arms†of the probe are complementary to the target sequence, which allows the miRNA to bind antiparallel to the padlock arms (see Fig. 1). We designed these ends to have similar annealing temperatures to maximize their binding efficiency with the miRNA. In between the ends is the reporter sequence, which can be utilized to quantify the initial miRNA concentration (see Fig. 1). Furthermore, we added a phosphate group modification to the 5’ end of the padlock sequence (Jonstrup et al., 2006).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-1-21.png" style="max-width:[250px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. Padlock probe formation and arrangement with miRNA</div></div>
+<p>The following steps are for generating a padlock probe by hand. Users will need a software tool such as SnapGene that displays melting temperatures of sequences.</p>
+<ol>
+<li>Paste target sequence in software of choice.</li>
+<li>Take the reverse complementary sequence of the target.</li>
+<li>Split the sequence in half. Use the first half of the sequence as the end of the padlock probe and the second half as the beginning of the probe.</li>
+<li>Insert the desired reporter sequence in between the two halves of the sequence.</li>
+<li>Calculate the annealing temperature of both halves and move nucleotides one at a time from one end of a half to the end of the other half until the difference between the annealing temperatures of the two arms is minimal.</li>
+</ol>
+<p>Last year, Lambert iGEM developed Probebuilder: a novel, intuitive, and user-friendly program for designing padlock probes. After validating the hsa-miR-451a padlock probe generated by Probebuilder, the team has continued to use the software to design padlocks for RCA reactions (see <a href="https://2022.igem.wiki/lambert-ga/software/">Lambert iGEM Wiki Software, 2022</a>).</p>
+<p>Probebuilder’s source code is available on the 2022 <a href="https://gitlab.igem.org/2022/software-tools/lambert-ga/">Gitlab repository</a></p>
+<h2 id="process">Process</h2>
+<p>The RCA process relies on the continuous replication of padlock probes to produce RCP that can be quantified and correlated to the target miRNA concentration. There are three main steps in the RCA process detailed by Jonstrup et al. in their 2006 paper: hybridization, ligation, and amplification. By designing the padlock probe to detect a specific miRNA target, the miRNA and padlock probe arms form a DNA-RNA hybridization (see Fig. 2a, 2b). The arms are then in closer proximity, and the enzyme SplintR ligase (which only ligates near RNA-DNA hybridization) utilizes a phosphate modification on the 5’ arm and ATP to circularize the padlock probe (Avantor Staff) (see Fig. 2c). After ligation, phi29 DNA polymerase uses the miRNA hybridized to the padlock arms as a primer to initiate and perform amplification (see Fig. 2d, 2e). The resultant rolling circle product (RCP) contains interspaced repeats of the middle sequence of the padlock probe (see Fig. 2f). This sequence of the RCP is complementary to the reporters (linear probes or lettuce) used to correlate fluorescence to varying concentrations of the target miRNA.</p>
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RCA process: (a, b) hybridization, (c) ligation, (d, e) amplification, (f) rolling circle product"],"images":[1,[[0,"https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-6-1.png"],[0,"https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-2.jpeg"],[0,"https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-3.jpeg"],[0,"https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-4.png"],[0,"https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-5.png"],[0,"https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-8.jpeg"]]],"size":[0,"[348px]"]}" ssr="" client="load" opts="{"name":"Image","value":true}" await-children=""><div class="flex w-full flex-col items-center justify-center p-1"><div class="carousel-root" tabindex="0"><div class="carousel carousel-slider" style="width:100%"><ul class="control-dots"><li class="dot selected" value="0" role="button" tabindex="0" aria-label="slide item 1"></li><li class="dot" value="1" role="button" tabindex="0" aria-label="slide item 2"></li><li class="dot" value="2" role="button" tabindex="0" aria-label="slide item 3"></li><li class="dot" value="3" role="button" tabindex="0" aria-label="slide item 4"></li><li class="dot" value="4" role="button" tabindex="0" aria-label="slide item 5"></li><li class="dot" value="5" role="button" tabindex="0" aria-label="slide item 6"></li></ul><button type="button" aria-label="previous slide / item" class="control-arrow control-prev control-disabled"></button><div class="slider-wrapper axis-horizontal"><ul class="slider animated" style="-webkit-transform:translate3d(0,0,0);-ms-transform:translate3d(0,0,0);-o-transform:translate3d(0,0,0);transform:translate3d(0,0,0);-webkit-transition-duration:350ms;-moz-transition-duration:350ms;-o-transition-duration:350ms;transition-duration:350ms;-ms-transition-duration:350ms"><li class="slide selected previous"><div class="slide"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-6-1.png"/></div></li><li class="slide"><div class="slide"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-2.jpeg"/></div></li><li class="slide"><div class="slide"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-3.jpeg"/></div></li><li class="slide"><div class="slide"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-4.png"/></div></li><li class="slide"><div class="slide"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-5.png"/></div></li><li class="slide"><div class="slide"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-8.jpeg"/></div></li></ul></div><button type="button" aria-label="next slide / item" class="control-arrow control-next"></button><p class="carousel-status">1 of 6</p></div><div class="carousel"><div class="thumbs-wrapper axis-vertical"><button type="button" class="control-arrow control-prev control-disabled" aria-label="previous slide / item"></button><ul class="thumbs animated" style="-webkit-transform:translate3d(0,0,0);-moz-transform:translate3d(0,0,0);-ms-transform:translate3d(0,0,0);-o-transform:translate3d(0,0,0);transform:translate3d(0,0,0);-ms-transform:translate3d(0,0,0);-webkit-transition-duration:350ms;-moz-transition-duration:350ms;-ms-transition-duration:350ms;-o-transition-duration:350ms;transition-duration:350ms;-ms-transition-duration:350ms"><li class="thumb selected" aria-label="slide item 1" style="width:80px" role="button" tabindex="0"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-6-1.png"/></li><li class="thumb" aria-label="slide item 2" style="width:80px" role="button" tabindex="0"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-2.jpeg"/></li><li class="thumb" aria-label="slide item 3" style="width:80px" role="button" tabindex="0"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-3.jpeg"/></li><li class="thumb" aria-label="slide item 4" style="width:80px" role="button" tabindex="0"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-4.png"/></li><li class="thumb" aria-label="slide item 5" style="width:80px" role="button" tabindex="0"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-5.png"/></li><li class="thumb" aria-label="slide item 6" style="width:80px" role="button" tabindex="0"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-8.jpeg"/></li></ul><button type="button" class="control-arrow control-next control-disabled" aria-label="next slide / item"></button></div></div></div><div class="text-center italic">Figure 2. RCA process: (a, b) hybridization, (c) ligation, (d, e) amplification, (f) rolling circle product</div></div></astro-island>
+<h1 id="2022-results">2022 Results</h1>
+<p>In 2022, we conducted multiple rounds of experimentation to generate quantifiable results with our product. First, we conducted initial experiments to assess how well our hsa-miR-1-3p biosensor (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245200">BBa_K4245200</a>) performs in a controlled laboratory setting by evaluating the effectiveness of rolling circle amplification (RCA) and various reporter mechanisms. All results in Figures 4-11 and13 were quantified in a plate reader at exciation wavelength of 480 nm and emission intensity at 518 nm.</p>
+<ol>
+<li>
+<p>To quantify the rolling circle product (RCP), we first used the fluorescence dye SYBRâ„¢ Safe dye which fluoresces when bound to ssDNA. The results were inconsistent, so we tested the potential rolling circle product (RCP) through gel electrophoresis. The gel showed a band near the wells, indicating a long strand of DNA was produced, likely our RCP (see Fig. 3). <div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-updated-figure-10.png" style="max-width:[250px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. Gel electrophoresis of RCA reactions, depicting bands greater than 1 kB; 2% agarose gel ran for 1 hour at 48V</div></div></p>
+</li>
+<li>
+<p>Through a literature review, we found a lettuce aptamer (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245133">BBa_K4245133</a>) and tested it with simulated RCP (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245131">BBa_K4245131</a>). This showed an increase in fluorescence compared to the controls (see Fig. 4), indicating that the lettuce aptamer is able to fluoresce when bound to the simulated RCP (VarnBuhler et al., 2022). <div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-18.png" style="max-width:[250px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Lettuce aptamer with simulated RCP.</div></div></p>
+</li>
+<li>
+<p>We performed a series of experiments of the split Lettuce aptamer (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245134">BBa_K4245134</a>; <a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245135">BBa_K4245135</a>) with actual RCP. The results display a significant increase in fluorescence with RCP and split lettuce as compared to the controls, indicating that the lettuce aptamer was able to successfully bind to the RCP and induce fluorescence of DFHBI-1T (see Fig. 5). <div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rcp-lettuce.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 5. Graph of fluorescence from left to right: 1) water + dye control, 2) split lettuce + dye control, 3) split lettuce + RCP + dye, 4) split lettuce + padlock&miRNA control + dye. </div></div></p>
+</li>
+<li>
+<p>After consultation with other experts on linear DNA probes, we decided to test these with simulated RCP (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245131">BBa_K4245131</a>) to ensure linear probes were an effective and characterizable means of quantifying miRNA (Zhou et al., 2015). The FAM dye tagged probe (<a href="%5BBBa_K4245131%5D(http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245130)">BBa_K4245130</a>) and BHQ-1 quencher tagged probe (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245132">BBa_K4245132</a>)) are complementary to our RCP. When the linear DNA probes are bound to the RCP, the fluorescence produced by the FAM Dye is quenched by the BHQ-1 quencher which significantly reduces fluorescent intensity (Zhou et al., 2015). As shown in Figure 6, linear DNA probes with simulated RCP have significantly less fluorescence than just the FAM dye, indicating the mechanism would be efficient with actual RCP. <div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-11.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 6. Linear DNA probes with simulated RCP.</div></div></p>
+</li>
+<li>
+<p>We further characterized the linear DNA probes with various simulated RCP concentrations. Figure 7 below shows a negative logarithmic correlation between the complement concentrations and the relative fluorescence units, a parallel relationship to our ODE model (see Fig. 8). <div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-12.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 7. Linear DNA probes with various concentrations of simulated RCP.</div></div> <div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-13.png
+" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 8. Deterministic ODE Model Simulation of RFU output dependent on concentration of linear DNA probe complement concentration.</div></div></p>
+</li>
+<li>
+<p>After we determined the vitality of using linear DNA probes with simulated RCP, we tested these probes with actual RCP. As shown in Figure 9 below, there is a statistically significant decrease in the fluorescence output of the probes with RCP as compared to that of just the FAM-tagged probe. This confirms that we produced our desired RCP.</p>
+</li>
+</ol>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-14.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 9. Fluorescent readout of RCP for miRNA-1-3p and miRNA-133a-3p using linear DNA probes.</div></div>
+<ol start="7">
+<li>We further characterized linear DNA probes to quantify the relationship between various miRNA concentrations and fluorescence. There is a negative logarithmic correlation between the miRNA concentrations and the RFU (see Fig. 10), a parallel relationship to our ODE model (see Fig. 11), validating that RCA coupled with linear probes are an effective means of quantifying miRNA.</li>
+</ol>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-15.png
+" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 10. Characterization curve for showing a negative logarithmic relationship between RFU from linear DNA probes and various miRNA concentrations.</div></div>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-16.png
+" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 11. Deterministic ODE Model Simulation of RFU output dependent on miRNA concentration</div></div>
+<h2 id="serum-testing">Serum Testing</h2>
+<p>To assess the feasibility of our biosensors in real-world applications, Lambert iGEM experimented with pooled human serum. Upon meeting with Dr. Charles Searles from the Emory University School of Medicine, we tested the ability of our RCA sensors to detect miRNAs in blood serum by spiking pooled human serum with hsa-miR-1-3p and RNase inhibitors (to prevent native RNases from degrading the added miRNA).
+The gel electrophoresis results show clear bands close to the wells (see Fig. 12), indicating our biosensors were able to produce a long strand, likely the RCP.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/proof-of-concept-figure-9.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 12. Agarose gel with RCP from RCA reactions and negative control reactions that had no added miRNA from serum treated with 10 nM miRNA; 2% agarose gel ran for 1 hour at 48V</div></div>
+<p>To experimentally validate that the long DNA strand that was produced is our desired RCP, we utilized linear DNA probes. As shown in Figure 13, there is a statistically significant decrease in the fluorescent output of the linear DNA probes with RCP as compared to that of just FAM tagged probes, validating the success of the RCA reaction with hsa-miR-1-3p in spiked serum.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-17-updated.png
+" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 13. Linear DNA probes fluorescence output from RCA with serum extracted miRNA-1-3p.</div></div>
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+ Copyright (c) 2018 Jed Watson.
+ Licensed under the MIT License (MIT), see
+ http://jedwatson.github.io/classnames
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o=e.getInitialImage();o&&!o.complete?o.addEventListener("load",e.setMountState):e.setMountState()})}},{key:"destroyCarousel",value:function(){this.state.initialized&&(this.unbindEvents(),this.destroyAutoPlay())}},{key:"setupAutoPlay",value:function(){this.autoPlay();var e=this.carouselWrapperRef;this.props.stopOnHover&&e&&(e.addEventListener("mouseenter",this.stopOnHover),e.addEventListener("mouseleave",this.startOnLeave))}},{key:"destroyAutoPlay",value:function(){this.clearAutoPlay();var 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g.onSwipeUp=this.props.verticalSwipe==="natural"?this.onSwipeBackwards:this.onSwipeForward,g.onSwipeDown=this.props.verticalSwipe==="natural"?this.onSwipeForward:this.onSwipeBackwards,g.style=M(M({},g.style),{},{height:this.state.itemSize}),E.height=this.state.itemSize;return y.default.createElement("div",{"aria-label":this.props.ariaLabel,className:D.default.ROOT(this.props.className),ref:this.setCarouselWrapperRef,tabIndex:this.props.useKeyboardArrows?0:void 0},y.default.createElement("div",{className:D.default.CAROUSEL(!0),style:{width:this.props.width}},this.renderControls(),this.props.renderArrowPrev(this.onClickPrev,f,this.props.labels.leftArrow),y.default.createElement("div",{className:D.default.WRAPPER(!0,this.props.axis),style:E},o?y.default.createElement(lt.default,le({tagName:"ul",innerRef:this.setListRef},g,{allowMouseEvents:this.props.emulateTouch}),this.props.infiniteLoop&&_,this.renderItems(),this.props.infiniteLoop&&v):y.default.createElement("ul",{className:D.default.SLIDER(!0,this.state.swiping),ref:function(z){return e.setListRef(z)},style:this.state.itemListStyle||{}},this.props.infiniteLoop&&_,this.renderItems(),this.props.infiniteLoop&&v)),this.props.renderArrowNext(this.onClickNext,h,this.props.labels.rightArrow),this.renderStatus()),this.renderThumbs())}}]),r}(y.default.Component);te.default=de;c(de,"displayName","Carousel");c(de,"defaultProps",{ariaLabel:void 0,axis:"horizontal",centerSlidePercentage:80,interval:3e3,labels:{leftArrow:"previous slide / item",rightArrow:"next slide / item",item:"slide item"},onClickItem:x.noop,onClickThumb:x.noop,onChange:x.noop,onSwipeStart:function(){},onSwipeEnd:function(){},onSwipeMove:function(){return!1},preventMovementUntilSwipeScrollTolerance:!1,renderArrowPrev:function(n,r,i){return y.default.createElement("button",{type:"button","aria-label":i,className:D.default.ARROW_PREV(!r),onClick:n})},renderArrowNext:function(n,r,i){return y.default.createElement("button",{type:"button","aria-label":i,className:D.default.ARROW_NEXT(!r),onClick:n})},renderIndicator:function(n,r,i,e){return y.default.createElement("li",{className:D.default.DOT(r),onClick:n,onKeyDown:n,value:i,key:i,role:"button",tabIndex:0,"aria-label":"".concat(e," ").concat(i+1)})},renderItem:function(n){return n},renderThumbs:function(n){var r=y.Children.map(n,function(i){var e=i;if(i.type!=="img"&&(e=y.Children.toArray(i.props.children).find(function(o){return o.type==="img"})),!!e)return e});return r.filter(function(i){return i}).length===0?(console.warn("No images found! Can't build the thumb list without images. If you don't need thumbs, set showThumbs={false} in the Carousel. Note that it's not possible to get images rendered inside custom components. More info at https://github.com/leandrowd/react-responsive-carousel/blob/master/TROUBLESHOOTING.md"),[]):r},statusFormatter:x.defaultStatusFormatter,selectedItem:0,showArrows:!0,showIndicators:!0,showStatus:!0,showThumbs:!0,stopOnHover:!0,swipeScrollTolerance:5,swipeable:!0,transitionTime:350,verticalSwipe:"standard",width:"100%",animationHandler:"slide",swipeAnimationHandler:Z.slideSwipeAnimationHandler,stopSwipingHandler:Z.slideStopSwipingHandler});var wt={};(function(t){Object.defineProperty(t,"__esModule",{value:!0}),Object.defineProperty(t,"Carousel",{enumerable:!0,get:function(){return n.default}}),Object.defineProperty(t,"CarouselProps",{enumerable:!0,get:function(){return r.CarouselProps}}),Object.defineProperty(t,"Thumbs",{enumerable:!0,get:function(){return i.default}});var n=e(te),r=wt,i=e(K);function e(o){return o&&o.__esModule?o:{default:o}}})(_e);function Pt(t){return t.images.map(n=>({source:n})),H.jsxs("div",{className:"flex w-full flex-col items-center justify-center p-1",children:[H.jsx(_e.Carousel,{useKeyboardArrows:!0,children:t.images.map((n,r)=>H.jsx("div",{className:"slide",children:H.jsx("img",{alt:"sample_file",src:n},r)}))}),H.jsx("div",{className:"text-center italic",children:t.caption})]})}export{Pt as default};
diff --git a/public/_astro/Nav.5d7e3ea6.js b/public/_astro/Nav.5d7e3ea6.js
new file mode 100644
index 0000000..27d7f8f
--- /dev/null
+++ b/public/_astro/Nav.5d7e3ea6.js
@@ -0,0 +1 @@
+import{a as e}from"./Nav.d61123f0.js";import"./extends.98964cd2.js";import"./index.453e6029.js";import"./index.c95c1985.js";import"./index.76dc11fb.js";import"./jsx-runtime.4844db2a.js";export{e as NavHome};
diff --git a/public/_astro/Nav.d61123f0.js b/public/_astro/Nav.d61123f0.js
new file mode 100644
index 0000000..0409359
--- /dev/null
+++ b/public/_astro/Nav.d61123f0.js
@@ -0,0 +1 @@
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diff --git a/public/_astro/PageLayoutComp.a397ad4e.js b/public/_astro/PageLayoutComp.a397ad4e.js
new file mode 100644
index 0000000..acb6721
--- /dev/null
+++ b/public/_astro/PageLayoutComp.a397ad4e.js
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+prose max-w-3xl
+prose-h1:font-serif prose-h1:text-4xl
+prose-h2:font-sans prose-h2:text-2xl
+prose-h3:font-sans prose-h3:text-lg prose-h3:font-semibold prose-h3:text-yellow-800
+prose-p:font-sans prose-p:text-gray-900
+prose-a:font-sans prose-a:text-gray-900 prose-a:underline prose-a:font-normal prose-a:text-yellow-800
+prose-quoteless
+prose-strong
+prose-em
+prose-ol
+prose-ul
+prose-li:text-gray-800
+prose-table
+prose-th
+prose-tr
+prose-td
+prose-img
+
+`,u=({children:t,header:s,title:r})=>(o.useState(!1),e.jsx(e.Fragment,{children:e.jsxs("body",{className:"w-screen",children:[s,e.jsx("div",{className:"bg-cover absolute w-screen left-0 top-0 p-0 m-0 z-10 h-1/2",style:{background:`linear-gradient(to bottom, rgba(255, 255, 255, 0) 36.46%, #FFFFFF 100%), url(${s})`,backgroundSize:"cover"}}),e.jsx(a,{}),e.jsx("div",{className:"w-full flex justify-center items-center",children:e.jsxs("div",{className:"flex justify-between m-5 z-50 max-w-6xl grow",children:[e.jsx("a",{href:"https://2023.igem.wiki/lambert-ga/",className:"font-serif z-30 text-white ",children:e.jsx("p",{className:"bg-blackA-11 text-xl rounded-full p-2",children:"Lambert-GA"})}),e.jsx(l,{})]})}),e.jsx("div",{className:"flex justify-center w-full items-center",children:e.jsxs("div",{className:"w-full m-5 max-w-3xl ",children:[e.jsx("p",{className:"font-serif font-bold z-30 absolute top-1/2 -translate-y-1/2 lg:-translate-x-20 text-5xl sm:text-6xl md:text-7xl",children:r}),e.jsx("div",{className:"absolute top-1/2 translate-y-20 h-32 lg:-translate-x-20 lg:border-l-2 border-gray-900 z-50"}),e.jsx("div",{className:n+"mt-[50vh] break-words grow sm:mt-[44vh] md:mt-[40vh]",children:t})]})})]})}));export{u as default};
diff --git a/public/_astro/PageLayoutCompPDF.0f9b8991.js b/public/_astro/PageLayoutCompPDF.0f9b8991.js
new file mode 100644
index 0000000..9f67b94
--- /dev/null
+++ b/public/_astro/PageLayoutCompPDF.0f9b8991.js
@@ -0,0 +1,20 @@
+import{r as o}from"./index.453e6029.js";import{j as e}from"./jsx-runtime.4844db2a.js";import{P as l}from"./ProgressBar.be6b89f5.js";import{N as a}from"./Nav.d61123f0.js";import"./use-scroll.c1cc80d7.js";import"./motion.13ff9c9b.js";import"./extends.98964cd2.js";import"./index.c95c1985.js";import"./index.76dc11fb.js";const n=`
+prose max-w-5xl
+prose-h1:font-serif prose-h1:text-4xl
+prose-h2:font-sans prose-h2:text-2xl
+prose-h3:font-sans prose-h3:text-lg prose-h3:font-semibold prose-h3:text-yellow-800
+prose-p:font-sans prose-p:text-gray-900
+prose-a:font-sans prose-a:text-gray-900 prose-a:underline prose-a:font-normal prose-a:text-yellow-800
+prose-quoteless
+prose-strong
+prose-em
+prose-ol
+prose-ul
+prose-li:text-gray-800
+prose-table
+prose-th
+prose-tr
+prose-td
+prose-img
+
+`,g=({children:t,header:s,title:r})=>(o.useState(!1),e.jsx(e.Fragment,{children:e.jsxs("body",{className:"w-screen",children:[s,e.jsx("div",{className:"bg-cover absolute w-screen left-0 top-0 p-0 m-0 z-10 h-1/2 ",style:{background:`linear-gradient(to bottom, rgba(255, 255, 255, 0) 36.46%, #FFFFFF 100%), url(${s})`,backgroundSize:"cover"}}),e.jsx(l,{}),e.jsx("div",{className:"w-full flex justify-center items-center",children:e.jsxs("div",{className:"flex justify-between m-5 z-50 max-w-6xl grow",children:[e.jsx("a",{href:"https://2023.igem.wiki/lambert-ga/",className:"font-serif z-30 text-white ",children:e.jsx("p",{className:"bg-blackA-11 text-xl rounded-full p-2",children:"Lambert-GA"})}),e.jsx(a,{})]})}),e.jsx("div",{className:"flex justify-center w-full items-center",children:e.jsxs("div",{className:"w-full m-5 max-w-5xl ",children:[e.jsx("p",{className:"font-serif text-5xl sm:text-6xl md:text-7xl font-bold z-30 absolute top-1/2 -translate-y-1/2 ",children:r}),e.jsx("div",{className:n+"mt-[50vh] break-words grow",children:t})]})})]})}));export{g as default};
diff --git a/public/_astro/ProgressBar.be6b89f5.js b/public/_astro/ProgressBar.be6b89f5.js
new file mode 100644
index 0000000..265d618
--- /dev/null
+++ b/public/_astro/ProgressBar.be6b89f5.js
@@ -0,0 +1 @@
+import{j as s}from"./jsx-runtime.4844db2a.js";import{u as r}from"./use-scroll.c1cc80d7.js";import{m as t}from"./motion.13ff9c9b.js";function a(){const{scrollY:e,scrollYProgress:o}=r();return s.jsx("div",{className:"bg-slate-100 fixed top-0 left-0 right-0 pt-8",style:{marginTop:-40},children:s.jsx(t.div,{className:"bottom-0 left-0 pb-1 mt-2 right-0 bg-yellow-400 origin-left",style:{scaleX:o}})})}export{a as P};
diff --git a/public/_astro/TabReact.a7adacc1.js b/public/_astro/TabReact.a7adacc1.js
new file mode 100644
index 0000000..5ab0a5b
--- /dev/null
+++ b/public/_astro/TabReact.a7adacc1.js
@@ -0,0 +1 @@
+import{_ as I}from"./extends.98964cd2.js";import{r as t}from"./index.453e6029.js";import{$ as Z,a as G,b as ee,c as O,d as j,e as te,f as h,g as x,h as V,i as oe}from"./index.76dc11fb.js";import{j as v}from"./jsx-runtime.4844db2a.js";import"./index.c95c1985.js";const A="rovingFocusGroup.onEntryFocus",ne={bubbles:!1,cancelable:!0},N="RovingFocusGroup",[S,k,ae]=Z(N),[re,L]=G(N,[ae]),[ce,se]=re(N),de=t.forwardRef((e,n)=>t.createElement(S.Provider,{scope:e.__scopeRovingFocusGroup},t.createElement(S.Slot,{scope:e.__scopeRovingFocusGroup},t.createElement(fe,I({},e,{ref:n}))))),fe=t.forwardRef((e,n)=>{const{__scopeRovingFocusGroup:a,orientation:o,loop:c=!1,dir:i,currentTabStopId:r,defaultCurrentTabStopId:u,onCurrentTabStopIdChange:l,onEntryFocus:s,...f}=e,d=t.useRef(null),p=ee(n,d),w=O(i),[$=null,T]=j({prop:r,defaultProp:u,onChange:l}),[y,g]=t.useState(!1),m=te(s),q=k(a),R=t.useRef(!1),[H,P]=t.useState(0);return t.useEffect(()=>{const b=d.current;if(b)return b.addEventListener(A,m),()=>b.removeEventListener(A,m)},[m]),t.createElement(ce,{scope:a,orientation:o,dir:w,loop:c,currentTabStopId:$,onItemFocus:t.useCallback(b=>T(b),[T]),onItemShiftTab:t.useCallback(()=>g(!0),[]),onFocusableItemAdd:t.useCallback(()=>P(b=>b+1),[]),onFocusableItemRemove:t.useCallback(()=>P(b=>b-1),[])},t.createElement(h.div,I({tabIndex:y||H===0?-1:0,"data-orientation":o},f,{ref:p,style:{outline:"none",...e.style},onMouseDown:x(e.onMouseDown,()=>{R.current=!0}),onFocus:x(e.onFocus,b=>{const J=!R.current;if(b.target===b.currentTarget&&J&&!y){const D=new CustomEvent(A,ne);if(b.currentTarget.dispatchEvent(D),!D.defaultPrevented){const C=q().filter(E=>E.focusable),Q=C.find(E=>E.active),W=C.find(E=>E.id===$),X=[Q,W,...C].filter(Boolean).map(E=>E.ref.current);K(X)}}R.current=!1}),onBlur:x(e.onBlur,()=>g(!1))})))}),ie="RovingFocusGroupItem",be=t.forwardRef((e,n)=>{const{__scopeRovingFocusGroup:a,focusable:o=!0,active:c=!1,tabStopId:i,...r}=e,u=V(),l=i||u,s=se(ie,a),f=s.currentTabStopId===l,d=k(a),{onFocusableItemAdd:p,onFocusableItemRemove:w}=s;return t.useEffect(()=>{if(o)return p(),()=>w()},[o,p,w]),t.createElement(S.ItemSlot,{scope:a,id:l,focusable:o,active:c},t.createElement(h.span,I({tabIndex:f?0:-1,"data-orientation":s.orientation},r,{ref:n,onMouseDown:x(e.onMouseDown,$=>{o?s.onItemFocus(l):$.preventDefault()}),onFocus:x(e.onFocus,()=>s.onItemFocus(l)),onKeyDown:x(e.onKeyDown,$=>{if($.key==="Tab"&&$.shiftKey){s.onItemShiftTab();return}if($.target!==$.currentTarget)return;const T=$e($,s.orientation,s.dir);if(T!==void 0){$.preventDefault();let g=d().filter(m=>m.focusable).map(m=>m.ref.current);if(T==="last")g.reverse();else if(T==="prev"||T==="next"){T==="prev"&&g.reverse();const m=g.indexOf($.currentTarget);g=s.loop?ve(g,m+1):g.slice(m+1)}setTimeout(()=>K(g))}})})))}),ue={ArrowLeft:"prev",ArrowUp:"prev",ArrowRight:"next",ArrowDown:"next",PageUp:"first",Home:"first",PageDown:"last",End:"last"};function le(e,n){return n!=="rtl"?e:e==="ArrowLeft"?"ArrowRight":e==="ArrowRight"?"ArrowLeft":e}function $e(e,n,a){const o=le(e.key,a);if(!(n==="vertical"&&["ArrowLeft","ArrowRight"].includes(o))&&!(n==="horizontal"&&["ArrowUp","ArrowDown"].includes(o)))return ue[o]}function K(e){const n=document.activeElement;for(const a of e)if(a===n||(a.focus(),document.activeElement!==n))return}function ve(e,n){return e.map((a,o)=>e[(n+o)%e.length])}const pe=de,ge=be,U="Tabs",[me,De]=G(U,[L]),B=L(),[xe,M]=me(U),Ie=t.forwardRef((e,n)=>{const{__scopeTabs:a,value:o,onValueChange:c,defaultValue:i,orientation:r="horizontal",dir:u,activationMode:l="automatic",...s}=e,f=O(u),[d,p]=j({prop:o,onChange:c,defaultProp:i});return t.createElement(xe,{scope:a,baseId:V(),value:d,onValueChange:p,orientation:r,dir:f,activationMode:l},t.createElement(h.div,I({dir:f,"data-orientation":r},s,{ref:n})))}),Te="TabsList",Ee=t.forwardRef((e,n)=>{const{__scopeTabs:a,loop:o=!0,...c}=e,i=M(Te,a),r=B(a);return t.createElement(pe,I({asChild:!0},r,{orientation:i.orientation,dir:i.dir,loop:o}),t.createElement(h.div,I({role:"tablist","aria-orientation":i.orientation},c,{ref:n})))}),he="TabsTrigger",Fe=t.forwardRef((e,n)=>{const{__scopeTabs:a,value:o,disabled:c=!1,...i}=e,r=M(he,a),u=B(a),l=z(r.baseId,o),s=Y(r.baseId,o),f=o===r.value;return t.createElement(ge,I({asChild:!0},u,{focusable:!c,active:f}),t.createElement(h.button,I({type:"button",role:"tab","aria-selected":f,"aria-controls":s,"data-state":f?"active":"inactive","data-disabled":c?"":void 0,disabled:c,id:l},i,{ref:n,onMouseDown:x(e.onMouseDown,d=>{!c&&d.button===0&&d.ctrlKey===!1?r.onValueChange(o):d.preventDefault()}),onKeyDown:x(e.onKeyDown,d=>{[" ","Enter"].includes(d.key)&&r.onValueChange(o)}),onFocus:x(e.onFocus,()=>{const d=r.activationMode!=="manual";!f&&!c&&d&&r.onValueChange(o)})})))}),_e="TabsContent",we=t.forwardRef((e,n)=>{const{__scopeTabs:a,value:o,forceMount:c,children:i,...r}=e,u=M(_e,a),l=z(u.baseId,o),s=Y(u.baseId,o),f=o===u.value,d=t.useRef(f);return t.useEffect(()=>{const p=requestAnimationFrame(()=>d.current=!1);return()=>cancelAnimationFrame(p)},[]),t.createElement(oe,{present:c||f},({present:p})=>t.createElement(h.div,I({"data-state":f?"active":"inactive","data-orientation":u.orientation,role:"tabpanel","aria-labelledby":l,hidden:!p,id:s,tabIndex:0},r,{ref:n,style:{...e.style,animationDuration:d.current?"0s":void 0}}),p&&i))});function z(e,n){return`${e}-trigger-${n}`}function Y(e,n){return`${e}-content-${n}`}const ye=Ie,Re=Ee,F=Fe,_=we;function Ge({a:e,b:n,c:a,d:o,e:c}){return v.jsxs(ye,{defaultValue:"a",className:"w-full ",children:[v.jsxs(Re,{className:" h-[54px] bg-neutral-50 rounded-[33px] shadow border border-gray-6 flex flex-shrink mb-10",children:[v.jsx(F,{value:"a",className:"grow text-center text-gray-11 font-sans text-lg font-medium hover:bg-gray-3 rounded-full m-1 data-[state=active]:bg-gray-4 data-[state=active]:text-gray-12",children:"2022 Overview"}),v.jsx(F,{value:"b",className:"grow text-center text-gray-11 font-sans text-lg 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diff --git a/public/_astro/client.4f88357e.js b/public/_astro/client.4f88357e.js
new file mode 100644
index 0000000..4aa0e1e
--- /dev/null
+++ b/public/_astro/client.4f88357e.js
@@ -0,0 +1 @@
+import{r as e}from"./index.453e6029.js";import{r as E}from"./index.c95c1985.js";var f,l,u=E;l=u.createRoot,f=u.hydrateRoot;const n=({value:t,name:r,hydrate:a=!0})=>{if(!t)return null;const o=a?"astro-slot":"astro-static-slot";return e.createElement(o,{name:r,suppressHydrationWarning:!0,dangerouslySetInnerHTML:{__html:t}})};n.shouldComponentUpdate=()=>!1;function x(t){for(const r in t)if(r.startsWith("__reactContainer"))return r}const v=t=>(r,a,{default:o,...d},{client:m})=>{if(!t.hasAttribute("ssr"))return;const p={identifierPrefix:t.getAttribute("prefix")};for(const[c,y]of Object.entries(d))a[c]=e.createElement(n,{value:y,name:c});const s=e.createElement(r,a,o!=null?e.createElement(n,{value:o}):o),i=x(t);return i&&delete t[i],m==="only"?e.startTransition(()=>{l(t).render(s)}):e.startTransition(()=>{f(t,s,p)})};export{v as default};
diff --git a/public/_astro/components.41e5cae5.js b/public/_astro/components.41e5cae5.js
new file mode 100644
index 0000000..ceee290
--- /dev/null
+++ b/public/_astro/components.41e5cae5.js
@@ -0,0 +1,44 @@
+import{r as Ir,c as Bl}from"./index.453e6029.js";import{j as g}from"./jsx-runtime.4844db2a.js";import{u as Yl}from"./use-scroll.c1cc80d7.js";import{u as Ta,e as Vl,M as ql,a as Xl,g as Wl,c as Ul,f as Hl,h as Rs,m as Gl}from"./motion.13ff9c9b.js";function $l(l){const e=Ta(()=>Vl(l)),{isStatic:t}=Ir.useContext(ql);if(t){const[,r]=Ir.useState(l);Ir.useEffect(()=>e.on("change",r),[])}return e}const Zl=l=>typeof l=="object"&&l.mix,Kl=l=>Zl(l)?l.mix:void 0;function Ql(...l){const e=!Array.isArray(l[0]),t=e?0:-1,r=l[0+t],i=l[1+t],n=l[2+t],s=l[3+t],o=Xl(i,n,{mixer:Kl(n[0]),...s});return e?o(r):o}function Ca(l,e){const t=$l(e()),r=()=>t.set(e());return r(),Wl(()=>{const i=()=>Hl.update(r,!1,!0),n=l.map(s=>s.on("change",i));return()=>{n.forEach(s=>s()),Ul(r)}}),t}function Jl(l){Rs.current=[],l();const e=Ca(Rs.current,l);return Rs.current=void 0,e}function ec(l,e,t,r){if(typeof l=="function")return Jl(l);const i=typeof e=="function"?e:Ql(e,t,r);return Array.isArray(l)?oa(l,i):oa([l],([n])=>i(n))}function oa(l,e){const t=Ta(()=>[]);return Ca(l,()=>{t.length=0;const r=l.length;for(let i=0;i<r;i++)t[i]=l[i].get();return e(t)})}function zr(l){if(l===void 0)throw new ReferenceError("this hasn't been initialised - super() hasn't been called");return l}function Sa(l,e){l.prototype=Object.create(e.prototype),l.prototype.constructor=l,l.__proto__=e}/*!
+ * GSAP 3.12.2
+ * https://greensock.com
+ *
+ * @license Copyright 2008-2023, GreenSock. All rights reserved.
+ * Subject to the terms at https://greensock.com/standard-license or for
+ * Club GreenSock members, the agreement issued with that membership.
+ * @author: Jack Doyle, jack@greensock.com
+*/var Gt={autoSleep:120,force3D:"auto",nullTargetWarn:1,units:{lineHeight:""}},Gi={duration:.5,overwrite:!1,delay:0},so,mt,je,nr=1e8,ye=1/nr,qs=Math.PI*2,tc=qs/4,rc=0,Pa=Math.sqrt,ic=Math.cos,nc=Math.sin,nt=function(e){return typeof e=="string"},Fe=function(e){return typeof e=="function"},Br=function(e){return typeof e=="number"},oo=function(e){return typeof e>"u"},Pr=function(e){return typeof e=="object"},jt=function(e){return e!==!1},ao=function(){return typeof window<"u"},Kn=function(e){return Fe(e)||nt(e)},Ma=typeof ArrayBuffer=="function"&&ArrayBuffer.isView||function(){},xt=Array.isArray,Xs=/(?:-?\.?\d|\.)+/gi,Oa=/[-+=.]*\d+[.e\-+]*\d*[e\-+]*\d*/g,qi=/[-+=.]*\d+[.e-]*\d*[a-z%]*/g,Es=/[-+=.]*\d+\.?\d*(?:e-|e\+)?\d*/gi,Aa=/[+-]=-?[.\d]+/,Da=/[^,'"\[\]\s]+/gi,sc=/^[+\-=e\s\d]*\d+[.\d]*([a-z]*|%)\s*$/i,De,ir,Ws,lo,$t={},ts={},Na,Ra=function(e){return(ts=Mi(e,$t))&&Lt},co=function(e,t){return console.warn("Invalid property",e,"set to",t,"Missing plugin? 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index 0000000..2fc9398
--- /dev/null
+++ b/public/_astro/extends.98964cd2.js
@@ -0,0 +1 @@
+function e(){return e=Object.assign?Object.assign.bind():function(a){for(var n=1;n<arguments.length;n++){var t=arguments[n];for(var r in t)Object.prototype.hasOwnProperty.call(t,r)&&(a[r]=t[r])}return a},e.apply(this,arguments)}export{e as _};
diff --git a/public/_astro/index.08c7c3df.js b/public/_astro/index.08c7c3df.js
new file mode 100644
index 0000000..a4f8866
--- /dev/null
+++ b/public/_astro/index.08c7c3df.js
@@ -0,0 +1 @@
+var n={exports:{}},p,u;function f(){if(u)return p;u=1;var t="SECRET_DO_NOT_PASS_THIS_OR_YOU_WILL_BE_FIRED";return p=t,p}var a,y;function m(){if(y)return a;y=1;var t=f();function i(){}function c(){}return c.resetWarningCache=i,a=function(){function e(R,l,P,S,v,T){if(T!==t){var s=new Error("Calling PropTypes validators directly is not supported by the `prop-types` package. Use PropTypes.checkPropTypes() to call them. Read more at http://fb.me/use-check-prop-types");throw s.name="Invariant Violation",s}}e.isRequired=e;function r(){return e}var o={array:e,bigint:e,bool:e,func:e,number:e,object:e,string:e,symbol:e,any:e,arrayOf:r,element:e,elementType:e,instanceOf:r,node:e,objectOf:r,oneOf:r,oneOfType:r,shape:r,exact:r,checkPropTypes:c,resetWarningCache:i};return o.PropTypes=o,o},a}var h;function g(){return h||(h=1,n.exports=m()()),n.exports}export{g as r};
diff --git a/public/_astro/index.453e6029.js b/public/_astro/index.453e6029.js
new file mode 100644
index 0000000..2bf3cb7
--- /dev/null
+++ b/public/_astro/index.453e6029.js
@@ -0,0 +1,9 @@
+function A(e,t){for(var n=0;n<t.length;n++){const u=t[n];if(typeof u!="string"&&!Array.isArray(u)){for(const o in u)if(o!=="default"&&!(o in e)){const c=Object.getOwnPropertyDescriptor(u,o);c&&Object.defineProperty(e,o,c.get?c:{enumerable:!0,get:()=>u[o]})}}}return Object.freeze(Object.defineProperty(e,Symbol.toStringTag,{value:"Module"}))}var X=typeof globalThis<"u"?globalThis:typeof window<"u"?window:typeof global<"u"?global:typeof self<"u"?self:{};function D(e){return e&&e.__esModule&&Object.prototype.hasOwnProperty.call(e,"default")?e.default:e}var k={exports:{}},r={};/**
+ * @license React
+ * react.production.min.js
+ *
+ * Copyright (c) Facebook, Inc. and its affiliates.
+ *
+ * This source code is licensed under the MIT license found in the
+ * LICENSE file in the root directory of this source tree.
+ */var y=Symbol.for("react.element"),V=Symbol.for("react.portal"),F=Symbol.for("react.fragment"),M=Symbol.for("react.strict_mode"),U=Symbol.for("react.profiler"),q=Symbol.for("react.provider"),L=Symbol.for("react.context"),N=Symbol.for("react.forward_ref"),z=Symbol.for("react.suspense"),B=Symbol.for("react.memo"),H=Symbol.for("react.lazy"),g=Symbol.iterator;function G(e){return e===null||typeof e!="object"?null:(e=g&&e[g]||e["@@iterator"],typeof e=="function"?e:null)}var R={isMounted:function(){return!1},enqueueForceUpdate:function(){},enqueueReplaceState:function(){},enqueueSetState:function(){}},j=Object.assign,O={};function p(e,t,n){this.props=e,this.context=t,this.refs=O,this.updater=n||R}p.prototype.isReactComponent={};p.prototype.setState=function(e,t){if(typeof e!="object"&&typeof e!="function"&&e!=null)throw Error("setState(...): takes an object of state variables to update or a function which returns an object of state variables.");this.updater.enqueueSetState(this,e,t,"setState")};p.prototype.forceUpdate=function(e){this.updater.enqueueForceUpdate(this,e,"forceUpdate")};function C(){}C.prototype=p.prototype;function m(e,t,n){this.props=e,this.context=t,this.refs=O,this.updater=n||R}var S=m.prototype=new C;S.constructor=m;j(S,p.prototype);S.isPureReactComponent=!0;var E=Array.isArray,P=Object.prototype.hasOwnProperty,b={current:null},x={key:!0,ref:!0,__self:!0,__source:!0};function I(e,t,n){var u,o={},c=null,s=null;if(t!=null)for(u in t.ref!==void 0&&(s=t.ref),t.key!==void 0&&(c=""+t.key),t)P.call(t,u)&&!x.hasOwnProperty(u)&&(o[u]=t[u]);var f=arguments.length-2;if(f===1)o.children=n;else if(1<f){for(var i=Array(f),a=0;a<f;a++)i[a]=arguments[a+2];o.children=i}if(e&&e.defaultProps)for(u in f=e.defaultProps,f)o[u]===void 0&&(o[u]=f[u]);return{$$typeof:y,type:e,key:c,ref:s,props:o,_owner:b.current}}function W(e,t){return{$$typeof:y,type:e.type,key:t,ref:e.ref,props:e.props,_owner:e._owner}}function w(e){return typeof e=="object"&&e!==null&&e.$$typeof===y}function J(e){var t={"=":"=0",":":"=2"};return"$"+e.replace(/[=:]/g,function(n){return t[n]})}var $=/\/+/g;function v(e,t){return typeof e=="object"&&e!==null&&e.key!=null?J(""+e.key):t.toString(36)}function _(e,t,n,u,o){var c=typeof e;(c==="undefined"||c==="boolean")&&(e=null);var s=!1;if(e===null)s=!0;else switch(c){case"string":case"number":s=!0;break;case"object":switch(e.$$typeof){case y:case V:s=!0}}if(s)return s=e,o=o(s),e=u===""?"."+v(s,0):u,E(o)?(n="",e!=null&&(n=e.replace($,"$&/")+"/"),_(o,t,n,"",function(a){return a})):o!=null&&(w(o)&&(o=W(o,n+(!o.key||s&&s.key===o.key?"":(""+o.key).replace($,"$&/")+"/")+e)),t.push(o)),1;if(s=0,u=u===""?".":u+":",E(e))for(var f=0;f<e.length;f++){c=e[f];var i=u+v(c,f);s+=_(c,t,n,i,o)}else if(i=G(e),typeof i=="function")for(e=i.call(e),f=0;!(c=e.next()).done;)c=c.value,i=u+v(c,f++),s+=_(c,t,n,i,o);else if(c==="object")throw t=String(e),Error("Objects are not valid as a React child (found: "+(t==="[object Object]"?"object with keys {"+Object.keys(e).join(", ")+"}":t)+"). 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diff --git a/public/_astro/index.76dc11fb.js b/public/_astro/index.76dc11fb.js
new file mode 100644
index 0000000..37b6611
--- /dev/null
+++ b/public/_astro/index.76dc11fb.js
@@ -0,0 +1 @@
+import{_ as A}from"./extends.98964cd2.js";import{r as c,$ as _,R as h}from"./index.453e6029.js";import{r as T}from"./index.c95c1985.js";function w(e,n=[]){let t=[];function o(s,l){const u=c.createContext(l),a=t.length;t=[...t,l];function i(d){const{scope:p,children:m,...f}=d,C=p?.[e][a]||u,v=c.useMemo(()=>f,Object.values(f));return c.createElement(C.Provider,{value:v},m)}function $(d,p){const m=p?.[e][a]||u,f=c.useContext(m);if(f)return f;if(l!==void 0)return l;throw new Error(`\`${d}\` must be used within \`${s}\``)}return i.displayName=s+"Provider",[i,$]}const r=()=>{const s=t.map(l=>c.createContext(l));return function(u){const a=u?.[e]||s;return c.useMemo(()=>({[`__scope${e}`]:{...u,[e]:a}}),[u,a])}};return r.scopeName=e,[o,U(r,...n)]}function U(...e){const n=e[0];if(e.length===1)return n;const t=()=>{const o=e.map(r=>({useScope:r(),scopeName:r.scopeName}));return function(s){const l=o.reduce((u,{useScope:a,scopeName:i})=>{const d=a(s)[`__scope${i}`];return{...u,...d}},{});return 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diff --git a/public/_astro/index.c95c1985.js b/public/_astro/index.c95c1985.js
new file mode 100644
index 0000000..8f9046a
--- /dev/null
+++ b/public/_astro/index.c95c1985.js
@@ -0,0 +1,24 @@
+import{r as za,g as Pa}from"./index.453e6029.js";var Co={exports:{}},ve={},xo={exports:{}},_o={};/**
+ * @license React
+ * scheduler.production.min.js
+ *
+ * Copyright (c) Facebook, Inc. and its affiliates.
+ *
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diff --git a/public/_astro/jsx-runtime.4844db2a.js b/public/_astro/jsx-runtime.4844db2a.js
new file mode 100644
index 0000000..5e7f611
--- /dev/null
+++ b/public/_astro/jsx-runtime.4844db2a.js
@@ -0,0 +1,9 @@
+import{r as l}from"./index.453e6029.js";var f={exports:{}},n={};/**
+ * @license React
+ * react-jsx-runtime.production.min.js
+ *
+ * Copyright (c) Facebook, Inc. and its affiliates.
+ *
+ * This source code is licensed under the MIT license found in the
+ * LICENSE file in the root directory of this source tree.
+ */var u=l,m=Symbol.for("react.element"),x=Symbol.for("react.fragment"),y=Object.prototype.hasOwnProperty,a=u.__SECRET_INTERNALS_DO_NOT_USE_OR_YOU_WILL_BE_FIRED.ReactCurrentOwner,v={key:!0,ref:!0,__self:!0,__source:!0};function i(t,r,p){var e,o={},s=null,_=null;p!==void 0&&(s=""+p),r.key!==void 0&&(s=""+r.key),r.ref!==void 0&&(_=r.ref);for(e in r)y.call(r,e)&&!v.hasOwnProperty(e)&&(o[e]=r[e]);if(t&&t.defaultProps)for(e in r=t.defaultProps,r)o[e]===void 0&&(o[e]=r[e]);return{$$typeof:m,type:t,key:s,ref:_,props:o,_owner:a.current}}n.Fragment=x;n.jsx=i;n.jsxs=i;f.exports=n;var d=f.exports;export{d as j};
diff --git a/public/_astro/motion.13ff9c9b.js b/public/_astro/motion.13ff9c9b.js
new file mode 100644
index 0000000..df50f3d
--- /dev/null
+++ b/public/_astro/motion.13ff9c9b.js
@@ -0,0 +1 @@
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r&&r.getProps().transformTemplate}willUpdate(r=!0){if(this.root.hasTreeAnimated=!0,this.root.isUpdateBlocked()){this.options.onExitComplete&&this.options.onExitComplete();return}if(!this.root.isUpdating&&this.root.startUpdate(),this.isLayoutDirty)return;this.isLayoutDirty=!0;for(let u=0;u<this.path.length;u++){const h=this.path[u];h.shouldResetTransform=!0,h.updateScroll("snapshot"),h.options.layoutRoot&&h.willUpdate(!1)}const{layoutId:a,layout:c}=this.options;if(a===void 0&&!c)return;const l=this.getTransformTemplate();this.prevTransformTemplateValue=l?l(this.latestValues,""):void 0,this.updateSnapshot(),r&&this.notifyListeners("willUpdate")}update(){if(this.updateScheduled=!1,this.isUpdateBlocked()){this.unblockUpdate(),this.clearAllSnapshots(),this.nodes.forEach(qn);return}this.isUpdating||this.nodes.forEach(oc),this.isUpdating=!1,this.nodes.forEach(ac),this.nodes.forEach(ec),this.nodes.forEach(nc),this.clearAllSnapshots();const 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r&&(c=this.removeTransform(c)),pc(c),{animationId:this.root.animationId,measuredBox:a,layoutBox:c,latestValues:{},source:this.id}}measurePageBox(){const{visualElement:r}=this.options;if(!r)return R();const a=r.measureViewportBox(),{scroll:c}=this.root;return c&&(Q(a.x,c.offset.x),Q(a.y,c.offset.y)),a}removeElementScroll(r){const a=R();U(a,r);for(let c=0;c<this.path.length;c++){const l=this.path[c],{scroll:u,options:h}=l;if(l!==this.root&&u&&h.layoutScroll){if(u.isRoot){U(a,r);const{scroll:f}=this.root;f&&(Q(a.x,-f.offset.x),Q(a.y,-f.offset.y))}Q(a.x,u.offset.x),Q(a.y,u.offset.y)}}return a}applyTransform(r,a=!1){const c=R();U(c,r);for(let l=0;l<this.path.length;l++){const u=this.path[l];!a&&u.options.layoutScroll&&u.scroll&&u!==u.root&&pt(c,{x:-u.scroll.offset.x,y:-u.scroll.offset.y}),ot(u.latestValues)&&pt(c,u.latestValues)}return ot(this.latestValues)&&pt(c,this.latestValues),c}removeTransform(r){const a=R();U(a,r);for(let c=0;c<this.path.length;c++){const l=this.path[c];if(!l.instance||!ot(l.latestValues))continue;Se(l.latestValues)&&l.updateSnapshot();const u=R(),h=l.measurePageBox();U(u,h),$n(a,l.latestValues,l.snapshot?l.snapshot.layoutBox:void 0,u)}return ot(this.latestValues)&&$n(a,this.latestValues),a}setTargetDelta(r){this.targetDelta=r,this.root.scheduleUpdateProjection(),this.isProjectionDirty=!0}setOptions(r){this.options={...this.options,...r,crossfade:r.crossfade!==void 0?r.crossfade:!0}}clearMeasurements(){this.scroll=void 0,this.layout=void 0,this.snapshot=void 0,this.prevTransformTemplateValue=void 0,this.targetDelta=void 0,this.target=void 0,this.isLayoutDirty=!1}forceRelativeParentToResolveTarget(){this.relativeParent&&this.relativeParent.resolvedRelativeTargetAt!==E.timestamp&&this.relativeParent.resolveTargetDelta(!0)}resolveTargetDelta(r=!1){var a;const c=this.getLead();this.isProjectionDirty||(this.isProjectionDirty=c.isProjectionDirty),this.isTransformDirty||(this.isTransformDirty=c.isTransformDirty),this.isSharedProjectionDirty||(this.isSharedProjectionDirty=c.isSharedProjectionDirty);const l=!!this.resumingFrom||this!==c;if(!(r||l&&this.isSharedProjectionDirty||this.isProjectionDirty||!((a=this.parent)===null||a===void 0)&&a.isProjectionDirty||this.attemptToResolveRelativeTarget))return;const{layout:h,layoutId:f}=this.options;if(!(!this.layout||!(h||f))){if(this.resolvedRelativeTargetAt=E.timestamp,!this.targetDelta&&!this.relativeTarget){const d=this.getClosestProjectingParent();d&&d.layout&&this.animationProgress!==1?(this.relativeParent=d,this.forceRelativeParentToResolveTarget(),this.relativeTarget=R(),this.relativeTargetOrigin=R(),Ct(this.relativeTargetOrigin,this.layout.layoutBox,d.layout.layoutBox),U(this.relativeTarget,this.relativeTargetOrigin)):this.relativeParent=this.relativeTarget=void 0}if(!(!this.relativeTarget&&!this.targetDelta)){if(this.target||(this.target=R(),this.targetWithTransforms=R()),this.relativeTarget&&this.relativeTargetOrigin&&this.relativeParent&&this.relativeParent.target?(this.forceRelativeParentToResolveTarget(),ya(this.target,this.relativeTarget,this.relativeParent.target)):this.targetDelta?(this.resumingFrom?this.target=this.applyTransform(this.layout.layoutBox):U(this.target,this.layout.layoutBox),pi(this.target,this.targetDelta)):U(this.target,this.layout.layoutBox),this.attemptToResolveRelativeTarget){this.attemptToResolveRelativeTarget=!1;const d=this.getClosestProjectingParent();d&&!!d.resumingFrom==!!this.resumingFrom&&!d.options.layoutScroll&&d.target&&this.animationProgress!==1?(this.relativeParent=d,this.forceRelativeParentToResolveTarget(),this.relativeTarget=R(),this.relativeTargetOrigin=R(),Ct(this.relativeTargetOrigin,this.target,d.target),U(this.relativeTarget,this.relativeTargetOrigin)):this.relativeParent=this.relativeTarget=void 0}at.resolvedTargetDeltas++}}}getClosestProjectingParent(){if(!(!this.parent||Se(this.parent.latestValues)||mi(this.parent.latestValues)))return this.parent.isProjecting()?this.parent:this.parent.getClosestProjectingParent()}isProjecting(){return!!((this.relativeTarget||this.targetDelta||this.options.layoutRoot)&&this.layout)}calcProjection(){var r;const a=this.getLead(),c=!!this.resumingFrom||this!==a;let l=!0;if((this.isProjectionDirty||!((r=this.parent)===null||r===void 0)&&r.isProjectionDirty)&&(l=!1),c&&(this.isSharedProjectionDirty||this.isTransformDirty)&&(l=!1),this.resolvedRelativeTargetAt===E.timestamp&&(l=!1),l)return;const{layout:u,layoutId:h}=this.options;if(this.isTreeAnimating=!!(this.parent&&this.parent.isTreeAnimating||this.currentAnimation||this.pendingAnimation),this.isTreeAnimating||(this.targetDelta=this.relativeTarget=void 0),!this.layout||!(u||h))return;U(this.layoutCorrected,this.layout.layoutBox);const f=this.treeScale.x,d=this.treeScale.y;Ca(this.layoutCorrected,this.treeScale,this.path,c),a.layout&&!a.target&&(this.treeScale.x!==1||this.treeScale.y!==1)&&(a.target=a.layout.layoutBox);const{target:m}=a;if(!m){this.projectionTransform&&(this.projectionDelta=mt(),this.projectionTransform="none",this.scheduleRender());return}this.projectionDelta||(this.projectionDelta=mt(),this.projectionDeltaWithTransform=mt());const p=this.projectionTransform;At(this.projectionDelta,this.layoutCorrected,m,this.latestValues),this.projectionTransform=_n(this.projectionDelta,this.treeScale),(this.projectionTransform!==p||this.treeScale.x!==f||this.treeScale.y!==d)&&(this.hasProjected=!0,this.scheduleRender(),this.notifyListeners("projectionUpdate",m)),at.recalculatedProjection++}hide(){this.isVisible=!1}show(){this.isVisible=!0}scheduleRender(r=!0){if(this.options.scheduleRender&&this.options.scheduleRender(),r){const a=this.getStack();a&&a.scheduleRender()}this.resumingFrom&&!this.resumingFrom.instance&&(this.resumingFrom=void 0)}setAnimationOrigin(r,a=!1){const c=this.snapshot,l=c?c.latestValues:{},u={...this.latestValues},h=mt();(!this.relativeParent||!this.relativeParent.options.layoutRoot)&&(this.relativeTarget=this.relativeTargetOrigin=void 0),this.attemptToResolveRelativeTarget=!a;const f=R(),d=c?c.source:void 0,m=this.layout?this.layout.source:void 0,p=d!==m,x=this.getStack(),V=!x||x.members.length<=1,y=!!(p&&!V&&this.options.crossfade===!0&&!this.path.some(dc));this.animationProgress=0;let g;this.mixTargetDelta=P=>{const b=P/1e3;Qn(h.x,r.x,b),Qn(h.y,r.y,b),this.setTargetDelta(h),this.relativeTarget&&this.relativeTargetOrigin&&this.layout&&this.relativeParent&&this.relativeParent.layout&&(Ct(f,this.layout.layoutBox,this.relativeParent.layout.layoutBox),fc(this.relativeTarget,this.relativeTargetOrigin,f,b),g&&Ka(this.relativeTarget,g)&&(this.isProjectionDirty=!1),g||(g=R()),U(g,this.relativeTarget)),p&&(this.animationValues=u,Na(u,l,this.latestValues,b,y,V)),this.root.scheduleUpdateProjection(),this.scheduleRender(),this.animationProgress=b},this.mixTargetDelta(this.options.layoutRoot?1e3:0)}startAnimation(r){this.notifyListeners("animationStart"),this.currentAnimation&&this.currentAnimation.stop(),this.resumingFrom&&this.resumingFrom.currentAnimation&&this.resumingFrom.currentAnimation.stop(),this.pendingAnimation&&(_(this.pendingAnimation),this.pendingAnimation=void 0),this.pendingAnimation=A.update(()=>{Nt.hasAnimatedSinceResize=!0,this.currentAnimation=Ja(0,Yn,{...r,onUpdate:a=>{this.mixTargetDelta(a),r.onUpdate&&r.onUpdate(a)},onComplete:()=>{r.onComplete&&r.onComplete(),this.completeAnimation()}}),this.resumingFrom&&(this.resumingFrom.currentAnimation=this.currentAnimation),this.pendingAnimation=void 0})}completeAnimation(){this.resumingFrom&&(this.resumingFrom.currentAnimation=void 0,this.resumingFrom.preserveOpacity=void 0);const r=this.getStack();r&&r.exitAnimationComplete(),this.resumingFrom=this.currentAnimation=this.animationValues=void 0,this.notifyListeners("animationComplete")}finishAnimation(){this.currentAnimation&&(this.mixTargetDelta&&this.mixTargetDelta(Yn),this.currentAnimation.stop()),this.completeAnimation()}applyTransformsToTarget(){const r=this.getLead();let{targetWithTransforms:a,target:c,layout:l,latestValues:u}=r;if(!(!a||!c||!l)){if(this!==r&&this.layout&&l&&Vi(this.options.animationType,this.layout.layoutBox,l.layoutBox)){c=this.target||R();const h=I(this.layout.layoutBox.x);c.x.min=r.target.x.min,c.x.max=c.x.min+h;const f=I(this.layout.layoutBox.y);c.y.min=r.target.y.min,c.y.max=c.y.min+f}U(a,c),pt(a,u),At(this.projectionDeltaWithTransform,this.layoutCorrected,a,u)}}registerSharedNode(r,a){this.sharedNodes.has(r)||this.sharedNodes.set(r,new _a),this.sharedNodes.get(r).add(a);const l=a.options.initialPromotionConfig;a.promote({transition:l?l.transition:void 0,preserveFollowOpacity:l&&l.shouldPreserveFollowOpacity?l.shouldPreserveFollowOpacity(a):void 0})}isLead(){const r=this.getStack();return r?r.lead===this:!0}getLead(){var r;const{layoutId:a}=this.options;return a?((r=this.getStack())===null||r===void 0?void 0:r.lead)||this:this}getPrevLead(){var r;const{layoutId:a}=this.options;return a?(r=this.getStack())===null||r===void 0?void 0:r.prevLead:void 0}getStack(){const{layoutId:r}=this.options;if(r)return this.root.sharedNodes.get(r)}promote({needsReset:r,transition:a,preserveFollowOpacity:c}={}){const l=this.getStack();l&&l.promote(this,c),r&&(this.projectionDelta=void 0,this.needsReset=!0),a&&this.setOptions({transition:a})}relegate(){const r=this.getStack();return r?r.relegate(this):!1}resetRotation(){const{visualElement:r}=this.options;if(!r)return;let a=!1;const{latestValues:c}=r;if((c.rotate||c.rotateX||c.rotateY||c.rotateZ)&&(a=!0),!a)return;const l={};for(let u=0;u<Xn.length;u++){const h="rotate"+Xn[u];c[h]&&(l[h]=c[h],r.setStaticValue(h,0))}r.render();for(const u in l)r.setStaticValue(u,l[u]);r.scheduleRender()}getProjectionStyles(r={}){var a,c;const l={};if(!this.instance||this.isSVG)return l;if(this.isVisible)l.visibility="";else return{visibility:"hidden"};const u=this.getTransformTemplate();if(this.needsReset)return this.needsReset=!1,l.opacity="",l.pointerEvents=Ut(r.pointerEvents)||"",l.transform=u?u(this.latestValues,""):"none",l;const h=this.getLead();if(!this.projectionDelta||!this.layout||!h.target){const p={};return this.options.layoutId&&(p.opacity=this.latestValues.opacity!==void 0?this.latestValues.opacity:1,p.pointerEvents=Ut(r.pointerEvents)||""),this.hasProjected&&!ot(this.latestValues)&&(p.transform=u?u({},""):"none",this.hasProjected=!1),p}const f=h.animationValues||h.latestValues;this.applyTransformsToTarget(),l.transform=_n(this.projectionDeltaWithTransform,this.treeScale,f),u&&(l.transform=u(f,l.transform));const{x:d,y:m}=this.projectionDelta;l.transformOrigin=`${d.origin*100}% ${m.origin*100}% 0`,h.animationValues?l.opacity=h===this?(c=(a=f.opacity)!==null&&a!==void 0?a:this.latestValues.opacity)!==null&&c!==void 0?c:1:this.preserveOpacity?this.latestValues.opacity:f.opacityExit:l.opacity=h===this?f.opacity!==void 0?f.opacity:"":f.opacityExit!==void 0?f.opacityExit:0;for(const p in Gt){if(f[p]===void 0)continue;const{correct:x,applyTo:V}=Gt[p],y=l.transform==="none"?f[p]:x(f[p],h);if(V){const g=V.length;for(let P=0;P<g;P++)l[V[P]]=y}else l[p]=y}return this.options.layoutId&&(l.pointerEvents=h===this?Ut(r.pointerEvents)||"":"none"),l}clearSnapshot(){this.resumeFrom=this.snapshot=void 0}resetTree(){this.root.nodes.forEach(r=>{var a;return(a=r.currentAnimation)===null||a===void 0?void 0:a.stop()}),this.root.nodes.forEach(qn),this.root.sharedNodes.clear()}}}function ec(t){t.updateLayout()}function nc(t){var e;const n=((e=t.resumeFrom)===null||e===void 0?void 0:e.snapshot)||t.snapshot;if(t.isLead()&&t.layout&&n&&t.hasListeners("didUpdate")){const{layoutBox:s,measuredBox:i}=t.layout,{animationType:o}=t.options,r=n.source!==t.layout.source;o==="size"?N(h=>{const f=r?n.measuredBox[h]:n.layoutBox[h],d=I(f);f.min=s[h].min,f.max=f.min+d}):Vi(o,n.layoutBox,s)&&N(h=>{const f=r?n.measuredBox[h]:n.layoutBox[h],d=I(s[h]);f.max=f.min+d,t.relativeTarget&&!t.currentAnimation&&(t.isProjectionDirty=!0,t.relativeTarget[h].max=t.relativeTarget[h].min+d)});const a=mt();At(a,s,n.layoutBox);const c=mt();r?At(c,t.applyTransform(i,!0),n.measuredBox):At(c,s,n.layoutBox);const l=!Pi(a);let u=!1;if(!t.resumeFrom){const h=t.getClosestProjectingParent();if(h&&!h.resumeFrom){const{snapshot:f,layout:d}=h;if(f&&d){const m=R();Ct(m,n.layoutBox,f.layoutBox);const p=R();Ct(p,s,d.layoutBox),Ti(m,p)||(u=!0),h.options.layoutRoot&&(t.relativeTarget=p,t.relativeTargetOrigin=m,t.relativeParent=h)}}}t.notifyListeners("didUpdate",{layout:s,snapshot:n,delta:c,layoutDelta:a,hasLayoutChanged:l,hasRelativeTargetChanged:u})}else if(t.isLead()){const{onExitComplete:s}=t.options;s&&s()}t.options.transition=void 0}function sc(t){at.totalNodes++,t.parent&&(t.isProjecting()||(t.isProjectionDirty=t.parent.isProjectionDirty),t.isSharedProjectionDirty||(t.isSharedProjectionDirty=!!(t.isProjectionDirty||t.parent.isProjectionDirty||t.parent.isSharedProjectionDirty)),t.isTransformDirty||(t.isTransformDirty=t.parent.isTransformDirty))}function ic(t){t.isProjectionDirty=t.isSharedProjectionDirty=t.isTransformDirty=!1}function rc(t){t.clearSnapshot()}function qn(t){t.clearMeasurements()}function oc(t){t.isLayoutDirty=!1}function ac(t){const{visualElement:e}=t.options;e&&e.getProps().onBeforeLayoutMeasure&&e.notify("BeforeLayoutMeasure"),t.resetTransform()}function Zn(t){t.finishAnimation(),t.targetDelta=t.relativeTarget=t.target=void 0,t.isProjectionDirty=!0}function cc(t){t.resolveTargetDelta()}function lc(t){t.calcProjection()}function uc(t){t.resetRotation()}function hc(t){t.removeLeadSnapshot()}function Qn(t,e,n){t.translate=D(e.translate,0,n),t.scale=D(e.scale,1,n),t.origin=e.origin,t.originPoint=e.originPoint}function Jn(t,e,n,s){t.min=D(e.min,n.min,s),t.max=D(e.max,n.max,s)}function fc(t,e,n,s){Jn(t.x,e.x,n.x,s),Jn(t.y,e.y,n.y,s)}function dc(t){return t.animationValues&&t.animationValues.opacityExit!==void 0}const mc={duration:.45,ease:[.4,0,.1,1]},ts=t=>typeof navigator<"u"&&navigator.userAgent.toLowerCase().includes(t),es=ts("applewebkit/")&&!ts("chrome/")?Math.round:L;function ns(t){t.min=es(t.min),t.max=es(t.max)}function pc(t){ns(t.x),ns(t.y)}function Vi(t,e,n){return t==="position"||t==="preserve-aspect"&&!be(Kn(e),Kn(n),.2)}const gc=bi({attachResizeListener:(t,e)=>$(t,"resize",e),measureScroll:()=>({x:document.documentElement.scrollLeft||document.body.scrollLeft,y:document.documentElement.scrollTop||document.body.scrollTop}),checkIsScrollRoot:()=>!0}),me={current:void 0},Si=bi({measureScroll:t=>({x:t.scrollLeft,y:t.scrollTop}),defaultParent:()=>{if(!me.current){const t=new 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diff --git a/public/_astro/teamMembers.55c4f4e7.js b/public/_astro/teamMembers.55c4f4e7.js
new file mode 100644
index 0000000..662ffc8
--- /dev/null
+++ b/public/_astro/teamMembers.55c4f4e7.js
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In 2021 I completed my MAT in Secondary Education Chemistry and started teaching at Lambert High School that fall. When I came in to set up my classroom before the semester began, I was surprised by the buzz of activity in the room across the hall. I peeked into Janet Standeven’s Biotechnology classroom and saw high schoolers doing real science...on their summer break...with a passion and depth of knowledge that I hadn’t been around since working in those university labs. As a child, I wanted to run research projects out of the garage like Meg’s parents in A Wrinkle in Time. Getting to collaborate with student scientists every day and take part in their journey may be even better :)",role:"Primary Investigator"},{name:"Dr. Brittney Denier Cantrell",imgSrc:"https://static.igem.wiki/teams/4683/wiki/teampage/cantrell.jpg",description:"It has been my privilege to support Lambert iGEM since 2012 and advise teams with my amazing counterpart, Janet Standeven, since 2015. 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diff --git a/public/attributions/index.html b/public/attributions/index.html
new file mode 100644
index 0000000..beab992
--- /dev/null
+++ b/public/attributions/index.html
@@ -0,0 +1,45 @@
+<!DOCTYPE html>
+<div>
+
+ <html lang="en">
+ <head>
+ <meta charset="UTF-8">
+ <meta name="viewport" content="width=device-width">
+ <link rel="icon" type="image/svg+xml" href="/favicon.svg">
+ <meta name="generator" content="Astro v2.10.15">
+ <!-- <link rel="preconnect" href="https://fonts.googleapis.com" />
+ <link rel="preconnect" href="https://fonts.gstatic.com" crossorigin /> -->
+
+ <link href="../components/fonts.css" rel="stylesheet">
+ <title></title>
+ <link rel="stylesheet" href="/lambert-ga/lambert-ga/_astro/RCAERCA.96298b81.css" />
+<link rel="stylesheet" href="/lambert-ga/lambert-ga/_astro/attributions.b36d46b4.css" /></head>
+ <div class="w-full flex justify-center">
+ <div class="w-full max-w-5xl mx-16 mt-8">
+ <div class="flex justify-between items-center">
+ <a href="https://2023.igem.wiki/lambert-ga/" class="font-serif z-30 ">
+ Lambert-GA
+ </a>
+ <style>astro-island,astro-slot,astro-static-slot{display:contents}</style><script>(()=>{var e=async t=>{await(await t())()};(self.Astro||(self.Astro={})).load=e;window.dispatchEvent(new Event("astro:load"));})();;(()=>{var d;{let p={0:t=>u(t),1:t=>l(t),2:t=>new RegExp(t),3:t=>new Date(t),4:t=>new Map(l(t)),5:t=>new Set(l(t)),6:t=>BigInt(t),7:t=>new URL(t),8:t=>new Uint8Array(t),9:t=>new Uint16Array(t),10:t=>new Uint32Array(t)},h=t=>{let[e,n]=t;return e in p?p[e](n):void 0},l=t=>t.map(h),u=t=>typeof t!="object"||t===null?t:Object.fromEntries(Object.entries(t).map(([e,n])=>[e,h(n)]));customElements.get("astro-island")||customElements.define("astro-island",(d=class extends HTMLElement{constructor(){super(...arguments);this.hydrate=async()=>{var i;if(!this.hydrator||!this.isConnected)return;let e=(i=this.parentElement)==null?void 0:i.closest("astro-island[ssr]");if(e){e.addEventListener("astro:hydrate",this.hydrate,{once:!0});return}let 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renderer-url="/lambert-ga/_astro/client.4f88357e.js" props="{}" ssr="" client="load" opts="{"name":"NavHome","value":true}" await-children=""><div class="flex justify-end"><link href="../components/fonts.css" rel="stylesheet"/><nav aria-label="Main" data-orientation="horizontal" dir="ltr" class="relative "><div style="position:relative"><ul data-orientation="horizontal" class="flex flex-row rounded-lg bg-white dark:bg-gray-800 p-2 space-x-2" dir="ltr"><div><li><button id="radix-:r2R2:-trigger-radix-:r2R1e:" data-state="closed" aria-expanded="false" aria-controls="radix-:r2R2:-content-radix-:r2R1e:" class="px-3 py-2 rounded-md hover:bg-gray-100 dark:hover:bg-gray-900 font-medium text-gray-700 dark:text-gray-100 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Team</button></li></div><div><li><button id="radix-:r2R2:-trigger-radix-:r2R2e:" data-state="closed" aria-expanded="false" aria-controls="radix-:r2R2:-content-radix-:r2R2e:" class="px-3 py-2 rounded-md hover:bg-gray-100 dark:hover:bg-gray-900 font-medium text-gray-700 dark:text-gray-100 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Project</button></li></div><div><li><button id="radix-:r2R2:-trigger-radix-:r2R3e:" data-state="closed" aria-expanded="false" aria-controls="radix-:r2R2:-content-radix-:r2R3e:" class="px-3 py-2 rounded-md hover:bg-gray-100 dark:hover:bg-gray-900 font-medium text-gray-700 dark:text-gray-100 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Wetlab</button></li></div><div><li><button id="radix-:r2R2:-trigger-radix-:r2R4e:" data-state="closed" aria-expanded="false" aria-controls="radix-:r2R2:-content-radix-:r2R4e:" class="px-3 py-2 rounded-md hover:bg-gray-100 dark:hover:bg-gray-900 font-medium text-gray-700 dark:text-gray-100 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Drylab</button></li></div><div><li><button id="radix-:r2R2:-trigger-radix-:r2R5e:" data-state="closed" aria-expanded="false" aria-controls="radix-:r2R2:-content-radix-:r2R5e:" class="px-3 py-2 rounded-md hover:bg-gray-100 dark:hover:bg-gray-900 font-medium text-gray-700 dark:text-gray-100 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Human Practices</button></li></div></ul></div></nav></div></astro-island>
+ </div>
+
+ <div class="mt-8">
+
+ <iframe style="width: 100%; height: 100vh;" className="w-screen h-screen p-5" src="https://attributions.igem.org?team=Lambert-GA&year=2023"></iframe>
+ <div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/attributions/untitled-design.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic"></div></div>
+
+
+ </div>
+
+ </div>
+
+ </div>
+
+
+
+
+
+</html>
+
+ <footer class="relative bg-[#292524] pt-8 pb-6 w-screen"><div class="w-full max-w-5xl mx-16 mt-8 container mx-auto px-4 py-10"><div class="flex flex-wrap text-left lg:text-left"><div class="w-full lg:w-6/12 px-4"><h1 class="text-4xl text-white font-serif font-light">Lambert-GA</h1><br/><h3 class="text-xs w-3/4 mt-0 mb-2 font-sans text-white">© <span id="get-current-year">2023</span> - Content on this site is licensed under a<a href="https://creativecommons.org/licenses/by/4.0/" class="underline hover:text-gray-300" target="_blank">Creative Commons Attribution 4.0 International license.</a><br/><br/>The repository used to create this website is available at<a href="https://gitlab.igem.org/2023/lambert-ga" class="underline hover:text-gray-300" target="_blank">https://gitlab.igem.org/2023/lambert-ga</a></h3></div><div class="w-full lg:w-5/12 px-4"><div class="flex flex-wrap items-top mb-6"><div class="w-full lg:w-4/12 px-4 ml-auto"><ul class="w-full list-unstyled"><li class="flex"><svg width="25" height="25" viewBox="0 0 25 25" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M18.7498 2.0835H15.6248C14.2435 2.0835 12.9187 2.63223 11.942 3.60898C10.9652 4.58573 10.4165 5.91049 10.4165 7.29183V10.4168H7.2915V14.5835H10.4165V22.9168H14.5832V14.5835H17.7082L18.7498 10.4168H14.5832V7.29183C14.5832 7.01556 14.6929 6.75061 14.8883 6.55526C15.0836 6.35991 15.3486 6.25016 15.6248 6.25016H18.7498V2.0835Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">Lambert iGEM</a></li><li class="flex"><svg width="25" height="28" viewBox="0 0 25 28" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M22.9168 4.52889C22.9168 4.52889 22.1877 6.90661 20.8335 8.37853C22.5002 19.701 11.0418 27.9664 2.0835 21.5126C4.37516 21.6258 6.66683 20.8332 8.3335 19.2481C3.12516 17.5497 0.520996 10.8695 3.12516 5.66114C5.41683 8.60498 8.9585 10.3033 12.5002 10.1901C11.5627 5.43469 16.6668 2.7173 19.7918 5.88759C20.9377 5.88759 22.9168 4.52889 22.9168 4.52889Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">@lamberths_iGEM</a></li><li class="flex "><svg width="25" height="25" viewBox="0 0 25 25" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M17.7085 2.21484H7.29183C4.41535 2.21484 2.0835 4.49911 2.0835 7.31688V17.521C2.0835 20.3387 4.41535 22.623 7.29183 22.623H17.7085C20.585 22.623 22.9168 20.3387 22.9168 17.521V7.31688C22.9168 4.49911 20.585 2.21484 17.7085 2.21484Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path><path d="M16.6667 11.7759C16.7952 12.6251 16.6472 13.4924 16.2435 14.2545C15.8399 15.0165 15.2012 15.6345 14.4184 16.0205C13.6355 16.4064 12.7484 16.5408 11.8831 16.4044C11.0179 16.268 10.2185 15.8678 9.59881 15.2608C8.9791 14.6537 8.57057 13.8707 8.43134 13.0231C8.29211 12.1754 8.42926 11.3064 8.82328 10.5395C9.2173 9.77269 9.84813 9.14706 10.626 8.75165C11.404 8.35625 12.2893 8.21119 13.1563 8.33712C14.0406 8.46558 14.8592 8.86923 15.4914 9.48846C16.1235 10.1077 16.5356 10.9096 16.6667 11.7759Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">@lambertigem</a></li><li class="flex"><svg width="25" height="24" viewBox="0 0 25 24" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M12.5002 18.2922C10.1043 18.2922 5.8335 18.1028 4.06266 17.7241C3.3335 17.5347 2.81266 17.0612 2.60433 16.3983C2.29183 15.3566 2.0835 13.1786 2.0835 11.6635C2.0835 10.1483 2.29183 7.97027 2.60433 6.92861C2.81266 6.26573 3.3335 5.79224 4.06266 5.60285C5.8335 5.22406 10.1043 5.03467 12.5002 5.03467C14.896 5.03467 19.1668 5.22406 20.9377 5.60285C21.6668 5.79224 22.1877 6.26573 22.396 6.92861C22.7085 7.97027 22.9168 10.1483 22.9168 11.6635C22.9168 13.1786 22.7085 15.3566 22.396 16.3983C22.1877 17.0612 21.6668 17.5347 20.9377 17.7241C19.1668 18.1028 14.896 18.2922 12.5002 18.2922Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path><path d="M10.4165 14.5046L15.6248 11.6637L10.4165 8.82275V14.5046Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">Lambert iGEM</a></li></ul></div></div></div></div></div></footer>
+</div>
\ No newline at end of file
diff --git a/public/contribution/index.html b/public/contribution/index.html
new file mode 100644
index 0000000..25aa913
--- /dev/null
+++ b/public/contribution/index.html
@@ -0,0 +1,167 @@
+<!DOCTYPE html>
+<html lang="en">
+ <head>
+ <meta charset="UTF-8">
+ <meta name="viewport" content="width=device-width">
+ <link rel="icon" type="image/svg+xml" href="/favicon.svg">
+ <meta name="generator" content="Astro v2.10.15">
+ <link href="../components/fonts.css" rel="stylesheet">
+ <!-- <link
+ rel="stylesheet"
+ href="https://cdn.jsdelivr.net/npm/katex@0.16.9/dist/katex.min.css"
+ integrity="sha384-n8MVd4RsNIU0tAv4ct0nTaAbDJwPJzDEaqSD1odI+WdtXRGWt2kTvGFasHpSy3SV"
+ crossorigin="anonymous"
+ />
+ <link
+ rel="stylesheet"
+ href="https://cdn.jsdelivr.net/npm/katex@0.16.9/dist/katex.min.css"
+ integrity="sha384-n8MVd4RsNIU0tAv4ct0nTaAbDJwPJzDEaqSD1odI+WdtXRGWt2kTvGFasHpSy3SV"
+ crossorigin="anonymous"
+ /> -->
+ <title>Contribution</title>
+ <link rel="stylesheet" href="/lambert-ga/lambert-ga/_astro/RCAERCA.96298b81.css" />
+<link rel="stylesheet" href="/lambert-ga/lambert-ga/_astro/RCAOverview2022.5440303d.css" />
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+ <h1 id="labpilot">LabPilot</h1>
+<h2 id="overview">Overview</h2>
+<p>In previous experiments, Lambert iGEM experienced firsthand
+difficulties of tedious pipetting and inaccurate results. Last year, Rolling Circle
+Amplification (RCA) reactions we ran took the majority of our time. This took important
+research time away, limiting us from our full potential. Issues like these inspired
+us to develop LabPilot, a frugal automated liquid handler that allows researchers
+to focus time elsewhere from running lengthy reactions like RCA. Pipetting is a fundamental laboratory technique used to transfer precise volumes of liquid from one container to another. Consistent pipetting is essential for achieving accurate results, maintaining data integrity, and facilitating advancements in synthetic biology research. However, manual pipetting, the primary method utilized in most labs, limits throughput (the amount of liquid passing through a pipette) and is easily susceptible to human error.
+To resolve these issues, automated liquid handlers are designed to automate and streamline the process of pipetting liquids in laboratory settings. Liquid handlers can be programmed to perform a wide range of pipetting tasks with high precision, accuracy, and efficiency, enabling high throughput experiments (Liquid Handling Automation Benefits). However, current automated liquid handlers costing between $5000 and $300,000 are difficult to use (Retisoft, 2021). To provide underfunded labs access to liquid handlers, Lambert iGEM developed LabPilot, a frugal liquid handler made with 3D printable and accessible parts that can accurately pipette based on direct user input (Hentz & Knaide, 2014). Therefore, it enables research to be conducted in an efficient manner, filling an essential gap in lab settings.</p>
+<h2 id="parts-list">Parts list</h2>
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+<table><thead><tr><th>Part:</th><th>Seller:</th></tr></thead><tbody><tr><td>Arduino Mega 2560</td><td>Yanmis (Amazon) <a href="https://tinyurl.com/2p8647ah">https://tinyurl.com/2p8647ah</a></td></tr><tr><td>CNC Shield</td><td>Yanmis (Amazon) <a href="https://tinyurl.com/2p8647ah">https://tinyurl.com/2p8647ah</a></td></tr><tr><td>A4988 Motor Drivers</td><td>Yanmis (Amazon) <a href="https://tinyurl.com/2p8647ah">https://tinyurl.com/2p8647ah</a></td></tr><tr><td>12 Volt 5 amp Power Supply and Cable</td><td>Alitove Store (Amazon) <a href="https://tinyurl.com/mb4rs42t">https://tinyurl.com/mb4rs42t</a></td></tr><tr><td>Five Nema 17 Motors</td><td>SIMAX3D (Amazon) <a href="https://tinyurl.com/389ssrd9">https://tinyurl.com/389ssrd9</a></td></tr><tr><td>Timing Belt And Pulleys Combo</td><td>Kee Yees Store (Amazon) <a href="https://tinyurl.com/2bkfz445">https://tinyurl.com/2bkfz445</a></td></tr><tr><td>Bearings</td><td>Yosoo Health Gear (Amazon) <a href="https://tinyurl.com/2vwxwvzv">https://tinyurl.com/2vwxwvzv</a></td></tr><tr><td>Limit Switches</td><td>Octagon Star (Amazon) <a href="https://tinyurl.com/dv65fkhv">https://tinyurl.com/dv65fkhv</a></td></tr><tr><td>Two 8mmx300mm Linear Motion Steel Rods</td><td>Amazon <a href="https://tinyurl.com/5e4hfrf6">https://tinyurl.com/5e4hfrf6</a></td></tr><tr><td>Two 8mmx100mm Threaded Steel Rods</td><td>Uxcell (Amazon) <a href="https://tinyurl.com/mr2ft89z">https://tinyurl.com/mr2ft89z</a></td></tr><tr><td>Four 8mmx350mm Linear Motion Steel Rods</td><td>Amazon <a href="https://tinyurl.com/ywsbncdf">https://tinyurl.com/ywsbncdf</a></td></tr><tr><td>Four 8mmx150mm Linear Motion Steel Rods</td><td>Amazon <a href="https://tinyurl.com/mu6ujt7s">https://tinyurl.com/mu6ujt7s</a></td></tr></tbody></table>
+<h2 id="design">Design</h2>
+<p>LabPilot is an automated liquid handler compatible with micropipettes. It utilizes an XYZ layout for movement, with most of its structural parts designed on Fusion 360, a computer-aided design (CAD) application. LabPilot’s design can be split into the frame, detachable molds for the bed, and XYZ planes with motors.</p>
+<h3 id="frame">Frame</h3>
+<p>Our team designed Labpilot’s frame to be small enough to fit inside a flow hood and large enough to fit standard laboratory containers on its bed (e.g. microcentrifuge tube racks, micropipette tip boxes, and well plates). We designed the frame by creating a linear rail extrusion, as shown in Figure W, typically found on commercial 3D printers. We used linear extrusions to make the cage-like design of LabPilot, inspired by Ender 3D printers like the Ender 5 (see Table 1) (Ender 5 S1 3D Printer). The finalized XYZ dimensions of LabPilot are 366mm x 296mm x 356mm (see Fig. 2). To provide stability to LabPilot’s frame, we added angle brackets that connect two of the extrusions that run perpendicular to each other at all 16 corners of the frame.
+We spoke to Priya Soneji, an undergraduate researcher at the Georgia Institute of Technology, who is a part of the Bhamla Lab. For the future, she recommended the switch to metal frames if the 3D-printed parts were imprecise. She confirmed the validity of our overall plan for controlling each motor axis on the XYZ plane and methods for motor movement.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/hardware/linearrailextrusion.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. CAD model of linear rail extrusion used to make the structural frame.</div></div>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/hardware/labpilotframe.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 2. CAD model of LabPilot frame.</div></div>
+<h3 id="molds">Molds</h3>
+<p>Lambert iGEM created molds that snap onto the bed plate of LabPilot so that the user can attach their desired components to the bed. The molds have pegs (see Fig. 3) that can be placed inside engraved indentations on the bed plate (see Fig. 4), which fixes the molds in place and enables the LabPilot app to calculate their position. LabPilot has a mold that fits every micropipette tip box, beaker, microcentrifuge tube rack, PCR tube rack, and well plate.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/hardware/labpilotbeakermold.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. Pegs placed on the bottom of the molds to fit into the bed plate.</div></div>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/hardware/labpilotbase.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Indentations on the bed plate for the molds to rest on.</div></div>
+<h3 id="xyz-motor-axis">XYZ Motor Axis</h3>
+<p>LabPilot utilizes five Nema 17 motors (Nema 17 Stepper Motor) (see Table 1) to control the three axes of movement needed for the pipetting mechanism. Nema 17 motors have a micro-stepping feature, enabling small and precise movements. Each motor is controlled by an Arduino Mega 2560 with a Ramps 1.4 CNC shield containing five A4988 motor drivers (see Table 1). The CNC shield uses two motors to control the vertical movement of the bed plate, as it is the heaviest moving part. The left/right movement and the forward/backward movement of the micropipette are controlled by one motor each. The last motor controls the pipetting mechanism (see Fig. 5) and is placed above the micropipette. Additionally, we placed limit switches for each axis to prevent over-rotation of the motors and calibration to home coordinates similar to 3D printers. Each axis has linear steel rods that act as supports, with bearings in place to ensure the pipette mechanism slides smoothly across the rods. We attached timing belts (thin rubber belts) with teeth (see Table 1) to the motor to carry the pipette left, right, forwards, and backwards across the X and Y axes.
+In addition to the linearly moving axes, LabPilot requires a motor to press the micropipette to the first and second stops. The pipetting mechanism motor converts the rotational motion of the motor into a linear force that can push down on the micropipette button. The motor applies a fine-tuned force to achieve first and second stops. Currently, LabPilot is designed to be used with Eppendorf micropipettes to ensure everything fits correctly. We attached a stationary arm to the bed plate to eject the pipette tips. The micropipette moves into the arm while sequentially traveling upwards. This way, the tip is pulled off the micropipette and discarded underneath.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/pipettemechanismsarvajith.jpeg" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 5. Pipette mechanism</div></div>
+<h2 id="app">App</h2>
+<p>To make LabPilot easier for researchers, Lambert iGEM designed a web app to control the liquid handler. Unlike the complex software that operates commercial liquid handlers such as Opentrons, the LabPilot app has a simple, human-centric user interface designed to be used immediately with simple and intuitive commands. This design enables users to quickly start using LabPilot and efficiently simplify their tasks without any difficulties.</p>
+<h3 id="how-it-works">How it Works</h3>
+<p>To intelligently control its actions, LabPilot utilizes an Arduino microcontroller connected to a laptop running the LabPilot web app via a USB cable. The microcontroller is in charge of controlling the movement of the pipette mechanism and the action of pipetting. The microcontroller moves the micropipette to a specific location by using a virtual coordinate system, which defines different areas of the surface with numbers. The LabPilot app knows what actions to take in order to move the desired axes to their coordinate destination.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/hardware/labpilotmicrocontroller.png" style="max-width:500" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 6. shows how the user input on the app controls the microcontroller.</div></div>
+<p>The app provides instructions to the microcontroller via the Arduino serial USB connection (see Fig. 6). By utilizing a system where the app executes high-level operations by compiling pipette actions into low-level instructions for the microcontroller, users have complete control over their LabPilot device. They can stop or pause it at any time.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/img-9427.jpeg" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 7. Shows how the user input controls LabPilot.</div></div>
+<h3 id="how-to-use">How to use</h3>
+<p>To use LabPilot, users must connect a laptop to LabPilot via a USB cable and open its web dashboard. Once the dashboard is open, there is a simple setup process where users can drag and drop components from the sidebar to recreate the physical layout of their LabPilot device, as seen in Figure Y. After setup, the app digitally recreates the layout of this device from data gathered in the setup process, enabling adequate simulation and control. Then, the users can queue and perform pipetting actions.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/image-10-10-23-at-8-43-pm.jpg" style="max-width:600" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 8. LabPilot’s setup interface.</div></div>
+<p>All pipetting actions consist of a reagent source and a dispensing source. Users can click on any well or beaker to select a reagent source. After choosing a reagent source, users can select a dispensing source by clicking on any well/beaker or multiple dispensing sources (by clicking and dragging or holding “shift†while they select multiple sources). Afterward, users will select the amount they want to dispense to each dispensing source and can add it to the queue (see Fig. 7). All pipetting actions in the queue are performed in the order they were assigned but can be dragged around to reorder in the queue.</p>
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+<h2 id="conclusion-and-future-directions">Conclusion and Future Directions</h2>
+<p>Researchers can leverage LabPilot’s capabilities to streamline other critical processes such as DNA sequencing, sample preparation, high-throughput screening, and assay development. By automating these tasks, LabPilot enables scientists to focus on higher-level analysis and interpretation of results, accelerating the pace of scientific discoveries. Next year, Lambert iGEM hopes to switch to an aluminum frame for its cost-effective, rigid nature and increase compatibility with various laboratory equipment. Additionally, we plan to test with polymerase chain reaction (PCR) as a proof of concept experiment. A successful PCR will have LabPilot demonstrate accuracy in pipetting volumes, with substantial precision and a significant reduction in PCR time. Once we can prove that LabPilot upholds the accuracy and efficiency of a human through a proof of concept, we plan on using LabPilot to automate a portion of our rolling circle amplification biosensors to improve efficiency and reduce the potential for human error. Our end goal is to develop a kit available for purchase and make LabPilot commercially available. LabPilot’s cost-effectiveness, user-friendly interface, and open-source nature make it accessible and adaptable to all laboratory settings.</p>
+<iframe title="Lambert-GA: LabPilot Prototype 1 Axis (2023) [English]" width="560" height="315" src="https://video.igem.org/videos/embed/5e82a2a5-8e47-4ae9-b7c9-545f0e3cabc7" frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups"></iframe>
+<h2 id="references">References</h2>
+<div class="pl-6 -indent-6"><p>Amazon.com: Usongshine Nema 17 Stepper Motor Bipolar Step Motor for …
+(n.d.). <a href="https://www.amazon.com/Usongshine-Stepper-Motor/dp/B0BPL725TN">https://www.amazon.com/Usongshine-Stepper-Motor/dp/B0BPL725TN</a></p></div>
+<div class="pl-6 -indent-6"><p>Choosing An Automated Liquid Handler. (2021, August 26). Retisoft.
+<a href="https://retisoft.com/choosing-a-liquid-handler/">https://retisoft.com/choosing-a-liquid-handler/</a></p></div>
+<div class="pl-6 -indent-6"><p>Ender-5 S1 3D printer. Crealitystore. (n.d.).
+<a href="https://store.creality.com/products/ender-5-s1-3d-printer?spm=..collection_90778a1d-d845-4ff0-a8c4-48fded4b7d74.albums_2.1&spm_prev=..index.header_1.1">https://store.creality.com/products/ender-5-s1-3d-printer?spm=..collection_90778a1d-d845-4ff0-a8c4-48fded4b7d74.albums_2.1&spm_prev=..index.header_1.1</a></p></div>
+<div class="pl-6 -indent-6"><p>Liquid Handling Automation Benefits. (n.d.). Retrieved October 1, 2023, from
+<a href="https://dispendix.com/blog/liquid-handling-automation-benefits">https://dispendix.com/blog/liquid-handling-automation-benefits</a></p></div>
+<div class="pl-6 -indent-6"><p>Hentz, N. G., & Knaide, T. R. (2014). Effect of Liquid-Handling Accuracy on
+Assay Performance. SLAS Technology, 19(2), 153–162.
+<a href="https://doi.org/10.1177/2211068213504095">https://doi.org/10.1177/2211068213504095</a></p></div>
+<div class="pl-6 -indent-6"><p>Eppendorf Research Plus Adjustable Volume, Single Channel Pipette, 2—20 uL,
+Yellow. Retrieved October 1, 2023, from
+<a href="https://www.pipettes.com/pipettes/single-channel-pipettes/manual/adjustable-volume/eppendorf-research-plus-single-channel-pipette-adjustable-2-20-ul">https://www.pipettes.com/pipettes/single-channel-pipettes/manual/adjustable-volume/eppendorf-research-plus-single-channel-pipette-adjustable-2-20-ul</a></p></div>
+<div class="pl-6 -indent-6"><p>OT-2 Robot Opentrons Labworks Inc. Retrieved October 1, 2023, from
+<a href="https://shop.opentrons.com/ot-2-robot/">https://shop.opentrons.com/ot-2-robot/</a></p></div>
+<div class="pl-6 -indent-6"><p>Ender 5 S1 3D Printer. (n.d.). Retrieved October 2, 2023, from
+<a href="https://store.creality.com/products/ender-5-s1-3d-printer?spm=..collection_90778a1d-d845-4ff0-a8c4-48fded4b7d74.albums_2.1&spm_prev=..index.header_1.1">https://store.creality.com/products/ender-5-s1-3d-printer?spm=..collection_90778a1d-d845-4ff0-a8c4-48fded4b7d74.albums_2.1&spm_prev=..index.header_1.1</a></p></div>
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+
+<h1 id="abstract">Abstract</h1>
+<p>In 2021, coronary artery disease (CAD) was responsible for 375,476 fatalities. Many of these deaths could be prevented by improved diagnosis, cost-effectiveness, and less invasive procedures. Through CADlock, we promoted early diagnostics and inclusive care for underrepresented demographics. We met this goal by emphasizing accessibility, affordability, and accuracy through procedures providing preliminary care to systematically marginalized groups before disease progression. By adding a nicking endonuclease, we enhanced rolling circle amplification (RCA) into exponential RCA, improving reaction accuracy. Additionally, we improved Micro-Q by utilizing a white LED to test various fluorescence wavelengths in a single frugal device. To further accommodate the accessibility of miRNA research, we created CADmir: a comprehensive search engine utilizing large language models to organize miRNA information. Finally, we educated our peers and teachers on the applications of biotechnology, inspiring future generations to make healthy, proactive lifestyle choices.</p>
+<h2 id="defining-the-problem">Defining the problem</h2>
+<p>Coronary artery disease (CAD) currently ranks as the third highest cause of death worldwide. CAD was responsible for 375,476 fatalities (CDC, 2019). It is also the number one cause of death in the United States, specifically in the southeastern region including our home state: Georgia (CDC, 2019) (see Fig. 1).</p>
+<p>CAD exhibits various symptoms, some of which cannot be visible (CDC, 2019). Moreover, women typically show symptoms 7 to 10 years later than men due to the high levels of estrogen in their bodies premenopause (Brown, 2022). The following displays various common symptoms linked to CAD (Maas & Appelman, 2010).</p>
+<ul>
+<li>Chest pain (angina), fullness or pressure</li>
+<li>Fatigue</li>
+<li>Heart palpitations, or sensations of a racing heartbeat</li>
+<li>Shortness of breath (dyspnea)</li>
+<li>Swelling in the hands or feet</li>
+</ul>
+<p>Currently, various detection and diagnosis methods are used for CAD. These include angiograms, computed tomography angiogram scans, echocardiograms, electrocardiograms, and nuclear stress tests (Nuclear Cardiac Stress Test: What to Expect, n.d., 2022). These tests, however, are not affordable, and some are highly invasive. Moreover, there are certain harmful side effects of certain stress tests, such as chest pain, which can increase a patient’s risk for a sudden heart attack ( Nuclear Cardiac Stress Test: What to Expect, n.d., 2022). To mitigate this, Lambert iGEM 2023 further emphasizes the importance of inclusivity by pursuing a more accurate and precise detection mechanism by broadening the scope of accessibility.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/homepage/usa.png" style="max-width:lg" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. Map of coronary artery disease distribution in the United States. </div></div>
+<h2 id="pathophysiology">Pathophysiology</h2>
+<p>Coronary artery disease is caused by plaque buildup which is initially clustered in fat and blood cells in the body (see Fig. 2). As the collection worsens, arteries narrow in a process called atherosclerosis. The following describes the key process in three crucial steps from the normal condition of the artery (Shahjehan, 2023).</p>
+<div class="pl-2"><p>Formation - The first stages of the formation of atherosclerosis include the deposition of foam cells, also known as lipid macrophages. They disrupt the tunica intima, which is the innermost layer of the artery. Due to this disruption, a “fatty streak†is formed (CDC, 2021).</p><p>Progression - The progression of low-density lipoprotein (LDL) particles and foam cells activate T cells, a type of white blood cell that leads to immune responses promoting the nourishment of the plaque. Thus, the plaque’s size increases and this process is known as angiogenesis (CDC, 2021).</p><p>Blockage - As the plaque continues to grow, it undergoes calcification which leads to the narrowing of the coronary arteries and restriction of blood flow. If there is complete blockage in the arteries, there is a severe chance of myocardial infarction, or a heart attack (CDC, 2021).</p></div>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/homepage/pathophys.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 2. The progression of CAD</div></div>
+<h1 id="microrna2022-project">MicroRNA/2022 Project</h1>
+<p>MicroRNAs (miRNAs) are a class of non-coding, port-transcriptional RNA molecules that play essential roles in regulating gene expression. miRNAs are about 20-23 nucleotides in length, and they enhance or inhibit protein production via translational repression, or the cleaving of mRNA sequences. miRNAs are first transcribed from DNA sequences into primary miRNAs, then processed into precursor miRNAs, and finally transcripted into mature miRNAs (see Fig. 3). Most miRNAs are identified by their 5p position (forward 5’ - 3’) and their 3p position reverse (3’ - 5’). miRNAs interact with target mRNAs with 3′untranslated regions (3′ UTR) to induce cleavage and translational repression (O’Brien et al., 2018). However, miRNAs also interact with other regions, including the 5′ UTR, coding sequences, and gene promoters (NIH, 2022).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/homepage/untitled-artwork.jpeg" style="max-width:lg" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. Mature miRNA editing</div></div>
+<p>In 2022, we tested miRNA hsa-miR-1-3p, which is upregulated in coronary artery disease (CAD), as a biomarker for screening the disease. This means it can detect the miRNAs at necessary concentrations in conjunction with padlock probes, which are long strands of DNA with ends (or arms) complementary to the target miRNA (See Fig. 4). By utilizing a process known as rolling circle amplification (RCA), we amplified the padlock sequence creating a product up to 10 kilobases long. Then, using the reporting mechanism called linear DNA probes, we successfully measured the fluorescence output with Micro-Q, our frugal fluorometer.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/homepage/group-6-1.png" style="max-width:lg" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Image description of hybridization of miRNA and padlock probes</div></div>
+<h2 id="barriers">Barriers</h2>
+<p>Lambert iGEM had certain concerns in their 2022 project to which solutions and alternatives are being approached and pursued in 2023. The 2022 project held concerns about the lack of accuracy with the padlock probes and microRNAs (miRNAs) and inefficient performance from the aptamers. Taking these factors into consideration, the team has developed a second phase of CADLOCK which consists of improved wetlab and hardware aspects that increase the efficiency of proactive care through innovations with accessibility, accuracy, and affordability.</p>
+<p><strong>Capillary tube:</strong> To improve the point of care and accessibility aspects of the 2022 project, the team began the development of capillary RCA, which negates the need for a fluorometer by using capillary tubes for visual, counting-based quantification. By removing the necessity of a fluorometer and expensive diagnostic equipment, capillary RCA could potentially enable more accessible and cost-effective testing of CAD.</p>
+<p><strong>MircroQ:</strong> After extensive in-field testing in Thailand, researchers determined two main issues to address while developing our new iteration of Micro-Q: its inability to fluoresce multiple samples simultaneously and fluorescing samples at different wavelengths. With this in mind, the team developed Micro-Q Pro, a device capable of exciting 8 PCR tubes containing samples that fluoresce anywhere from 400 to 700 nm. Utilizing a white LED strip, a 3D printed holster, and an ESP-32 Camera we were able to accurately detect and quantify fluorescence.</p>
+<p><strong>Exponential RCA:</strong> Although RCA is highly sensitive to the target miRNAs, it cannot differentiate between various concentrations of miRNA. To address this, the Lambert iGEM team is pursuing exponential RCA (eRCA), an adaptation of the rolling circle amplification model that produces exponentially greater fluorescence per unit of miRNA, therefore increasing between miRNA concentration and margin of error.</p>
+<p><strong>Estrogen:</strong> Females are more susceptible to CAD due to their fluctuating levels of estrogen. Essentially, estrogen acts as a CAD suppressor, and so when females undergo menopause with a drastic estrogen drop, they have an increased risk of developing CAD. Lambert iGEM expanded its target population to men as well. Since men have stable levels of estrogen, they would be an accurate population to test for consistent results. Lambert iGEM experimented with mir-20-b which, with a high level in men, indicates CAD, while in premenopausal women, indicates protection from CAD.</p>
+<p><strong>Protein Purification:</strong> While RCA provides a more frugal alternative to existing methods, costing $8.98, phi2DNA polymerase makes up approximately 56% of the cost. In addition to this, multiple RCA reactions needed to be performed in order for an accurate diagnosis, increasing the total cost significantly. To increase the preventative aspect of healthcare and decrease the cost of the overall assay, we produced phi29 DNA polymerase through protein purification, a method to cheaply produce phi29.</p>
+<p><strong>CADmir:</strong> To address the need for a database that extracts functional miRNA information and organizes the vast volume of scientific literature regarding miRNAs, we developed CADmir: a miRNA search engine utilizing large language models to process and organize the extensive information about miRNAs. With CADmir, most of the relevant data about specific miRNAs are consolidated in one place, improving information accessibility and reducing the time barrier associated with obtaining high-quality scientific information. CADmir works alongside existing databases such as miRBase to provide links to relevant scientific literature and incorporate sequence and genomic data. CADmir expands research in miRNAs and creates a new precedent for the organization of vital scientific information.</p>
+<p><strong>Lab Pilot:</strong> Pipetting is a fundamental laboratory technique used to transfer precise volumes of liquid from one container to another. Consistent pipetting is essential for achieving accurate results, maintaining data integrity, and facilitating advancements in synthetic biology research. However, manual pipetting limits throughput and is easily susceptible to human error. To resolve these issues, automated liquid handlers are designed to automate and streamline the process of pipetting liquids in laboratory settings. However, current automated liquid handlers costing between $5000 and $300,000 are difficult to use. Because of this, the majority of current labs don’t have easy access to an automated liquid handler. To provide underfunded labs access to liquid handlers, Lambert iGEM developed LabPilot, a frugal liquid handler made with 3D printable and accessible parts that can accurately pipette based on direct user input. LabPilot is an automated liquid handler compatible with a variety of micropipettes. It utilizes an XYZ layout for movement, with most of its structural parts designed on Fusion 360, a computer-aided design application.</p>
+<h2 id="our-approach">Our Approach</h2>
+<p>2023 Lambert iGEM has undertaken a new approach with the project’s primary focus area as inclusivity in order to enable 2023 CADlock to be readily available regardless of sex, ethnicity, or socioeconomic status.
+To address this initiative, Lambert iGEM implemented several tactics, including an exponential approach to improve the accuracy of RCA, a sex inclusive wetlab initiative, a cost-effective fluorometer that accommodates various wavelengths, a prototype for an automated pipetter, an accessible database for CAD microRNAs, a STEM inclusivity podcast, and a “Science for All†webinar.
+We spoke with multiple researchers and cardiologists who aided our decisions regarding the improvement of our hardware and wetlab systems.
+Our team builds upon CADlock by creating improved wetlab and frugal hardware devices and taking a holistically proactive approach through accuracy, affordability, and accessibility.</p>
+<h2 id="references">References</h2>
+<div class="pl-6 -indent-6"><p>‌Brown, J. C., Gerhardt, T. E., & Kwon, E. (2022). Risk Factors For Coronary
+Artery Disease. PubMed; StatPearls Publishing.
+<a href="https://pubmed.ncbi.nlm.nih.gov/32119297/">https://pubmed.ncbi.nlm.nih.gov/32119297/</a></p></div>
+<div class="pl-6 -indent-6"><p>CDC. (2019, December 2). Heart Disease Facts. Centers for Disease Control and
+Prevention. <a href="https://www.cdc.gov/heartdisease/facts.htm">https://www.cdc.gov/heartdisease/facts.htm</a></p></div>
+<div class="pl-6 -indent-6"><p>CDC. (2021, July 19). Coronary artery disease. Centers for Disease Control and
+Prevention. <a href="https://www.cdc.gov/heartdisease/coronary_ad.htm">https://www.cdc.gov/heartdisease/coronary_ad.htm</a></p></div>
+<div class="pl-6 -indent-6"><p>Maas, A. H. E. M., & Appelman, Y. E. A. (2010). Gender differences in coronary
+heart disease. Netherlands Heart Journal : Monthly Journal of the Netherlands
+Society of Cardiology and the Netherlands Heart Foundation, 18(12), 598–602.
+<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3018605/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3018605/</a></p></div>
+<div class="pl-6 -indent-6"><p>Nuclear Cardiac Stress Test: What to Expect. (n.d.). Cleveland Clinic.
+<a href="https://my.clevelandclinic.org/health/diagnostics/17276-nuclear-cardiac-stress-test">https://my.clevelandclinic.org/health/diagnostics/17276-nuclear-cardiac-stress-test</a></p></div>
+<div class="pl-6 -indent-6"><p>O’Brien, J., Hayder, H., Zayed, Y., & Peng, C. (2018, June 28). Overview
+of microrna biogenesis, mechanisms of actions, and circulation. Frontiers.
+<a href="https://www.frontiersin.org/articles/10.3389/fendo.2018.00402/full">https://www.frontiersin.org/articles/10.3389/fendo.2018.00402/full</a></p></div>
+<div class="pl-6 -indent-6"><p>Shahjehan, R. D. (2023, February 9). Coronary artery disease. StatPearls -
+NCBI Bookshelf. <a href="https://www.ncbi.nlm.nih.gov/books/NBK564304/">https://www.ncbi.nlm.nih.gov/books/NBK564304/</a></p></div>
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component-export="default" renderer-url="/lambert-ga/_astro/client.4f88357e.js" props="{"title":[0,"Experiments"],"header":[0,"https://static.igem.wiki/teams/4683/wiki/header-images/screen-shot-2023-10-11-at-1-10-13-am.png"],"file":[0,"/Users/ryandu/Desktop/iGEM/2023/wiki/src/pages/experiments.mdx"],"url":[0,"/lambert-ga/experiments"]}" ssr="" client="load" opts="{"name":"Layout","value":true}" await-children=""><body class="w-screen">https://static.igem.wiki/teams/4683/wiki/header-images/screen-shot-2023-10-11-at-1-10-13-am.png<div class="bg-cover absolute w-screen left-0 top-0 p-0 m-0 z-10 h-1/2" style="background:linear-gradient(to bottom, rgba(255, 255, 255, 0) 36.46%, #FFFFFF 100%), url(https://static.igem.wiki/teams/4683/wiki/header-images/screen-shot-2023-10-11-at-1-10-13-am.png);background-size:cover"></div><div class="bg-slate-100 fixed top-0 left-0 right-0 pt-8" style="margin-top:-40px"><div class="bottom-0 left-0 pb-1 mt-2 right-0 bg-yellow-400 origin-left" style="transform:scaleX(0) translateZ(0)"></div></div><div class="w-full flex justify-center items-center"><div class="flex justify-between m-5 z-50 max-w-6xl grow"><a href="https://2023.igem.wiki/lambert-ga/" class="font-serif z-30 text-white "><p class="bg-blackA-11 text-xl rounded-full p-2">Lambert-GA</p></a><div class="flex justify-end z-30"><nav aria-label="Main" data-orientation="horizontal" dir="ltr" class="relative "><div style="position:relative"><ul data-orientation="horizontal" class="flex flex-row rounded-full bg-blackA-11 p-2 space-x-2" dir="ltr"><div><li><button id="radix-:r1Rk:-trigger-radix-:r1Rbk:" data-state="closed" aria-expanded="false" aria-controls="radix-:r1Rk:-content-radix-:r1Rbk:" class="px-4 py-2 rounded-full hover:bg-whiteA-6 font-medium text-gray-3 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Team</button></li></div><div><li><button id="radix-:r1Rk:-trigger-radix-:r1Rjk:" data-state="closed" aria-expanded="false" aria-controls="radix-:r1Rk:-content-radix-:r1Rjk:" class="px-4 py-2 rounded-full hover:bg-whiteA-6 font-medium text-gray-3 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Project</button></li></div><div><li><button id="radix-:r1Rk:-trigger-radix-:r1Rrk:" data-state="closed" aria-expanded="false" aria-controls="radix-:r1Rk:-content-radix-:r1Rrk:" class="px-4 py-2 rounded-full hover:bg-whiteA-6 font-medium text-gray-3 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Wetlab</button></li></div><div><li><button id="radix-:r1Rk:-trigger-radix-:r1R13k:" data-state="closed" aria-expanded="false" aria-controls="radix-:r1Rk:-content-radix-:r1R13k:" class="px-4 py-2 rounded-full hover:bg-whiteA-6 font-medium text-gray-3 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Drylab</button></li></div><div><li><button id="radix-:r1Rk:-trigger-radix-:r1R1bk:" data-state="closed" aria-expanded="false" aria-controls="radix-:r1Rk:-content-radix-:r1R1bk:" class="px-4 py-2 rounded-full hover:bg-whiteA-6 font-medium text-gray-3 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Human Practices</button></li></div></ul></div></nav></div></div></div><div class="flex justify-center w-full items-center"><div class="w-full m-5 max-w-3xl "><p class="font-serif font-bold z-30 absolute top-1/2 -translate-y-1/2 lg:-translate-x-20 text-5xl sm:text-6xl md:text-7xl">Experiments</p><div class="absolute top-1/2 translate-y-20 h-32 lg:-translate-x-20 lg:border-l-2 border-gray-900 z-50"></div><div class="
+prose max-w-3xl
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+ <h1 id="rolling-circle-amplification-rca">Rolling Circle Amplification (RCA)</h1>
+<h2 id="rca--2022-protocol">RCA | 2022 Protocol</h2>
+<h3 id="dna-rna-hybridization">DNA-RNA Hybridization</h3>
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+<table><thead><tr><th>(A) - RCA</th><th>(B) - Negative Control (no enzymes)</th></tr></thead><tbody><tr><td>1 uL SplintR buffer</td><td>1 uL SplintR buffer</td></tr><tr><td>0.5 uL 100 nM padlock probe</td><td>0.5 uL 100 nM padlock probe</td></tr><tr><td>*1 uL 1000 pM miRNA</td><td>1 uL 1000 pM miRNA</td></tr><tr><td>*6 uL RNase free water</td><td>7.5 uL RNase free water</td></tr><tr><td>*changes according to the concentration of miRNA (see:miRNA Detection Limit Experimentation)</td><td></td></tr></tbody></table>
+<ol>
+<li>Heat the solution at 65°C for 3 minutes, and slowly cool it to room temperature over 10 minutes.</li>
+</ol>
+<h3 id="ligation">Ligation</h3>
+
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+<table><thead><tr><th>(A) - RCA</th><th>(B) - Negative Control (no enzymes)</th></tr></thead><tbody><tr><td>1 uL SplintR Ligase</td><td>0.5 uL RNase Inhibitor</td></tr></tbody></table>
+<ol>
+<li>Incubate the reaction for 2 hours at 37°C.</li>
+<li>Bring the reaction up to 65°C for 20 minutes to terminate.</li>
+</ol>
+<h3 id="amplification">Amplification</h3>
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+<table><thead><tr><th>(A) - RCA</th><th>(B) - Negative Control (no enzymes)</th></tr></thead><tbody><tr><td>2.5 uL phi29 buffer</td><td>12.5 uL RNase free water</td></tr><tr><td>1.25 uL phi29 DNA polymerase</td><td>2.5 uL phi29 buffer</td></tr><tr><td>1 uL albumin</td><td></td></tr><tr><td>6.76 uL dNTP mix</td><td></td></tr><tr><td>2.99 uL RNase free water</td><td></td></tr></tbody></table>
+<ol>
+<li>Incubate the reactions for 8 hours at 37°C.</li>
+<li>Bring the reactions up to 65°C for 10 minutes to inactivate the DNA polymerase.</li>
+</ol>
+<h2 id="mirna-detection-limit-experimentation--2022-protocol">miRNA Detection Limit Experimentation | 2022 Protocol</h2>
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+<table><thead><tr><th>miRNA</th><th>water</th><th>Concentration</th></tr></thead><tbody><tr><td>5 uL 1000 pM</td><td>2 uL</td><td>204 pM</td></tr><tr><td>1 uL 1000 pM h</td><td>6 uL</td><td>40.8 pM</td></tr><tr><td>0.5 uL 1000 pM</td><td>6.5 uL</td><td>20.4 pM</td></tr><tr><td>1 uL 100 pM h</td><td>6 uL</td><td>4.08 pM</td></tr><tr><td>0.8 uL 100 pM</td><td>6.2 uL</td><td>3.27 pM</td></tr><tr><td>0.5 uL 100 pM h</td><td>6.5 uL</td><td>2.04 pM</td></tr><tr><td>0.3 uL 100 pM</td><td>6.7 uL</td><td>1.22 pM</td></tr><tr><td>1 uL 10 pM h</td><td>6 uL</td><td>0.41 pM</td></tr><tr><td>0.5 uL 10 pM</td><td>6.5 uL</td><td>0.204 pM</td></tr><tr><td>3 uL 1 pM h</td><td>4 uL</td><td>0.122 pM</td></tr><tr><td>1 uL 1 pM</td><td>6 uL</td><td>40 fM</td></tr></tbody></table>
+<h2 id="optimized-rca">Optimized RCA</h2>
+<h3 id="dna-rna-hybridization-1">DNA-RNA Hybridization</h3>
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+<table><thead><tr><th>(A) - RCA</th><th>(B) - Negative Control (no enzymes)</th></tr></thead><tbody><tr><td>1 uL SplintR buffer</td><td>1 uL SplintR buffer</td></tr><tr><td>0.5 uL 100 nM padlock probe</td><td>0.5 uL 100 nM padlock probe</td></tr><tr><td>*1 uL 1000 pM miRNA</td><td>1 uL 1000 pM miRNA</td></tr><tr><td>*6 uL RNase free water</td><td>7.5 uL RNase free water</td></tr><tr><td>*changes according to the concentration of miRNA (see:miRNA Detection Limit Experimentation)</td><td></td></tr></tbody></table>
+<ol>
+<li>Heat the solution at 65°C for 3 minutes, and slowly cool it to room temperature over 10 minutes.</li>
+</ol>
+<h3 id="ligation-1">Ligation</h3>
+
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+<table><thead><tr><th>(A) - RCA</th><th>(B) - Negative Control (no enzymes)</th></tr></thead><tbody><tr><td>1 uL SplintR buffer</td><td></td></tr><tr><td>0.5 uL RNase Inhibitor</td><td></td></tr></tbody></table>
+<ol>
+<li>Incubate the reaction for 30 minutes at 37°C.</li>
+<li>Bring the reaction up to 65°C for 20 minutes to terminate.</li>
+</ol>
+<h3 id="amplification-1">Amplification</h3>
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+<table><thead><tr><th>(A) - RCA</th><th>(B) - Negative Control (no enzymes)</th></tr></thead><tbody><tr><td>2.5 uL phi29 buffer</td><td>12.5 uL RNase free water</td></tr><tr><td>1.25 uL phi29 DNA polymerase</td><td>2.5 uL phi29 buffer</td></tr><tr><td>1 uL albumin</td><td></td></tr><tr><td>6.76 uL dNTP mix</td><td></td></tr><tr><td>2.99 uL RNase free water</td><td></td></tr></tbody></table>
+<ol>
+<li>Incubate the reactions for 5 hours at 37°C.</li>
+<li>Bring the reactions up to 65°C for 10 minutes to inactivate the DNA polymerase.</li>
+</ol>
+<h2 id="amplification-with-phi29-xt">Amplification with Phi29-XT</h2>
+<h3 id="amplification-2">Amplification</h3>
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+
+<table><thead><tr><th>(A) - RCA</th><th>(B) - Negative Control (no enzymes)</th></tr></thead><tbody><tr><td>4 uL phi29-XT buffer</td><td>4 uL phi29-XT buffer</td></tr><tr><td>2 uL dNTP mix</td><td>6 uL RNase free water</td></tr><tr><td>2 uL RNase free water</td><td></td></tr><tr><td>2 uL phi29-XT DNA Polymerase</td><td></td></tr></tbody></table>
+<ol>
+<li>Incubate the reaction for 3 minutes at 95°C with the lid set at 100°C.</li>
+<li>Incubate the reaction for 2 hours at 42°C.</li>
+<li>Bring the reactions up to 65°C for 10 minutes to inactivate the DNA polymerase</li>
+</ol>
+<h2 id="bluegel-with-rcp--2022-protocol">BlueGelâ„¢ with RCP | 2022 Protocol</h2>
+<ol>
+<li>For a 2% agarose concentration, add 0.4 g of agarose to 20 mL of 1X BE buffer.</li>
+<li>Microwave gel mix for 30 seconds, or until clear.</li>
+<li>Allow the solution to cool until 65°C.</li>
+<li>Add 2 uL of SYBRâ„¢ Safe.</li>
+<li>Pour gel mixture into gel mold with combs.</li>
+<li>Let cool at room temperature and set.</li>
+</ol>
+<h3 id="readout-reaction">Readout Reaction</h3>
+<ol>
+<li>Add 10 μL of RCP + 2 μL of loading dye into the gel</li>
+<li>Add 12uL of the 2-log ladder into the gel</li>
+<li>Start the blueGelâ„¢ gel electrophoresis system.</li>
+<li>After 1 hour, use the UV illumination on the blueGelâ„¢ gel electrophoresis system to visualize the DNA.</li>
+</ol>
+<h2 id="linear-dna-probes-with-rcp--2022-protocol">Linear DNA Probes with RCP | 2022 Protocol</h2>
+<p>Add the following to an amber microcentrifuge tube to make FAM-Probe Mastermix:</p>
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+<table><thead><tr><th></th><th></th></tr></thead><tbody><tr><td>FAM Dye Tagged Linear DNA Probe Mastermix</td><td>Volume of 1 uM FAM dye: (# of reactions) x (0.4 µL 100 uM FAM Dye Tagged Linear DNA Probe) x 1.1</td></tr><tr><td></td><td>Volume of TE: (# of reactions) x (29 µL TE) x 1.1</td></tr></tbody></table>
+<p>In nuclease-free PCR tubes add the following triplicates:</p>
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+<table><thead><tr><th></th><th></th></tr></thead><tbody><tr><td>Tris-EDTA Buffer (Background)</td><td>34 µL TE Buffer</td></tr><tr><td>FAM Dye Tagged Linear DNA Probe + TE</td><td>29.4 µL Mastermix, 4.6 µL TE Buffer</td></tr><tr><td>BHQ-1 Quencher Tagged Linear DNA Probe + TE</td><td>1.6 µL 100 uM BHQ-1 Quencher Tagged Probe, 32.4 µL TE Buffer</td></tr><tr><td>FAM Dye Tagged Linear DNA Probe + BHQ-1 Quencher Tagged Linear DNA Probe + Padlock Probe + miRNA</td><td>29.4 µL Mastermix, 1.6 µL 100 uM BHQ-1 Quencher Tagged Probe, 3 µL Padlock + miRNA Solution</td></tr><tr><td>FAM Dye Tagged Linear DNA Probe + BHQ-1 Quencher Tagged Linear DNA Probe + Rolling Circle Product + TE</td><td>29.4 µL Mastermix, 1.6 µL 100 uM BHQ-1 Quencher Tagged Probe, 3 µL Linear Probe Rolling Circle Product</td></tr></tbody></table>
+<ol>
+<li>Flick and spin down tubes.</li>
+<li>Place tubes in the thermocycler at 41°C for 1 minute.</li>
+<li>Place tubes in thermocycler 37°C for 1 minute.</li>
+<li>Pipette all 32 μL of solution into a 384-well plate to measure fluorescence at an excitation wavelength of 480 nm and emission intensity at 518 nm using a plate reader.</li>
+</ol>
+<h2 id="lettuce-with-rcp--2022-protocol">Lettuce with RCP | 2022 Protocol</h2>
+<p>In nuclease-free PCR tubes add the following triplicates:</p>
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+<table><thead><tr><th></th><th></th></tr></thead><tbody><tr><td>Background</td><td>25 μL RNase-free water, 1 μL 10X Splint R Ligase Buffer, 2.5 μL 10X phi29 Reaction Buffer</td></tr><tr><td>Lettuce Left + Lettuce Right + Dye</td><td>19 μL RNase-free water, 1 μL 10X Splint R Ligase Buffer, 2.5 μL 10X phi29 Reaction Buffer, 3 μL Split Lettuce Left Sequence (100 uM stock), 3 μL Split Lettuce Right Sequence (100 uM stock)</td></tr><tr><td>RCP + Lettuce Left + Lettuce Right + Dye</td><td>19.5 μL RNase-free water, 3 μL Split Lettuce Left Sequence (100 uM stock), 3 μL Split Lettuce Right Sequence (100 uM stock), 3 μL RCP</td></tr><tr><td>Padlock + miRNA + Lettuce Left + Lettuce Right + Dye</td><td>19.5 μL RNase-free water, 3 μL Split Lettuce Left Sequence (100 uM stock), 3 μL Split Lettuce Right Sequence (100 uM stock), 3 μL Control: Padlock + miRNA</td></tr><tr><td>Lettuce Full</td><td>22.5 μL RNase-free water, 6 μL Lettuce Sequence (100 uM stock)</td></tr></tbody></table>
+<ol>
+<li>Centrifuge the reagents.</li>
+<li>On the sides of the centrifuged PCR tubes, carefully add 1.5 μL of DFHBI-1T (100 uM stock)</li>
+<li>For controls without the dye, steps 1-3 were repeated with 1.5 μL RNase-free water being added in place of the dye.</li>
+<li>Vortex and centrifuge reagents.</li>
+<li>Incubate the reaction at 70°C for 5 minutes, then cool to and incubate at 25°C for 1 hour.</li>
+<li>Read fluorescence on the plate reader. The emission spectra is 528 nm, and the excitation spectra is 480 nm.</li>
+</ol>
+<h2 id="sybr-safe-with-rcp">SYBRâ„¢ Safe with RCP</h2>
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+<table><thead><tr><th></th><th></th></tr></thead><tbody><tr><td>Background</td><td>4 μL SYBR™ Safe, 20 μL TE Buffer</td></tr><tr><td>Padlock + miRNA + SYBR™ Safe</td><td>20 μL Padlock + miRNA Solution, 4 μL SYBR™ Safe</td></tr><tr><td>RCP + SYBR™ Safe</td><td>20 μL RCP, 4 μL SYBR™ Safe</td></tr></tbody></table>
+<ol>
+<li>Vortex and centrifuge reagents.</li>
+<li>Incubate the reaction at 37°C for 1 hour.</li>
+<li>Read fluorescence on the plate reader. The emission spectra is 528 nm, and the excitation spectra is 480 nm.</li>
+</ol>
+<h2 id="capillary-rca">Capillary RCA</h2>
+<h3 id="dna-rna-hybridization-2">DNA-RNA Hybridization</h3>
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+<table><thead><tr><th>(A) - RCA</th><th>(B) - Negative Control (no enzymes)</th></tr></thead><tbody><tr><td>1 uL SplintR buffer</td><td>1 uL SplintR buffer</td></tr><tr><td>0.5 uL 100 nM padlock probe</td><td>0.5 uL 100 nM padlock probe</td></tr><tr><td>*1 uL 1.66 fM miRNA</td><td>1 uL 1.66 fM miRNA</td></tr><tr><td>*6 uL RNase free water</td><td>6.5 uL RNase free water</td></tr></tbody></table>
+<ol>
+<li>Heat the solution at 65°C for 3 minutes, and slowly cool it to room temperature over 10 minutes.</li>
+</ol>
+<h3 id="ligation-2">Ligation</h3>
+
+
+
+
+
+
+
+<table><thead><tr><th>(A) - RCA</th><th>(B) - Negative Control (no enzymes)</th></tr></thead></table>
+<p>|1 uL SplintR Ligase
+0.5 uL RNase Inhibitor|</p>
+<ol>
+<li>
+<p>Incubate the reaction for 2 hours at 37°C.</p>
+</li>
+<li>
+<p>Bring the reaction up to 65°C for 20 minutes to terminate.</p>
+</li>
+<li>
+<p>Orient PCR tubes horizontally and slide capillary tubes into each tube.</p>
+</li>
+<li>
+<p>Allow capillary action to draw up reaction mixture until there is about 1 centimeter of air left in the tube.</p>
+</li>
+<li>
+<p>Carefully slide the capillary tubes into the sigillum wax tray on both ends of the tube.</p>
+</li>
+<li>
+<p>Apply a thin coat of transparent nail polish to both sides of the capillary tube, and let dry for approximately 30 seconds.</p>
+</li>
+<li>
+<p>Place the capillary tubes in a tray and incubate at 37°C for 5 hours.</p>
+</li>
+<li>
+<p>Incubate the remainder of the reaction mixture in PCR tubes at 37°C for 5 hours.</p>
+</li>
+<li>
+<p>Carefully remove the tray from the incubator, and place the tray into a UV illuminator.</p>
+</li>
+<li>
+<p>Image the tubes with a phone camera and count the number of dots visualized in each capillary tube.</p>
+</li>
+</ol>
+<h3 id="gel">GEL:</h3>
+<ol>
+<li>Use an air pump to blow the liquid from the capillary tubes into PCR tubes.</li>
+<li>Add 2 uL loading dye to the PCR tubes and pipette into gel.</li>
+<li>For the RCA reactions run within PCR tubes:</li>
+<li>Pipette 10 uL of RCP + 2 uL loading dye into the gel.</li>
+<li>Add 12uL of the 2-log ladder into the gel.</li>
+<li>Start the blueGelâ„¢ gel electrophoresis system.</li>
+<li>After 1 hour, use the UV illumination on the blueGelâ„¢ gel electrophoresis system to visualize the DNA</li>
+</ol>
+<h2 id="erca-protocol">eRCA Protocol</h2>
+<h3 id="dna-rna-hybridization-3">DNA-RNA Hybridization</h3>
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+<table><thead><tr><th>(A) - RCA</th><th>(B) - no enzymes</th></tr></thead><tbody><tr><td>1 uL ligation buffer</td><td>1 uL ligation buffer</td></tr><tr><td>0.5 uL 100 nM padlock probe</td><td>0.5 uL 100 nM padlock probe</td></tr><tr><td>1 uL miRNA (1:10)</td><td>1 uL miRNA</td></tr><tr><td>6 uL water</td><td>7.5 uL water</td></tr></tbody></table>
+<ol>
+<li>Heat the solution at 65°C for 3 minutes, and slowly cool it to room temperature over 10 minutes.</li>
+</ol>
+<h3 id="ligation-3">Ligation</h3>
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+<table><thead><tr><th>(A) - RCA</th><th>(B) - no enzymes</th></tr></thead><tbody><tr><td>1 uL Splint R</td><td></td></tr><tr><td>0.5 uL RNase Inhibitor</td><td></td></tr></tbody></table>
+<ol>
+<li>Incubate the reaction for 2 hours at 37°C.</li>
+<li>Bring the reaction up to 65°C for 20 minutes to terminate.</li>
+</ol>
+<h3 id="amplification-3">Amplification</h3>
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+<table><thead><tr><th>(A) - RCA</th><th>(B) - Negative control (no enzymes)</th></tr></thead><tbody><tr><td>2.5 uL phi29 buffer</td><td>10.01 uL RNase free water</td></tr><tr><td>1.25 uL phi29 DNA polymerase</td><td>2.5 uL phi29 buffer</td></tr><tr><td>1.5 uL Nb.BbvCI</td><td>2.49 uL rCutSmart buffer</td></tr><tr><td>6.76 uL dNTPs</td><td></td></tr><tr><td>2.49 uL rCutSmart buffer</td><td></td></tr></tbody></table>
+<ol>
+<li>Incubate the reactions for 5 hours at 37°C</li>
+<li>Bring the reactions up to 65°C for 10 minutes to inactivate the DNA polymerase</li>
+</ol>
+<h2 id="erca-readout">eRCA Readout</h2>
+<p>In nuclease-free PCR tubes add the following triplicates:</p>
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+<table><thead><tr><th></th><th></th></tr></thead><tbody><tr><td>Background</td><td>22.51 uL RNase-free water, 1 uL 10X Splint R Ligase Buffer, 2.5 uL10X phi29 Reaction Buffer, 2.49 uL rCutSmart Buffer</td></tr><tr><td>RCP + Dye</td><td>19.5 uL RNase-free water, 3uL RCP</td></tr><tr><td>Control + Dye</td><td>19.5 RNase-free water, 3uL Control: Padlock + miRNA</td></tr><tr><td>Lettuce Full + Dye</td><td>22.5 uL RNase-free water, 6uL Lettuce Sequence (100 uM)</td></tr></tbody></table>
+<ol>
+<li>Centrifuge the reagents.</li>
+<li>On the sides of the centrifuged PCR tubes, carefully add 1.5 μL of DFHBI-1T (100 uM stock)
+<ul>
+<li>For controls without the dye, steps 1-3 were repeated with 1.5 μL RNase-free water being added in place of the dye.</li>
+</ul>
+</li>
+<li>Vortex and centrifuge reagents.</li>
+<li>Incubate the reaction at 70°C for 5 minutes, then cool to and incubate at 25°C for 1 hour.</li>
+<li>Read fluorescence on the plate reader. The emission spectra is 528 nm, and the excitation spectra is 480 nm.</li>
+</ol>
+<h1 id="protein-purification">Protein Purification</h1>
+<h2 id="nebexpress-ni-spin-column-protocol">NEBExpress Ni Spin Column Protocol</h2>
+<h3 id="to-prepare-buffers-for-ni-spin-columns-under-native-conditions">To prepare buffers for Ni Spin Columns under Native Conditions</h3>
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+<table><thead><tr><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td></td><td>Lysis/Binding Buffer: 20 mM sodium phosphate, 300mM NaCl, pH 7.4</td><td>Wash Buffer: 20mM sodium phosphate, 300mM NaCl, 5mM Imadazole pH 7.4</td><td>Elution Buffer: 20 mM sodium phosphate, 300 mM NaCl, 500 imidazole pH 7.4</td></tr><tr><td>2X IMAC Buffer</td><td>7.5 ml</td><td>5.0 ml</td><td>2.5 ml</td></tr><tr><td>2M Imidazole</td><td></td><td>0.025ml</td><td>1.25 ml</td></tr><tr><td>H2O</td><td>7.5 ml</td><td>5 ml</td><td>1.25 ml</td></tr><tr><td>Total</td><td>15.0 ml</td><td>10.0 ml</td><td>5.0 ml</td></tr></tbody></table>
+<h2 id="column-preparation">Column Preparation</h2>
+<ol>
+<li>Remove the bottom tab of the column by twisting, loosen the top cap and place the column in the collection tube provided.</li>
+<li>Centrifuge column at 800 x g for 1 minute to remove the storage buffer, discard the buffer.</li>
+<li>Add 250 μl of Lysis/Binding buffer to the column.</li>
+<li>Centrifuge column at 800 x g for 1 minute, discard the Lysis/Binding buffer.</li>
+<li>Place the column in a new 2 ml microcentrifuge tube</li>
+</ol>
+<h2 id="sample-cell-lysis">Sample Cell Lysis</h2>
+<ol>
+<li>Add 5ml of Luria Broth (LB) to a culture tube</li>
+<li>Add 5ul of antibiotic to the culture tube</li>
+<li>Collect a colony from the transformation plate using a pipette tip and insert it into the culture tube</li>
+<li>Let the tube shake in the incubator and allow growth for 24 hours</li>
+<li>Moved 2 ml of inoculation culture to a microcentrifuge tube</li>
+<li>Centrifuged the microcentrifuge tube for 5 min at 15600 rpm</li>
+<li>Removed supernatant (liquid LB) from the centrifuge tube, leaving only the cell pellet</li>
+<li>Add 250μL of TE buffer to the microcentrifuge tube containing the cell pellet</li>
+<li>Hydrated lyophilized lysozyme with 1mL TE buffer</li>
+<li>Added 50μL of lysozyme into the microcentrifuge tube for the final protein sample lysate</li>
+</ol>
+<h2 id="lysate-binding">Lysate Binding</h2>
+<ol>
+<li>Add up to 500 μl of the protein sample lysate to the column.
+<em>Note: If sample volume is greater than 500 μl multiple applications can be performed; collect the flow through in separate microcentrifuge tubes.</em></li>
+<li>Tap the column to mix the lysate with the resin and allow binding for 2 minutes.
+<em>Note: Binding of some His-tagged proteins can be increased with longer mixing times. Cap the column and seal the bottom using the plug provided. Mix end-over-end at 4°C for desired time (typically 5-15 minutes). Prolonged mixing may result in more non-specific binding.</em></li>
+<li>Centrifuge column at 800 x g for 1 minute, reserve flow through.</li>
+<li>Place the column in a new 2 ml microcentrifuge tube.</li>
+</ol>
+<h2 id="column-wash">Column Wash</h2>
+<ol>
+<li>Add 250 μl of Wash Buffer to the column and centrifuge at 800 x g for 1 minute.</li>
+<li>Repeat wash step twice, collect each wash in a separate 2 ml microcentrifuge tube.</li>
+</ol>
+<h2 id="protein-elution">Protein Elution</h2>
+<ol>
+<li>Place column in a new 2 ml microcentrifuge tube.</li>
+<li>Add 200 μl of Elution Buffer to the column. Mix the resin with the elution buffer thoroughly.
+<em>Note: Elution volume can be reduced to 100 μl if a more concentrated protein sample is desired, total target protein yield may be lower.</em></li>
+<li>Centrifuge at 800 x g for 1 minute, save eluted sample.</li>
+<li>Place column in a new 2 ml microcentrifuge tube and repeat elution step. Typically, >90% of the bound protein is eluted following the second elution.</li>
+<li>Analyze the clarified cell lysate (load), flow through, washes and eluates by SDS-PAGE.</li>
+</ol>
+ </astro-slot></div></div></div></body></astro-island>
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+ <title>Hardware</title>
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+ <h1 id="labpilot">LabPilot</h1>
+<h2 id="overview">Overview</h2>
+<p>In previous experiments, Lambert iGEM experienced firsthand
+difficulties of tedious pipetting and inaccurate results. Last year, Rolling Circle
+Amplification (RCA) reactions we ran took the majority of our time. This took important
+research time away, limiting us from our full potential. Issues like these inspired
+us to develop LabPilot, a frugal automated liquid handler that allows researchers
+to focus time elsewhere from running lengthy reactions like RCA. Pipetting is a fundamental laboratory technique used to transfer precise volumes of liquid from one container to another. Consistent pipetting is essential for achieving accurate results, maintaining data integrity, and facilitating advancements in synthetic biology research. However, manual pipetting, the primary method utilized in most labs, limits throughput (the amount of liquid passing through a pipette) and is easily susceptible to human error.
+To resolve these issues, automated liquid handlers are designed to automate and streamline the process of pipetting liquids in laboratory settings. Liquid handlers can be programmed to perform a wide range of pipetting tasks with high precision, accuracy, and efficiency, enabling high throughput experiments (Liquid Handling Automation Benefits). However, current automated liquid handlers costing between $5000 and $300,000 are difficult to use (Retisoft, 2021). To provide underfunded labs access to liquid handlers, Lambert iGEM developed LabPilot, a frugal liquid handler made with 3D printable and accessible parts that can accurately pipette based on direct user input (Hentz & Knaide, 2014). Therefore, it enables research to be conducted in an efficient manner, filling an essential gap in lab settings.</p>
+<h2 id="parts-list">Parts list</h2>
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+
+<table><thead><tr><th>Part:</th><th>Seller:</th></tr></thead><tbody><tr><td>Arduino Mega 2560</td><td>Yanmis (Amazon) <a href="https://tinyurl.com/2p8647ah">https://tinyurl.com/2p8647ah</a></td></tr><tr><td>CNC Shield</td><td>Yanmis (Amazon) <a href="https://tinyurl.com/2p8647ah">https://tinyurl.com/2p8647ah</a></td></tr><tr><td>A4988 Motor Drivers</td><td>Yanmis (Amazon) <a href="https://tinyurl.com/2p8647ah">https://tinyurl.com/2p8647ah</a></td></tr><tr><td>12 Volt 5 amp Power Supply and Cable</td><td>Alitove Store (Amazon) <a href="https://tinyurl.com/mb4rs42t">https://tinyurl.com/mb4rs42t</a></td></tr><tr><td>Five Nema 17 Motors</td><td>SIMAX3D (Amazon) <a href="https://tinyurl.com/389ssrd9">https://tinyurl.com/389ssrd9</a></td></tr><tr><td>Timing Belt And Pulleys Combo</td><td>Kee Yees Store (Amazon) <a href="https://tinyurl.com/2bkfz445">https://tinyurl.com/2bkfz445</a></td></tr><tr><td>Bearings</td><td>Yosoo Health Gear (Amazon) <a href="https://tinyurl.com/2vwxwvzv">https://tinyurl.com/2vwxwvzv</a></td></tr><tr><td>Limit Switches</td><td>Octagon Star (Amazon) <a href="https://tinyurl.com/dv65fkhv">https://tinyurl.com/dv65fkhv</a></td></tr><tr><td>Two 8mmx300mm Linear Motion Steel Rods</td><td>Amazon <a href="https://tinyurl.com/5e4hfrf6">https://tinyurl.com/5e4hfrf6</a></td></tr><tr><td>Two 8mmx100mm Threaded Steel Rods</td><td>Uxcell (Amazon) <a href="https://tinyurl.com/mr2ft89z">https://tinyurl.com/mr2ft89z</a></td></tr><tr><td>Four 8mmx350mm Linear Motion Steel Rods</td><td>Amazon <a href="https://tinyurl.com/ywsbncdf">https://tinyurl.com/ywsbncdf</a></td></tr><tr><td>Four 8mmx150mm Linear Motion Steel Rods</td><td>Amazon <a href="https://tinyurl.com/mu6ujt7s">https://tinyurl.com/mu6ujt7s</a></td></tr></tbody></table>
+<h2 id="design">Design</h2>
+<p>LabPilot is an automated liquid handler compatible with micropipettes. It utilizes an XYZ layout for movement, with most of its structural parts designed on Fusion 360, a computer-aided design (CAD) application. LabPilot’s design can be split into the frame, detachable molds for the bed, and XYZ planes with motors.</p>
+<h3 id="frame">Frame</h3>
+<p>Our team designed Labpilot’s frame to be small enough to fit inside a flow hood and large enough to fit standard laboratory containers on its bed (e.g. microcentrifuge tube racks, micropipette tip boxes, and well plates). We designed the frame by creating a linear rail extrusion, as shown in Figure W, typically found on commercial 3D printers. We used linear extrusions to make the cage-like design of LabPilot, inspired by Ender 3D printers like the Ender 5 (see Table 1) (Ender 5 S1 3D Printer). The finalized XYZ dimensions of LabPilot are 366mm x 296mm x 356mm (see Fig. 2). To provide stability to LabPilot’s frame, we added angle brackets that connect two of the extrusions that run perpendicular to each other at all 16 corners of the frame.
+We spoke to Priya Soneji, an undergraduate researcher at the Georgia Institute of Technology, who is a part of the Bhamla Lab. For the future, she recommended the switch to metal frames if the 3D-printed parts were imprecise. She confirmed the validity of our overall plan for controlling each motor axis on the XYZ plane and methods for motor movement.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/hardware/linearrailextrusion.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. CAD model of linear rail extrusion used to make the structural frame.</div></div>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/hardware/labpilotframe.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 2. CAD model of LabPilot frame.</div></div>
+<h3 id="molds">Molds</h3>
+<p>Lambert iGEM created molds that snap onto the bed plate of LabPilot so that the user can attach their desired components to the bed. The molds have pegs (see Fig. 3) that can be placed inside engraved indentations on the bed plate (see Fig. 4), which fixes the molds in place and enables the LabPilot app to calculate their position. LabPilot has a mold that fits every micropipette tip box, beaker, microcentrifuge tube rack, PCR tube rack, and well plate.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/hardware/labpilotbeakermold.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. Pegs placed on the bottom of the molds to fit into the bed plate.</div></div>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/hardware/labpilotbase.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Indentations on the bed plate for the molds to rest on.</div></div>
+<h3 id="xyz-motor-axis">XYZ Motor Axis</h3>
+<p>LabPilot utilizes five Nema 17 motors (Nema 17 Stepper Motor) (see Table 1) to control the three axes of movement needed for the pipetting mechanism. Nema 17 motors have a micro-stepping feature, enabling small and precise movements. Each motor is controlled by an Arduino Mega 2560 with a Ramps 1.4 CNC shield containing five A4988 motor drivers (see Table 1). The CNC shield uses two motors to control the vertical movement of the bed plate, as it is the heaviest moving part. The left/right movement and the forward/backward movement of the micropipette are controlled by one motor each. The last motor controls the pipetting mechanism (see Fig. 5) and is placed above the micropipette. Additionally, we placed limit switches for each axis to prevent over-rotation of the motors and calibration to home coordinates similar to 3D printers. Each axis has linear steel rods that act as supports, with bearings in place to ensure the pipette mechanism slides smoothly across the rods. We attached timing belts (thin rubber belts) with teeth (see Table 1) to the motor to carry the pipette left, right, forwards, and backwards across the X and Y axes.
+In addition to the linearly moving axes, LabPilot requires a motor to press the micropipette to the first and second stops. The pipetting mechanism motor converts the rotational motion of the motor into a linear force that can push down on the micropipette button. The motor applies a fine-tuned force to achieve first and second stops. Currently, LabPilot is designed to be used with Eppendorf micropipettes to ensure everything fits correctly. We attached a stationary arm to the bed plate to eject the pipette tips. The micropipette moves into the arm while sequentially traveling upwards. This way, the tip is pulled off the micropipette and discarded underneath.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/pipettemechanismsarvajith.jpeg" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 5. Pipette mechanism</div></div>
+<h2 id="app">App</h2>
+<p>To make LabPilot easier for researchers, Lambert iGEM designed a web app to control the liquid handler. Unlike the complex software that operates commercial liquid handlers such as Opentrons, the LabPilot app has a simple, human-centric user interface designed to be used immediately with simple and intuitive commands. This design enables users to quickly start using LabPilot and efficiently simplify their tasks without any difficulties.</p>
+<h3 id="how-it-works">How it Works</h3>
+<p>To intelligently control its actions, LabPilot utilizes an Arduino microcontroller connected to a laptop running the LabPilot web app via a USB cable. The microcontroller is in charge of controlling the movement of the pipette mechanism and the action of pipetting. The microcontroller moves the micropipette to a specific location by using a virtual coordinate system, which defines different areas of the surface with numbers. The LabPilot app knows what actions to take in order to move the desired axes to their coordinate destination.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/hardware/labpilotmicrocontroller.png" style="max-width:500" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 6. shows how the user input on the app controls the microcontroller.</div></div>
+<p>The app provides instructions to the microcontroller via the Arduino serial USB connection (see Fig. 6). By utilizing a system where the app executes high-level operations by compiling pipette actions into low-level instructions for the microcontroller, users have complete control over their LabPilot device. They can stop or pause it at any time.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/img-9427.jpeg" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 7. Shows how the user input controls LabPilot.</div></div>
+<h3 id="how-to-use">How to use</h3>
+<p>To use LabPilot, users must connect a laptop to LabPilot via a USB cable and open its web dashboard. Once the dashboard is open, there is a simple setup process where users can drag and drop components from the sidebar to recreate the physical layout of their LabPilot device, as seen in Figure Y. After setup, the app digitally recreates the layout of this device from data gathered in the setup process, enabling adequate simulation and control. Then, the users can queue and perform pipetting actions.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/image-10-10-23-at-8-43-pm.jpg" style="max-width:600" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 8. LabPilot’s setup interface.</div></div>
+<p>All pipetting actions consist of a reagent source and a dispensing source. Users can click on any well or beaker to select a reagent source. After choosing a reagent source, users can select a dispensing source by clicking on any well/beaker or multiple dispensing sources (by clicking and dragging or holding “shift†while they select multiple sources). Afterward, users will select the amount they want to dispense to each dispensing source and can add it to the queue (see Fig. 7). All pipetting actions in the queue are performed in the order they were assigned but can be dragged around to reorder in the queue.</p>
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+<h2 id="conclusion-and-future-directions">Conclusion and Future Directions</h2>
+<p>Researchers can leverage LabPilot’s capabilities to streamline other critical processes such as DNA sequencing, sample preparation, high-throughput screening, and assay development. By automating these tasks, LabPilot enables scientists to focus on higher-level analysis and interpretation of results, accelerating the pace of scientific discoveries. Next year, Lambert iGEM hopes to switch to an aluminum frame for its cost-effective, rigid nature and increase compatibility with various laboratory equipment. Additionally, we plan to test with polymerase chain reaction (PCR) as a proof of concept experiment. A successful PCR will have LabPilot demonstrate accuracy in pipetting volumes, with substantial precision and a significant reduction in PCR time. Once we can prove that LabPilot upholds the accuracy and efficiency of a human through a proof of concept, we plan on using LabPilot to automate a portion of our rolling circle amplification biosensors to improve efficiency and reduce the potential for human error. Our end goal is to develop a kit available for purchase and make LabPilot commercially available. LabPilot’s cost-effectiveness, user-friendly interface, and open-source nature make it accessible and adaptable to all laboratory settings.</p>
+<iframe title="Lambert-GA: LabPilot Prototype 1 Axis (2023) [English]" width="560" height="315" src="https://video.igem.org/videos/embed/5e82a2a5-8e47-4ae9-b7c9-545f0e3cabc7" frameborder="0" allowfullscreen="" sandbox="allow-same-origin allow-scripts allow-popups"></iframe>
+<h2 id="references">References</h2>
+<div class="pl-6 -indent-6"><p>Amazon.com: Usongshine Nema 17 Stepper Motor Bipolar Step Motor for …
+(n.d.). <a href="https://www.amazon.com/Usongshine-Stepper-Motor/dp/B0BPL725TN">https://www.amazon.com/Usongshine-Stepper-Motor/dp/B0BPL725TN</a></p></div>
+<div class="pl-6 -indent-6"><p>Choosing An Automated Liquid Handler. (2021, August 26). Retisoft.
+<a href="https://retisoft.com/choosing-a-liquid-handler/">https://retisoft.com/choosing-a-liquid-handler/</a></p></div>
+<div class="pl-6 -indent-6"><p>Ender-5 S1 3D printer. Crealitystore. (n.d.).
+<a href="https://store.creality.com/products/ender-5-s1-3d-printer?spm=..collection_90778a1d-d845-4ff0-a8c4-48fded4b7d74.albums_2.1&spm_prev=..index.header_1.1">https://store.creality.com/products/ender-5-s1-3d-printer?spm=..collection_90778a1d-d845-4ff0-a8c4-48fded4b7d74.albums_2.1&spm_prev=..index.header_1.1</a></p></div>
+<div class="pl-6 -indent-6"><p>Liquid Handling Automation Benefits. (n.d.). Retrieved October 1, 2023, from
+<a href="https://dispendix.com/blog/liquid-handling-automation-benefits">https://dispendix.com/blog/liquid-handling-automation-benefits</a></p></div>
+<div class="pl-6 -indent-6"><p>Hentz, N. G., & Knaide, T. R. (2014). Effect of Liquid-Handling Accuracy on
+Assay Performance. SLAS Technology, 19(2), 153–162.
+<a href="https://doi.org/10.1177/2211068213504095">https://doi.org/10.1177/2211068213504095</a></p></div>
+<div class="pl-6 -indent-6"><p>Eppendorf Research Plus Adjustable Volume, Single Channel Pipette, 2—20 uL,
+Yellow. Retrieved October 1, 2023, from
+<a href="https://www.pipettes.com/pipettes/single-channel-pipettes/manual/adjustable-volume/eppendorf-research-plus-single-channel-pipette-adjustable-2-20-ul">https://www.pipettes.com/pipettes/single-channel-pipettes/manual/adjustable-volume/eppendorf-research-plus-single-channel-pipette-adjustable-2-20-ul</a></p></div>
+<div class="pl-6 -indent-6"><p>OT-2 Robot Opentrons Labworks Inc. Retrieved October 1, 2023, from
+<a href="https://shop.opentrons.com/ot-2-robot/">https://shop.opentrons.com/ot-2-robot/</a></p></div>
+<div class="pl-6 -indent-6"><p>Ender 5 S1 3D Printer. (n.d.). Retrieved October 2, 2023, from
+<a href="https://store.creality.com/products/ender-5-s1-3d-printer?spm=..collection_90778a1d-d845-4ff0-a8c4-48fded4b7d74.albums_2.1&spm_prev=..index.header_1.1">https://store.creality.com/products/ender-5-s1-3d-printer?spm=..collection_90778a1d-d845-4ff0-a8c4-48fded4b7d74.albums_2.1&spm_prev=..index.header_1.1</a></p></div>
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+ <h1 id="human-practices">Human Practices</h1>
+<h2 id="overview">Overview</h2>
+<p>Last year, Lambert iGEM identified patients as the end goal of CADlock’s implementation. In order to accomplish this, we interviewed multiple leading microRNA researchers and cardiologists, who contributed to identifying the direction of our project as a point-of-care diagnostic screening tool to track the progression of coronary artery disease (CAD). Inspired to expand last year’s project with a focus on inclusivity, we improved and implemented wet lab mechanisms, including exponential rolling circle amplification (eRCA) from RCA <a href="https://2023.igem.wiki/lambert-ga/rca/">see RCA</a>. We also used protein purification to create our own phi29 DNA polymerase to minimize reaction costs. In addition, we expanded upon MicroQ, implementing an adaptation called MicroQ Pro. Furthermore, after talking with a graduate student, we developed LabPilot, an automated pipetter. We created a podcast and spoke to multiple doctors and professionals about the direction of our project. Drawing on the expertise of exceptional stakeholders, our journey transformed from conceptualization to implementation (see Fig. 1).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/hp-graphic-1.png" style="max-width:800" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. Overview graphic of the 2023 inclusivity approach.</div></div>
+<h2 id="doctors">Doctors</h2>
+<h3 id="dr-gentry">Dr. Gentry</h3>
+<p>We spoke to Dr. Mindy B. Gentry, a cardiologist in Marietta, Georgia, about sex inclusivity and CAD. We originally planned to use a gene called SVEP1, a gene that influences atherosclerosis. Specifically, SVEP1 expresses a protein that stimulates the development of plaque in arteries. This gene has a negative correlation with a miRNA named miR-1269b. Instead, she guided us to look at CAD correlation with an emphasis on estrogen-induced pathways. With her help, we were able to find a correlation between CAD and an estrogen-specific miRNA. Dr. Gentry, who runs a female cardiac health center, expressed the need for proactive measures to be taken to preserve heart health for females. The incorporation of Lambert iGEM’s estrogen initiative, guided by her aid, strengthens the proactive approach by making a diagnostic for a specialized risk group.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/gentry.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 2. Dr. Mindy B. Gentry</div></div>
+<h3 id="dr-fairweather">Dr. Fairweather</h3>
+<p>Dr. DeLisa Fairweather is a director of research at the Department of Cardiovascular Diseases at Mayo Clinic’s Florida campus. She conducts research on individualized therapies and improved diagnosis in the Cardiovascular field. Due to her specialized and intensive research on estrogen in cardiovascular disease, we spoke to her about our initiatives in trying to incorporate sex inclusivity in our project. She informed us that estrogen increases autoimmune disease in women, and inflammation associated with it leads to CAD in women. Initially, we had the understanding that miR-20b should protect women from CAD, but she clarified when estrogen drops with menopause, the Estrogen Receptors Alpha pathway would be so low that miR-20b would have no effect. On the other hand, miR-20b is a viable indicator for CAD in men. She guided us to expand our focus to this miR to encapsulate men, as well as have a specialized risk group for women who have symptoms of inflammation. Thus, we were able to not only target our proactive initiative towards women but men as well.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/fairweather.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. Dr. DeLisa Fairweather</div></div>
+<h2 id="researches">Researches</h2>
+<h3 id="dr-bhamla">Dr. Bhamla</h3>
+<p>Dr. Saad Bhamla, head of the Bhamla Lab at the Georgia Institute of Technology, has been an invaluable collaborator and supporter of Lambert iGEM. Through projects like the Frugal Science Academy (done in partnership with Lambert iGEM advisor Janet Standeven), Dr. Bhamla has provided funding, feedback, and opportunities to test designs in real-world settings. Last year, his input enabled Lambert iGEM to develop Micro-Q, a low-cost fluorometer, and test it in Thailand. This year, Dr. Bhamla’s insights allowed further iteration and adaptation of Micro-Q Pro to better its accuracy and facilitate the real-world impact of many Lambert iGEM projects.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/bhamla.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Dr. Saad Bhamla</div></div>
+<h3 id="rajas-poorna-a-grad-student-at-gt">Rajas Poorna, (a grad student at GT)</h3>
+<p>We spoke to Rajas Poorna, a graduate student working for the Bhamla Lab at the Georgia Institute of Technology, regarding alternatives to our existing RCA methodology to increase its accessibility in the field. Rajas informed us about existing capillary-tube-based diagnostics methods that used PCR and suggested modifying RCA to use capillary tubes. During our testing, we encountered problems with our experiments, such as capping the tubes and drawing up liquid. Some of his suggestions included using a biocompatible wax sealant to effectively seal the tubes and maintain the sample’s integrity by tilting the PCR tubes sideways to counteract the force of gravity on the liquid as it rose in the tube. Our conversations with Rajas gave us insight into developing a novel method of quantifying that does not require a fluorometer, enabling greater inclusivity and accessibility of our diagnostic tool.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/poorna.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 5. Rajas Poorna</div></div>
+<h3 id="dr-sliverman">Dr. Sliverman</h3>
+<p>This year, Lambert iGEM wanted to produce phi29 DNA Polymerase, a protein involved in the amplification process of RCA, by utilizing the Immobilized Metal Affinity Chromatography (IMAC) mechanism<a href="https://2023.igem.wiki/lambert-ga/protein-purification/">see Protein Purification</a>. However, after talking to professors at Georgia Tech, they informed us to consider obtaining it through the cell-free system, as the cells will be able to intake the plasmid better than in the in vivo system while creating the optimal amount of phi29 DNA Polymerase needed in our lab. We then spoke to Dr. Adam Sliverman, a Scientist at Sherlock Biosciences, who guided us in choosing either cellfree or IMAC. He informed us about the inability to scale large amounts of protein in vitro. Therefore, he suggested our team grow a liter of E. coli with phi29 DNA Polymerase in a pET expression vector, inducing it with IPTG and outgrowing it overnight. He also assured us that the purification of the protein from that point is relatively straightforward. With this information, we continued our research on the IMAC mechanism to selectively extract the his-tagged phi29 DNA polymerase from the lysate to obtain the purified phi29 DNA polymerase for the RCA mechanism.
+Aside from protein purification, we also wanted to pursue exponential rolling circle amplification (eRCA). Dr. Silverman has previously experimented with RCA and believed eRCA to be a sufficient mechanism for our sensor’s sensitivity. As eRCA exponentially increases output, he advised us to include additional controls to ensure we are not amplifying errors. He suggested using an ON-state reporter mechanism, as it would reduce error/noise by promoting signal gain. While we had been experimenting with the ON-state lettuce DNA aptamers, Dr. Silverman also proposed using G-quadruplexes or SYBR Green instead, as DNA aptamers are not very fluorescent. Although we considered these mechanisms, we found that the efficiency and cost of linear probes — an OFF-state reporter — was more suitable for our project.
+We also considered multiplexed detection of miRNAs using RCA, however, Dr. Silverman advised us against this due to the low efficiency of SplintR Ligase. He suggested attempting multiplexing with pre-ligated padlock probes first to eliminate the potential error created by the enzyme, but we plan to test this idea in the future.
+Dr. Silverman’s feedback in improving the affordability and accuracy of our biosensor enabled us to create a higher-quality assay that makes preliminary care more accessible for all patients.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/silverman.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 6. Dr. Adam Silverman</div></div>
+<h3 id="dr-charles-searles">Dr. Charles Searles</h3>
+<p>Dr. Charles Searles is an associate professor of medicine at Emory Healthcare Hospital and a cardiologist specializing in biomarkers correlated with CAD. Last year, he provided us with hsa-miR-451a — unrelated to the pathophysiology of CAD — as a control for our proof-of-concept experimentation. Dr. Searles has tested our biosensors in his lab and offered to test them with actual patient serum samples in the future. He also gave us helpful insights on what would be useful in a miRNA database for CAD and validated the need for a database like CADmir. After we built CADmir, we shared it with Dr. Searles, and he gave us valuable feedback on improvements we could make to the platform. We iterated on his input and modified CADmir with filtration and testing on our publications. With his help, we were able to develop a miRNA database for CAD that helps miRNA researchers find information faster.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/searles.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 7. Dr. Charles Searles</div></div>
+<h3 id="kimberly-ann-rooney">Kimberly Ann Rooney</h3>
+<p>Kimberly Ann Rooney, a lab technician at the Searles Cardiovascular Lab led by Dr. Charles Searles, ran RCA reactions at Emory utilizing our protocol to test whether our biosensor could be practical and applicable as a diagnostic tool. Ms. Rooney gave us feedback on our methodology, advising that we reduce the number of variables used as controls and use serial dilutions to obtain more precise miRNA concentrations. She guided us towards optimizing the overall time of our assay, allowing us to further emphasize proactive care by decreasing the time it takes for hospitals to communicate results to patients.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/rooney.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 8. Kimberly Ann Rooney</div></div>
+<h3 id="dr-mark-styczynski">Dr. Mark Styczynski</h3>
+<p>Dr. Mark Styczynski, a researcher from the Georgia Institute of Technology, told us that eRCA is a plausible mechanism. If the error with the RCA assay is from the technological aspect, eRCA should perform as expected. However, it could potentially amplify the discrepancy in our data if the error is due to the biological process of RCA. Dr. Styczinski also pointed out that using an OFF-state reporter, such as linear DNA probes, has a limited dynamic range as it is losing signal. He suggested using an ON-state sensor instead, as it would address this fundamental limitation. While we had been working with split lettuce DNA aptamers, Dr. Styczynski urged us away from this mechanism as they are more unreliable than other reporters. We then proposed producing whole aptamers through RCA. He was against this idea as the secondary structures of multiple aptamers’ would likely interact poorly on a single strand of DNA. Therefore, we utilized eRCA to produce whole lettuce aptamers, as this mechanism produces separate, isolated strands of DNA rather than a single, long strand.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/styczynski.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 9. Dr. Mark Styczynski</div></div>
+<h3 id="megan-mcsweeney">Megan McSweeney</h3>
+<p>Megan McSweeney is a graduate student at the Georgia Institute of Technology and part of the Styczynski lab. She stated that the efficiency of eRCA would depend on the source of the error, paralleling Dr. Styczynski’s input. eRCA could be more accurate than RCA if it does not amplify the error. She was also concerned that our discrepancy could be due to contamination, leading us to take extra precautions to ensure an aseptic and RNase-free environment. Her feedback helped us increase the accuracy of our biosensor, therefore increasing its proactiveness and applicability in point-of-care testing.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/mcsweeney.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 10. Megan McSweeney</div></div>
+<h3 id="priya-soneji">Priya Soneji</h3>
+<p>Priya Soneji is an undergraduate researcher at the Georgia Institute of Technology and part of the Bhamla Lab. She designed and built a device called Trackoscope, a frugal tracking microscope for tracking the movement of microorganisms. It has a similar frame and structural design we intended to use for LabPilot, which is the inexpensive, automated micropipetter we are creating to aid labwork. We proposed our design on Fusion 360, a computer-aided design (CAD) software tool, to Priya, who verified that this software was the best option for CAD design. She also recommended that we try aluminum supports in case the 3D printed parts were time-consuming and imprecise issues. Furthermore, Priya uses the Nema 17 motors in her Trackoscope design, which includes a micro-stepping feature that enables precise motions. We then asked her if using Nema 17 motors seemed appropriate for our build, and she said they are very reliable and accurate motors. She confirmed the validity of our overall plan for controlling each motor axis on the XYZ plane and methods for motor movement. Priya greatly improved our understanding of the accuracy of our pipette mechanism and also allowed us to look into the affordability of some elements.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/priya.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 11. Priya Soneji</div></div>
+<h2 id="institutionsbroader-community">Institutions/Broader Community</h2>
+<h3 id="dr-shana-scott">Dr. Shana Scott</h3>
+<p>We also spoke to Dr. Shana Scott, one of the leading members of the American Heart Association (AHA). She helped us establish and co-hosted the September Heart Health Month. She also guided us towards initiating an updated CERP (Cardiac Emergency Response Plan) at our school in order to cater to a more inclusive community. Dr. Shana Scott was also integral in promoting and increasing our outreach countywide. Lambert iGEM originally planned school-wide September Heart Health webinars and only planned CPR/First Aid education. With Dr. Scott’s guidance, we were able to increase our outreach and expand our CPR program to a policy.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/scott.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 12. Dr. Shana Scott</div></div>
+<h3 id="mrs-michelle-routhenstein">Mrs. Michelle Routhenstein</h3>
+<p>Mrs. Routhenstein, a nutritionist, inspired our initiative to shift towards a more proactive approach. As a guest on our podcast, she detailed the importance of maintaining a balanced diet and implementing exercise in a daily routine as crucial practices in preventing heart disease altogether. She discussed general trends she’d seen in her patients with CAD, such as a poor diet filled with saturated fats, and a lack of an active lifestyle. She explained how as a nutritionist, her minor changes to these patients’ diets improved their condition tremendously. She even inspired us to conduct our September Heart Health webinar to encourage the message of the importance of diet and nutrition in preventing CAD to a broad and diverse audience.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/routhenstein.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 13. Mrs. Michelle Routhenstein</div></div>
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+ <h1 id="overview">Overview</h1>
+<p>Healthcare is broken down into one of two categories: reactive and proactive. The reactive approach utilizes medical intervention to treat already present symptoms and conditions (Waldman, 2019). Unfortunately, the United States healthcare system focuses strictly on reactive treatment, accounting for more than 75% of healthcare spending in the U.S. (Mercury Healthcare, 2018). These reactive practices occur in coronary artery disease (CAD) diagnosis; patients undergo invasive tests after showing observable symptoms (Skelly, Hashimoto, & Buckley et al., 2016). Although there is a shift from reactive to proactive healthcare regarding age-related diseases, patient retention remains an issue (Mercury Healthcare, 2018). While people visit facilities for early testing, the delayed process of receiving lab results- whether it be from outsourcing samples to different labs, scheduling, etc.- has the effect of fewer people returning to healthcare facilities for preventative care (Mercury Healthcare, 2018) . Furthermore, four out of seven studies reported missed diagnoses of malignancy, hypothyroidism, hyperthyroidism, osteoporosis, and microbiological results (Callen et al., 2011). CADlock aims to increase the accessibility to preventative healthcare by emphasizing affordable screening, early diagnostics, and accessibility for all. This approach will ensure care for individuals of all demographics and socioeconomic statuses. Therefore, our team’s goal was a multifaceted approach using synthetic biology to develop diagnostic solutions, to educate our community about proactive healthcare, and to include underrepresented populations within our initiatives (see Fig. 1).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/inclusivity/hp-hierarchy.png" style="max-width:600" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. The expansion of our inclusivity focus this year, combining accessibility, affordability, and accuracy under proactivity.</div></div>
+<p>Our inclusive preventative approach begins by diagnosing CAD via microRNAs. We expanded Rolling Circle Amplification (RCA) to exponential RCA (eRCA) which increases signal strength and decreases time to detection from picomolar concentrations of miRNA in serum samples. This approach directly targets the issue of patient retention by increasing the accessibility of knowledge patients have about their condition. This early diagnosis and consultation brings patients back in since they know they may be susceptible to a disease, and thus may prevent it before their condition worsens. In addition to using eRCA, we also demonstrate using capillary tubes to perform RCA reactions with the goal of incorporating a future all-in-one reaction/visualization device for point-of-care testing. To address the high cost of phi29 polymerase we experimented with cloning and purifying our own proteins. Under the advice of Dr. Charles Searles, in conjuction to changing the RNA detection methods, we also expanded our library of miRNAs to include multiple novel sequences shown to be indicative of CAD in African American populations in Atlanta, GA (Dluzen et al., 2016). We envision an eventual library of miRNA padlocks available to clinicians to provide individualized medical diagnostics, thus enabling clinicians to directly provide individualized early diagnostics to their patient populations. Essentially, the increase in the rate at which patients receive their test results is positively correlated with proactive care because patients are more willing to take precautionary measures when they are given optimal accessibility to care (Mercury Healthcare, 2018).</p>
+<p>During our research into miRNAs, it was evident that a lack of a database for miRNAs specifically indicated in coronary diseases was needed. To address the gap, we developed CADmir, a large language model (LLM) powered web app that synthesizes scientific information backed up by open-source, peer reviewed miRNA research (see Fig. 2). This will allow anyone with computer access to quickly conduct a literature review on heart disease related miRNA research, increasing accessibility to scientific knowledge.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/inclusivity/cadmir.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 2. Image of the CADmir user interface</div></div>
+<p>Pipetting multiple reactions for trials in triplicate can be tedious and lead to errors that interfere with data analysis or worse, having to repeat costly experiments. We developed LabPilot, an automated pipette system to increase pipet reliability and increase the researcher’s time efficiency. According to multiple interviews with current Ph.D. and Postdoctoral researchers, pipetting is time consuming, error prone, and seen as a hindrance to their “real†work LabPilot is a low-cost alternative to commercial liquid handling systems that cost $10,000+, limiting their use to well funded labs (see Fig. 3).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/inclusivity/labpilot.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. LabPilot pipeline</div></div>
+<p>In our current use, RCA reactions output a fluorescent signal leading to the need for a method of quantifying the output. Last year, we developed Micro-Q, a low-cost fluorometer that utilized a photoresistor, cloud-based quantification algorithms, and an intuitive mobile app to quantify biotechnology reactions. This year, we improved Micro-Q to quantify most fluorescent materials by utilizing a 5000k LED strip and expanded the capabilities to quantify a strip of PCR tubes with a camera-based quantification method. Ultimately, this device increases accessibility to fluorescence based experiments and diagnostics worldwide.</p>
+<p>Preventative care is two-fold, relying on early detection and educating patients. By implementing different RNA detection methods, software, and novel hardware, we aim to increase early detection. Educating local and dispersed populations was another major initiative this year. We held a local Heart Healthy Awareness month and contributed to a state-wide teacher professional development. To reach a broader audience, we created a podcast called SynbioOutLoud, and collaborated with Flowers for the Future, an international organization dedicated to supplying resources in order to promote STEM education for girls in Kabul, Afghanistan.</p>
+<h1 id="wetlab">Wetlab</h1>
+<h2 id="rca">RCA</h2>
+<p>Furthering last year’s approach with accessibility, affordability, and accuracy, we continued utilizing and improving RCA to assess CAD risk groups while also addressing underrepresented demographics of people. Improvements to last year’s RCA mechanism include testing the biosensor’s specificity, comparing reporter mechanisms, and optimizing reaction times. In addition to RCA modifications, Lambert iGEM pursued a new wet-lab initiative called exponential rolling circle amplification (eRCA). eRCA provides a more sensitive reading of microRNAs with a faster reading time. The emphasis on additional accuracy through eRCA and affordability to end users enables CADlock to be accessible to a wide range of demographics, not limited to race, sex, and economic status (See <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: eRCA</a>).</p>
+<h2 id="capillary">Capillary</h2>
+<p>Lambert iGEM explored optimizing the reaction time and resources by running the reaction in capillary tubes instead of PCR tubes for DNA amplification and imaging. This quicker response time allows for more patients to be tested in a point-of-care setting. This increase in healthcare accessibility and thus patient retention time allows for an emphasis to be placed on proactive care. MicroRNAs (miRNAs) can easily diffuse through the capillary tube due to the tube’s small diameter. This results in high amounts of DNA in isolated locations (see Fig. 4). When exposed to a specific wavelength of light, individual specks of DNA can be seen by the naked eye. DNA can then be counted without the use of complex fluorescence quantification devices such as fluorometers or plate readers, which makes the test more accessible and lowers the technology barrier. Overall, capillary amplification is a reliable, efficient, and user-friendly assay. <a href="https://2023.igem.wiki/lambert-ga/rca/">See Wetlab: Capillary</a>.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/inclusivity/screenshot-2023-10-11-102911.png" style="max-width:500" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. A representation of the difference between the RCA reaction in the PCR tube vs. the capillary tube.</div></div>
+<h2 id="protein-purification">Protein Purification</h2>
+<p>In addition to frugal technologies, Lambert iGEM aimed to decrease the cost of Rolling Circle Amplification (RCA) reagents by producing the enzymes required through protein purification. Phi29 DNA polymerase, priced at $251.00 for 1,250 units from New England Biolabs, not only covers the majority of the cost of RCA reagents but also possesses invaluable qualities such as strand displacement and processive synthesis properties that are necessary for RCA (New England Biolabs, n.d). By producing phi29 DNA polymerase through protein purification, combined with the accuracy of exponential RCA, Lambert iGEM aims to increase the affordability and in turn accessibility, both of which are contributing factors to inclusive care. This allows for more tests because the proteins are much more affordable, increasing the preventative aspect of healthcare. <a href="https://2023.igem.wiki/lambert-ga/protein-purification/">See Wetlab: Protein Purification</a>.</p>
+<h2 id="estrogen">Estrogen</h2>
+<p>While researching CAD risk factors and statistics, we came across literature that informed us about disparities among the sexes concerning early CAD diagnosis. We first conferred with Dr. Mindy Gentry, a women’s cardiologist, to get her opinion on the sex differences in cardiovascular health. She confirmed our insights about estrogen being a factor in increased susceptibility to CAD in women. To further our scientific research, we spoke to Dr. DeLisa Fairweather, a director of translational research in the Department of Cardiovascular Medicine at Mayo Clinic’s Florida campus. She educated us on estrogen and its role as a CAD suppressor, and explained how women after menopause are more likely to develop heart disease because estrogen levels drastically decrease after menopause, causing women to be more likely to develop inflammation. Dr. Fairweather detailed that women who have inflammation in the heart are more likely to develop autoimmune disease and CAD. Additionally, she also guided us in the further development of our inclusivity by discussing how the hormone affects men as well. She told us that since men have a low estrogen level that also remains stable, they would be an ideal demographic to test for consistency in results, unlike women who have fluctuating levels of the hormone. Under the guidance of Dr. Fairweather, we tested miRNA-20b using Rolling Circle Amplification. In the presence of estrogen, CAD suppressors are activated and miRNA-20b is produced. miR-20b causes a negative feedback loop and suppresses estrogen, which will in turn inhibit the CAD suppressors. Dr. Fairweather explained how in men, a high level of miR-20b means that they are likely to develop CAD, however, in premenopausal women with inflammation symptoms, miR-20b protects them from developing CAD. She explained how if there is a high level of 20b, it will inhibit the estrogen from causing a catalyst between the developing autoimmune disease and CAD. We tested miR-20b with the RCA mechanism and ran a 1% gel to validate the presence of our product (see Fig. 5). Ultimately, increasing the factors that can be tested to signify CAD correlation encompasses a wider yet specified group, allowing for quality proactive care to be implemented. <a href="https://2023.igem.wiki/lambert-ga/rca/">See Wetlab: Inclusivity</a>.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/inclusivity/gel1.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 5. The working gel, two wells on the right of the ladder contains the estrogen RCP.</div></div>
+<h1 id="software">Software</h1>
+<h2 id="cadmir">CADmir</h2>
+<p>Lambert iGEM developed CADmir as a centralized microRNA (miRNA) database to enhance researchers’ access to functional information about miRNAs more efficiently. (see Fig. 6) Currently, valuable insights on miRNAs are dispersed throughout academic literature, posing a challenge for researchers seeking detailed information about specific miRNAs. CADmir aims to address these issues by offering a cohesive and easily accessible platform that organizes all of the current scientific literature about miRNAs, facilitating quick and efficient searches. Lambert iGEM hopes that with CADmir, more researchers will explore miRNA research and incorporate miRNAs into more projects without requiring substantial amounts of time dedicated to reading through papers. CADmir aims to advance the field of miRNA research by improving information accessibility and reducing the time barrier associated with obtaining high-quality scientific information on these topics. <a href="https://2023.igem.wiki/lambert-ga/software/">See Drylab: Software</a>.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/inclusivity/cadmir-time.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 6. Timed research trial for the research through CADmir, approximately four minutes to find the estrogen miRNA and resources.</div></div>
+<h2 id="labpilot">LabPilot</h2>
+<p>LabPilot was targeted towards underfunded laboratories and independent researchers to increase accessibility to this crucial equipment. The leading retailing liquid handler costs around $10,000, prohibiting less-privileged laboratories from purchasing liquid handlers and instead manually pipetting (OT-2 Robot, 2023). LabPilot is primarily made with 3D printed parts, making it affordable and easily replicable by any community with a 3D printer at a build cost of $250 and a pipette cost of $409 (Eppendorf Research Plus Adjustable Volume, Single Channel Pipette, 2 - 20 uL, Yellow)(See Parts List), and about 6.5% the cost of a commercial device (OT-2 Robot). LabPilot upholds the standard of accuracy found in other liquid handlers by having a significant difference when comparing the volume of liquid pipetted by LabPilot and a human (See Proof of Concept). LabPilot’s intuitive software utilizes critical user experience (UX) principles: a user can quickly transfer liquid from one location to another with just three taps. Additionally, LabPilot’s software conforms to the Web Content Accessibility Guidelines 2.1 standards, ensuring a quality user experience for people with disabilities. (See Hardware: LabPilot) <a href="https://2023.igem.wiki/lambert-ga/hardware/">See Drylab: Hardware</a>.</p>
+<h2 id="micro-q">Micro-Q</h2>
+<p>Many medical diagnostics utilize fluorescence reporter mechanisms with mathematical models to quantify substances. However, commercial fluorometers are often bulky and cost over $10,000, making these diagnostics inaccessible. Last year, Micro-Q exhibited single-tube green fluorescence quantification for $16 but was limited to one sample. This year, Micro-Q Plus allows multi-sample, multi-spectral quantification with camera based fluorescence detection enabling automatic generation of fluorescence/protein expression curves. All these capabilities fit in a pocket-sized portable box, increasing accessibility to underfunded labs and the public. (See Measurement: MicroQ)<a href="https://2023.igem.wiki/lambert-ga/measurement/">See Drylab: Measurement</a>.</p>
+<h1 id="education">Education</h1>
+<h2 id="summer-camps">Summer camps</h2>
+<p>Lambert iGEM held a summer Biotechnology Bootcamp for students between the ages of 12-14 where students participated in hands-on experiments while learning fundamental scientific concepts and skills needed in preparation for high school. The students learned about DNA structure and functions, acids and bases, proteins and enzymes, and more through a biotechnology application lens. The students were given pre and post surveys every day to gauge their learning progress. For example, we presented statements like “I can describe the function and purpose of a gel electrophoresis.†and the campers would respond with how confident they were. After speaking with our advisor, Dr. Brittney Cantrell, she provided us with a standards alignment chart that is used by the Forsyth County Board of Education. We reorganized the chart to include a link to all presentations, activities, and pre-planned labs that Lambert iGEM has made so that teachers are able to follow the new biotechnology curriculum and find easily accessible materials. Alongside the teaching materials, we linked competitions and events that younger students who are passionate about science can participate in.</p>
+<iframe src="https://static.igem.wiki/teams/4683/wiki/inclusivity/new-standards-alignment-1.pdf" height="1000px" width="120%"></iframe>
+<h2 id="teacher-training">Teacher training</h2>
+<p>To supplement the Standards Alignment teacher resources, Lambert iGEM attended the Georgia Teacher Training Conference at Athens Technical College. Teachers from around the state participated in learning how the new introductory synthetic biology curriculum ( which was created by following the Science Georgia Standards of Excellence) will embed itself within the current science curriculum, and specifically how teachers can make these new concepts and applications of science in the real-world engaging and understandable to their students. We led and assisted teachers through a lab that they could implement in their classrooms. These lessons will particularly be implemented at the middle school level, where students have enough background knowledge of cellular functions and will be able to understand synthetic biology. Additionally, we were able to conduct pre and post-surveys at the teacher training, which helped us evaluate the level of impact that students and teachers will have upon implementation of the synthetic biology curriculum using the Standards Alignment resources (see Fig. 7)</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/inclusivity/teacher-training.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 7. Images from the data collected from surveys at the teacher training workshop. </div></div>
+<h2 id="podcast">Podcast</h2>
+<p>Human Practices involves speaking with various professionals, doctors, and researchers to gain insight into the direction and impact of our project. This year, Lambert iGEM made the knowledge available to anyone interested by creating a podcast on Spotify called SynbioOutLoud (see Fig. 8). In this podcast, we invited a diverse set of several guests, including cardiologists and nutritionists, to discuss barriers they have experienced in the STEM field and how they handled prospective prejudice. We took their primary accounts and feedback to inspire our project direction for this year (see Fig. 9). Dr. Mindy Gentry, a women’s cardiac care specialist, expressed the lack of heart health awareness for women, enabling us to expand sex inclusivity to promote our ongoing research on estrogen’s correlation to CAD (see Estrogen, Inclusivity). Mrs. Saru Bhardwaj, Lambert High School’s AP chemistry teacher, expressed the lack of education regarding gender disparities, and the changes she encountered when she was a student. In addressing this, we conducted a meta-analysis on gender distributions spanning six years of iGEM teams and compiled the findings into a comprehensive spreadsheet. In the study, we found that although the demographic distributions on teams are equal, the specific occupations on the team are very segregated. For example, the females on the teams are primarily centered around human practices, while the majority of males work with hardware. We noticed this trend in our teams too, and so combatting this trend was an integral inspiration for our inclusivity this season. Dr. Cantrell, a member of the Forsyth County Schools Secondary Science Specialist, iGEM advisor, and guest on our podcast informed us about a lack of community involvement she has observed while trying to promote inclusivity in science education. She expressed the need for female role models in the current society. In order to synthesize and express what Lambert iGEM has learned from these conversations, we developed a final episode of our podcast where we talk about our personal account of being students and women, and what we have noted from this experience. We discussed the “why†of our initiatives this year and the ideas behind their implementations. Overall, the SynbioOutLoud podcast enabled Lambert iGEM to create products catering to the community’s education and awareness (see Fig. 10).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/inclusivity/podcast.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 8. SynbioOutLoud podcast cover image.</div></div>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/inclusivity/podcast-graph.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 9. The number of plays and published episodes of our podcast. </div></div>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/inclusivity/podcast-listens.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 10. The data per person who spoke on our podcast per listener interest. </div></div>
+<iframe src="https://static.igem.wiki/teams/4683/wiki/inclusivity/real-gender-breakdown-2013.pdf" height="1000px" width="120%"></iframe>
+<h2 id="heart-health-awareness-month">Heart Health Awareness month</h2>
+<p>Lambert iGEM planned to expand upon the SynbioOutLoud podcast by hosting an audience consisting of Lambert High School’s HOSA chapter, Science Olympiad, and Women in STEM club members to listen to highlighted guest speakers and professionals in the field. However, we wanted to expand the outreach by increasing the number of people who had access to this opportunity, so we converted the event to virtual webinars that focus on all aspects of heart health, both proactive and reactive, for the entire month of September. As the name suggests, the “Science for All†purpose was to access a wider audience, thus further creating an inclusive approach to education. Lambert iGEM hosted webinars discussing the importance of nutrition and exercise to prevent heart disease, and the process of First Aid/CPR/AED as a response system for when heart emergencies occur. Lambert iGEM was also accompanied by the American Heart Association (AHA), who co-hosted the webinar, teaching the audience about emergency response and CPR. While the members were not certified due to safety concerns, they were equipped with the proper knowledge to be an upstander rather than a bystander in the event of a cardiac emergency (see Fig. 11). To conclude the event, we specifically presented our gender research of all iGEM teams, along with the analysis of the impact created and the perceived trend for future demographic distribution in science roles. By advertising the “Science for All†webinars through the county for greater demographic participation, Lambert iGEM was able to address a larger number of people through social media connections, the involvement of the AHA in promoting the webinar, and outreach towards other STEM clubs such as Women in STEM, Science Olympiad, and HOSA. This event attempts to spread the knowledge of synthetic biology and STEM. We are also currently in the process of updating a formal inclusive Cardiac Emergency Response Plan to be implemented and piloted at Lambert High School.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/inclusivity/webinar-listenres.png" style="max-width:600" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 11. Webinar participants at our school receive prizes for answering questions correctly. </div></div>
+<h2 id="flowers-for-the-future">Flowers for the Future</h2>
+<p>After a discussion with some of our members who are part of the Lambert Women in STEM club, we decided to contact an organization called Flowers for the Future. They are a student-led organization that helps girls in Afghanistan by providing resources for STEM education for girls. Some of the topics covered include chemistry, biology, electricity, cellular pathways, computer science, coding, and mathematics. We noticed that their curriculum does not include resources for biotechnology concepts such as electrochemistry, application, lab equipment, basics of central dogma, DNA extraction, the purpose of pipetting, PCR, and Gel electrophoresis. After a discussion with the Flowers for the Future members, we agreed to send them multiple videos and presentations of these concepts with additional labs that they could do with household items, as they were not allowed access to many items due to Taliban restrictions.
+In order to maintain safety and confidentiality for our students and partners, Flowers For the Future makes no mention of any names, locations, and/or specificities anywhere on websites, social media, or the internet (e.g. “learning academy,†not “[name] schoolâ€) to avoid the potentially dangerous path to the academy. The organization also fundraises to provide the girls with laptops and wifi so they can learn from home and not be seen or have their safety undermined. They also do not publicize the organizations they work with, and make sure that any and all released video lessons are put on the Flowers for the Future YouTube channel, with no other information given than the instructional topics being presented.</p>
+<h1 id="references">References</h1>
+<div class="pl-6 -indent-6"><p>Callen, J., Georgiou, A., Li, J., & Westbrook, J. I. (2011). The safety implications of missed test results for hospitalized patients: a systematic review. BMJ quality & safety, 20(2), 194–199. <a href="https://doi.org/10.1136/bmjqs.2010.044339">https://doi.org/10.1136/bmjqs.2010.044339</a></p></div>
+<div class="pl-6 -indent-6"><p>Centers for Disease Control and Prevention. (2023, May 15). Women and heart disease. Centers for Disease Control and Prevention. <a href="https://www.cdc.gov/heartdisease/women.htm">https://www.cdc.gov/heartdisease/women.htm</a></p></div>
+<div class="pl-6 -indent-6"><p>Dluzen, D. F., Noren Hooten, N., Zhang, Y., Kim, Y., Glover, F. E., Tajuddin, S. M., Jacob, K. D., Zonderman, A. B., & Evans, M. K. (2016). Racial differences in microRNA and gene expression in hypertensive women. Scientific reports, 6, 35815. <a href="https://doi.org/10.1038/srep35815">https://doi.org/10.1038/srep35815</a></p></div>
+<div class="pl-6 -indent-6"><p>Introduction - noninvasive testing for coronary artery disease - NCBI … (n.d.-a). <a href="https://www.ncbi.nlm.nih.gov/books/NBK361133/">https://www.ncbi.nlm.nih.gov/books/NBK361133/</a></p></div>
+<div class="pl-6 -indent-6"><p>Moving away from Reactive Care Models | Mercury Healthcare. (n.d.). <a href="https://www.mercuryhealthcare.com/blog/from-reactive-to-proactive-health">https://www.mercuryhealthcare.com/blog/from-reactive-to-proactive-health</a></p></div>
+<div class="pl-6 -indent-6"><p>Waldman, S. A., & Terzic, A. (2019). Health Care Evolves From Reactive to Proactive. Clinical pharmacology and therapeutics, 105(1), 10–13. <a href="https://doi.org/10.1002/cpt.1295">https://doi.org/10.1002/cpt.1295</a></p></div>
+ </astro-slot></div></div></div></body></astro-island>
+ <footer class="relative bg-[#292524] pt-8 pb-6 w-screen"><div class="w-full max-w-5xl mx-16 mt-8 container mx-auto px-4 py-10"><div class="flex flex-wrap text-left lg:text-left"><div class="w-full lg:w-6/12 px-4"><h1 class="text-4xl text-white font-serif font-light">Lambert-GA</h1><br/><h3 class="text-xs w-3/4 mt-0 mb-2 font-sans text-white">© <span id="get-current-year">2023</span> - Content on this site is licensed under a<a href="https://creativecommons.org/licenses/by/4.0/" class="underline hover:text-gray-300" target="_blank">Creative Commons Attribution 4.0 International license.</a><br/><br/>The repository used to create this website is available at<a href="https://gitlab.igem.org/2023/lambert-ga" class="underline hover:text-gray-300" target="_blank">https://gitlab.igem.org/2023/lambert-ga</a></h3></div><div class="w-full lg:w-5/12 px-4"><div class="flex flex-wrap items-top mb-6"><div class="w-full lg:w-4/12 px-4 ml-auto"><ul class="w-full list-unstyled"><li class="flex"><svg width="25" height="25" viewBox="0 0 25 25" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M18.7498 2.0835H15.6248C14.2435 2.0835 12.9187 2.63223 11.942 3.60898C10.9652 4.58573 10.4165 5.91049 10.4165 7.29183V10.4168H7.2915V14.5835H10.4165V22.9168H14.5832V14.5835H17.7082L18.7498 10.4168H14.5832V7.29183C14.5832 7.01556 14.6929 6.75061 14.8883 6.55526C15.0836 6.35991 15.3486 6.25016 15.6248 6.25016H18.7498V2.0835Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">Lambert iGEM</a></li><li class="flex"><svg width="25" height="28" viewBox="0 0 25 28" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M22.9168 4.52889C22.9168 4.52889 22.1877 6.90661 20.8335 8.37853C22.5002 19.701 11.0418 27.9664 2.0835 21.5126C4.37516 21.6258 6.66683 20.8332 8.3335 19.2481C3.12516 17.5497 0.520996 10.8695 3.12516 5.66114C5.41683 8.60498 8.9585 10.3033 12.5002 10.1901C11.5627 5.43469 16.6668 2.7173 19.7918 5.88759C20.9377 5.88759 22.9168 4.52889 22.9168 4.52889Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">@lamberths_iGEM</a></li><li class="flex "><svg width="25" height="25" viewBox="0 0 25 25" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M17.7085 2.21484H7.29183C4.41535 2.21484 2.0835 4.49911 2.0835 7.31688V17.521C2.0835 20.3387 4.41535 22.623 7.29183 22.623H17.7085C20.585 22.623 22.9168 20.3387 22.9168 17.521V7.31688C22.9168 4.49911 20.585 2.21484 17.7085 2.21484Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path><path d="M16.6667 11.7759C16.7952 12.6251 16.6472 13.4924 16.2435 14.2545C15.8399 15.0165 15.2012 15.6345 14.4184 16.0205C13.6355 16.4064 12.7484 16.5408 11.8831 16.4044C11.0179 16.268 10.2185 15.8678 9.59881 15.2608C8.9791 14.6537 8.57057 13.8707 8.43134 13.0231C8.29211 12.1754 8.42926 11.3064 8.82328 10.5395C9.2173 9.77269 9.84813 9.14706 10.626 8.75165C11.404 8.35625 12.2893 8.21119 13.1563 8.33712C14.0406 8.46558 14.8592 8.86923 15.4914 9.48846C16.1235 10.1077 16.5356 10.9096 16.6667 11.7759Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">@lambertigem</a></li><li class="flex"><svg width="25" height="24" viewBox="0 0 25 24" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M12.5002 18.2922C10.1043 18.2922 5.8335 18.1028 4.06266 17.7241C3.3335 17.5347 2.81266 17.0612 2.60433 16.3983C2.29183 15.3566 2.0835 13.1786 2.0835 11.6635C2.0835 10.1483 2.29183 7.97027 2.60433 6.92861C2.81266 6.26573 3.3335 5.79224 4.06266 5.60285C5.8335 5.22406 10.1043 5.03467 12.5002 5.03467C14.896 5.03467 19.1668 5.22406 20.9377 5.60285C21.6668 5.79224 22.1877 6.26573 22.396 6.92861C22.7085 7.97027 22.9168 10.1483 22.9168 11.6635C22.9168 13.1786 22.7085 15.3566 22.396 16.3983C22.1877 17.0612 21.6668 17.5347 20.9377 17.7241C19.1668 18.1028 14.896 18.2922 12.5002 18.2922Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path><path d="M10.4165 14.5046L15.6248 11.6637L10.4165 8.82275V14.5046Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">Lambert iGEM</a></li></ul></div></div></div></div></div></footer>
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diff --git a/public/index.html b/public/index.html
new file mode 100644
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+++ b/public/index.html
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+<!DOCTYPE html>
+<div>
+<html lang="en">
+ <head>
+ <meta charset="UTF-8">
+ <meta name="viewport" content="width=device-width">
+ <link rel="icon" type="image/svg+xml" href="/favicon.svg">
+ <meta name="generator" content="Astro v2.10.15">
+ <!-- <link rel="preconnect" href="https://fonts.googleapis.com" />
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+ <link href="../components/fonts.css" rel="stylesheet">
+ <title>Lambert-GA</title>
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+ <div class="w-full flex justify-center">
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+ <div class="flex justify-between items-center">
+ <a href="https://2023.igem.wiki/lambert-ga/" class="font-serif z-30 ">
+ Lambert-GA
+ </a>
+ <style>astro-island,astro-slot,astro-static-slot{display:contents}</style><script>(()=>{var e=async t=>{await(await t())()};(self.Astro||(self.Astro={})).load=e;window.dispatchEvent(new Event("astro:load"));})();;(()=>{var d;{let p={0:t=>u(t),1:t=>l(t),2:t=>new RegExp(t),3:t=>new Date(t),4:t=>new Map(l(t)),5:t=>new Set(l(t)),6:t=>BigInt(t),7:t=>new URL(t),8:t=>new Uint8Array(t),9:t=>new Uint16Array(t),10:t=>new Uint32Array(t)},h=t=>{let[e,n]=t;return e in p?p[e](n):void 0},l=t=>t.map(h),u=t=>typeof t!="object"||t===null?t:Object.fromEntries(Object.entries(t).map(([e,n])=>[e,h(n)]));customElements.get("astro-island")||customElements.define("astro-island",(d=class extends HTMLElement{constructor(){super(...arguments);this.hydrate=async()=>{var i;if(!this.hydrator||!this.isConnected)return;let e=(i=this.parentElement)==null?void 0:i.closest("astro-island[ssr]");if(e){e.addEventListener("astro:hydrate",this.hydrate,{once:!0});return}let 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+
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+ </div>
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+ </div>
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.012);--crimsonA2: hsla(330, 100%, 51%, .032);--crimsonA3: hsla(332, 99.1%, 47.1%, .063);--crimsonA4: hsla(331, 99.9%, 44.3%, .102);--crimsonA5: hsla(333, 99.9%, 42.3%, .153);--crimsonA6: hsla(333, 99.5%, 40.5%, .224);--crimsonA7: hsla(335, 99.7%, 39.1%, .322);--crimsonA8: hsla(336, 99.5%, 38.5%, .44);--crimsonA9: hsla(336, 99.9%, 44.3%, .761);--crimsonA10: hsla(336, 100%, 42.5%, .808);--crimsonA11: hsla(336, 99.8%, 40.3%, .883);--crimsonA12: hsla(340, 99%, 10%, .95);--cyan1: 185deg 60% 98.7%;--cyan2: 185deg 73.3% 97.1%;--cyan3: 186deg 70.2% 94.4%;--cyan4: 186deg 63.8% 90.6%;--cyan5: 187deg 58.3% 85.4%;--cyan6: 188deg 54.6% 78.4%;--cyan7: 189deg 53.7% 68.7%;--cyan8: 189deg 60.3% 52.5%;--cyan9: 190deg 95% 39%;--cyan10: 191deg 91.2% 36.8%;--cyan11: 192deg 85% 31%;--cyan12: 192deg 88% 12.5%;--cyanA1: hsla(195, 95.2%, 41.2%, .02);--cyanA2: hsla(185, 99.9%, 42.3%, .051);--cyanA3: hsla(186, 97.8%, 42.2%, .095);--cyanA4: hsla(186, 99.9%, 38.5%, .153);--cyanA5: hsla(187, 99.3%, 36.6%, .232);--cyanA6: hsla(188, 99.4%, 35.4%, .334);--cyanA7: hsla(189, 99.6%, 35%, .483);--cyanA8: hsla(189, 99.9%, 37.6%, .761);--cyanA9: hsla(190, 100%, 37.8%, .98);--cyanA10: hsla(191, 99.9%, 34.6%, .969);--cyanA11: hsla(192, 100%, 27.6%, .953);--cyanA12: hsla(192, 100%, 11%, .98);--gold1: 50deg 20% 99.1%;--gold2: 47deg 52.9% 96.7%;--gold3: 46deg 38.2% 93.7%;--gold4: 44deg 32.7% 90.1%;--gold5: 43deg 29.9% 85.7%;--gold6: 41deg 28.3% 79.8%;--gold7: 39deg 27.6% 71.9%;--gold8: 36deg 27.2% 61.8%;--gold9: 36deg 20% 49.5%;--gold10: 36deg 19.8% 45.7%;--gold11: 36deg 20% 39%;--gold12: 36deg 16% 20%;--goldA1: hsla(60, 89.3%, 18.3%, .012);--goldA2: hsla(47, 99.9%, 34.6%, .051);--goldA3: hsla(45, 97%, 27.9%, .087);--goldA4: hsla(46, 98%, 25.4%, .134);--goldA5: hsla(43, 98.4%, 22.6%, .185);--goldA6: hsla(41, 99.7%, 22%, .259);--goldA7: hsla(38, 99.8%, 21.5%, .357);--goldA8: hsla(36, 99.3%, 21.5%, .487);--goldA9: hsla(36, 99.9%, 16.2%, .604);--goldA10: hsla(36, 99.2%, 14.6%, .636);--goldA11: hsla(35, 99.1%, 11.2%, .687);--goldA12: hsla(38, 98%, 3.8%, .832);--grass1: 116deg 50% 98.9%;--grass2: 120deg 60% 97.1%;--grass3: 120deg 53.6% 94.8%;--grass4: 121deg 47.5% 91.4%;--grass5: 122deg 42.6% 86.5%;--grass6: 124deg 39% 79.7%;--grass7: 126deg 37.1% 70.2%;--grass8: 131deg 38.1% 56.3%;--grass9: 131deg 41% 46.5%;--grass10: 132deg 43.1% 42.2%;--grass11: 133deg 50% 32.5%;--grass12: 130deg 30% 14.9%;--grassA1: hsla(120, 94.9%, 38.7%, .016);--grassA2: hsla(120, 94.9%, 38.7%, .048);--grassA3: hsla(120, 98%, 35.5%, .079);--grassA4: hsla(120, 98.7%, 31.5%, .126);--grassA5: hsla(122, 98.5%, 29.9%, .193);--grassA6: hsla(125, 99.2%, 27.9%, .283);--grassA7: hsla(125, 99.9%, 27%, .408);--grassA8: hsla(131, 100%, 27.6%, .604);--grassA9: hsla(131, 99.7%, 26.3%, .726);--grassA10: hsla(132, 99.9%, 24%, .761);--grassA11: hsla(133, 99.5%, 19.5%, .84);--grassA12: hsla(128, 98%, 4.9%, .895);--gray1: 0deg 0% 99%;--gray2: 0deg 0% 97.3%;--gray3: 0deg 0% 95.1%;--gray4: 0deg 0% 93%;--gray5: 0deg 0% 90.9%;--gray6: 0deg 0% 88.7%;--gray7: 0deg 0% 85.8%;--gray8: 0deg 0% 78%;--gray9: 0deg 0% 56.1%;--gray10: 0deg 0% 52.3%;--gray11: 0deg 0% 43.5%;--gray12: 0deg 0% 9%;--grayA1: hsla(0, 0%, 0%, .012);--grayA2: hsla(0, 0%, 0%, .027);--grayA3: hsla(0, 0%, 0%, .047);--grayA4: hsla(0, 0%, 0%, .071);--grayA5: hsla(0, 0%, 0%, .09);--grayA6: hsla(0, 0%, 0%, .114);--grayA7: hsla(0, 0%, 0%, .141);--grayA8: hsla(0, 0%, 0%, .22);--grayA9: hsla(0, 0%, 0%, .439);--grayA10: hsla(0, 0%, 0%, .478);--grayA11: hsla(0, 0%, 0%, .565);--grayA12: hsla(0, 0%, 0%, .91);--green1: 136deg 50% 98.9%;--green2: 138deg 62.5% 96.9%;--green3: 139deg 55.2% 94.5%;--green4: 140deg 48.7% 91%;--green5: 141deg 43.7% 86%;--green6: 143deg 40.3% 79%;--green7: 146deg 38.5% 69%;--green8: 151deg 40.2% 54.1%;--green9: 151deg 55% 41.5%;--green10: 152deg 57.5% 37.6%;--green11: 153deg 67% 28.5%;--green12: 155deg 40% 14%;--greenA1: hsla(140, 94.9%, 38.7%, .016);--greenA2: hsla(138, 99.9%, 38.5%, .051);--greenA3: hsla(139, 97.7%, 36.9%, .087);--greenA4: hsla(139, 98.5%, 32.7%, .134);--greenA5: hsla(141, 100%, 30.4%, .2);--greenA6: hsla(142, 99%, 28.9%, .295);--greenA7: hsla(146, 99.5%, 27.6%, .428);--greenA8: hsla(151, 99.5%, 28.8%, .644);--greenA9: hsla(151, 99.9%, 28%, .812);--greenA10: hsla(152, 99.6%, 25.8%, .84);--greenA11: hsla(153, 99.9%, 21%, .906);--greenA12: hsla(155, 99.4%, 6.2%, .918);--indigo1: 225deg 60% 99.4%;--indigo2: 223deg 100% 98.6%;--indigo3: 223deg 98.4% 97.1%;--indigo4: 223deg 92.9% 95%;--indigo5: 224deg 87.1% 92%;--indigo6: 224deg 81.9% 87.8%;--indigo7: 225deg 77.4% 82.1%;--indigo8: 226deg 75.4% 74.5%;--indigo9: 226deg 70% 55.5%;--indigo10: 226deg 58.6% 51.3%;--indigo11: 226deg 55% 45%;--indigo12: 226deg 62% 17%;--indigoA1: hsla(240, 92.6%, 26.5%, .008);--indigoA2: hsla(223, 100%, 51%, .028);--indigoA3: hsla(224, 100%, 50.1%, .059);--indigoA4: hsla(223, 98%, 48.5%, .099);--indigoA5: hsla(225, 98.6%, 46.4%, .15);--indigoA6: hsla(224, 99.5%, 44.9%, .224);--indigoA7: hsla(225, 99.7%, 43.9%, .318);--indigoA8: hsla(226, 99.5%, 43.1%, .448);--indigoA9: hsla(226, 100%, 41.2%, .757);--indigoA10: hsla(226, 99.8%, 37.1%, .773);--indigoA11: hsla(226, 99.6%, 31.1%, .797);--indigoA12: hsla(226, 99.3%, 11.4%, .938);--lime1: 85deg 50% 98.7%;--lime2: 85deg 66.7% 96.5%;--lime3: 85deg 76% 92.3%;--lime4: 84deg 75.3% 87.5%;--lime5: 84deg 71.5% 81.9%;--lime6: 82deg 65% 74.6%;--lime7: 79deg 53.2% 61.8%;--lime8: 76deg 61.7% 45.1%;--lime9: 81deg 67% 50%;--lime10: 80deg 68.3% 46.9%;--lime11: 75deg 80% 26%;--lime12: 78deg 70% 11.5%;--limeA1: hsla(80, 93.8%, 31.4%, .02);--limeA2: hsla(85, 99.3%, 40.2%, .059);--limeA3: hsla(84, 98.7%, 43.2%, .138);--limeA4: hsla(84, 99.6%, 43%, .22);--limeA5: hsla(85, 99.8%, 41.8%, .31);--limeA6: hsla(82, 99.8%, 39.3%, .42);--limeA7: hsla(79, 99.7%, 34.6%, .585);--limeA8: hsla(76, 99.8%, 33.7%, .828);--limeA9: hsla(81, 99.8%, 40.2%, .836);--limeA10: hsla(80, 100%, 37.6%, .851);--limeA11: hsla(75, 99.5%, 22%, .95);--limeA12: hsla(78, 99.6%, 8.4%, .965);--mauve1: 300deg 20% 99%;--mauve2: 300deg 7.7% 97.5%;--mauve3: 294deg 5.5% 95.3%;--mauve4: 289deg 4.7% 93.3%;--mauve5: 283deg 4.4% 91.3%;--mauve6: 278deg 4.1% 89.1%;--mauve7: 271deg 3.9% 86.3%;--mauve8: 255deg 3.7% 78.8%;--mauve9: 252deg 4% 57.3%;--mauve10: 253deg 3.5% 53.5%;--mauve11: 252deg 4% 44.8%;--mauve12: 260deg 25% 11%;--mauveA1: hsla(300, 89.3%, 18.3%, .012);--mauveA2: hsla(300, 78.1%, 9%, .028);--mauveA3: hsla(300, 99.5%, 7.7%, .051);--mauveA4: hsla(270, 90.5%, 6.1%, .071);--mauveA5: hsla(270, 83%, 5.2%, .091);--mauveA6: hsla(300, 93.5%, 3.7%, .114);--mauveA7: hsla(270, 82.6%, 3.3%, .142);--mauveA8: hsla(255, 95.2%, 3.7%, .22);--mauveA9: hsla(255, 94.8%, 3.7%, .444);--mauveA10: hsla(253, 96.5%, 3.8%, .483);--mauveA11: hsla(247, 97.9%, 3.2%, .569);--mauveA12: hsla(261, 98.7%, 3%, .918);--mint1: 165deg 80% 98.8%;--mint2: 164deg 88.2% 96.7%;--mint3: 164deg 76.6% 93.3%;--mint4: 165deg 68.8% 89.5%;--mint5: 165deg 60.6% 84.5%;--mint6: 165deg 53.5% 76.9%;--mint7: 166deg 50.7% 66.1%;--mint8: 168deg 52.8% 51%;--mint9: 167deg 65% 66%;--mint10: 167deg 59.3% 63.1%;--mint11: 172deg 72% 28.5%;--mint12: 172deg 70% 12%;--mintA1: hsla(168, 95.4%, 42.8%, .024);--mintA2: hsla(164, 99.1%, 47.1%, .063);--mintA3: hsla(164, 99.3%, 43.5%, .118);--mintA4: hsla(164, 99.3%, 41.3%, .177);--mintA5: hsla(165, 99%, 37.5%, .248);--mintA6: hsla(165, 100%, 35%, .353);--mintA7: hsla(166, 99.9%, 33.5%, .51);--mintA8: hsla(168, 99.6%, 34.6%, .75);--mintA9: hsla(167, 99.9%, 39.5%, .561);--mintA10: hsla(167, 99.7%, 37.4%, .589);--mintA11: hsla(172, 99.8%, 22.4%, .922);--mintA12: hsla(172, 99.7%, 8.8%, .965);--olive1: 110deg 20% 99%;--olive2: 120deg 16.7% 97.6%;--olive3: 119deg 10.1% 95.2%;--olive4: 118deg 8.1% 93%;--olive5: 117deg 7.1% 90.8%;--olive6: 115deg 6.4% 88.5%;--olive7: 114deg 5.9% 85.4%;--olive8: 110deg 5.2% 77.3%;--olive9: 110deg 3.5% 55.5%;--olive10: 111deg 2.8% 51.7%;--olive11: 110deg 3% 43%;--olive12: 110deg 25% 9.5%;--oliveA1: hsla(120, 89.3%, 18.3%, .012);--oliveA2: hsla(120, 87.7%, 16%, .028);--oliveA3: hsla(120, 99.5%, 7.7%, .051);--oliveA4: hsla(120, 92.3%, 8.5%, .075);--oliveA5: hsla(120, 86%, 6.9%, .099);--oliveA6: hsla(120, 94.8%, 6.8%, .122);--oliveA7: hsla(120, 99.3%, 5.2%, .153);--oliveA8: hsla(110, 93.8%, 5.2%, .24);--oliveA9: hsla(111, 98.7%, 3%, .459);--oliveA10: hsla(111, 93.5%, 2.9%, .499);--oliveA11: hsla(111, 95.2%, 2.5%, .585);--oliveA12: hsla(110, 97.6%, 2.6%, .93);--orange1: 24deg 70% 99%;--orange2: 24deg 83.3% 97.6%;--orange3: 24deg 100% 95.3%;--orange4: 25deg 100% 92.2%;--orange5: 25deg 100% 88.2%;--orange6: 25deg 100% 82.8%;--orange7: 24deg 100% 75.3%;--orange8: 24deg 94.5% 64.3%;--orange9: 24deg 94% 50%;--orange10: 24deg 100% 46.5%;--orange11: 24deg 100% 37%;--orange12: 15deg 60% 17%;--orangeA1: hsla(20, 94.9%, 38.7%, .016);--orangeA2: hsla(24, 95.8%, 46.5%, .044);--orangeA3: hsla(25, 100%, 50.5%, .095);--orangeA4: hsla(26, 100%, 50%, .157);--orangeA5: hsla(25, 100%, 50.1%, .236);--orangeA6: hsla(25, 100%, 50.1%, .346);--orangeA7: hsla(24, 100%, 50.1%, .495);--orangeA8: hsla(24, 99.7%, 48.7%, .695);--orangeA9: hsla(24, 99.9%, 48.4%, .969);--orangeA10: hsla(23, 100%, 46.4%, .98);--orangeA11: hsla(23, 100%, 36.8%, .98);--orangeA12: hsla(15, 99.4%, 11%, .934);--pink1: 322deg 100% 99.4%;--pink2: 323deg 100% 98.4%;--pink3: 323deg 86.3% 96.5%;--pink4: 323deg 78.7% 94.2%;--pink5: 323deg 72.2% 91.1%;--pink6: 323deg 66.3% 86.6%;--pink7: 323deg 62% 80.1%;--pink8: 323deg 60.3% 72.4%;--pink9: 322deg 65% 54.5%;--pink10: 322deg 63.9% 50.7%;--pink11: 322deg 75% 46%;--pink12: 320deg 70% 13.5%;--pinkA1: hsla(320, 100%, 51%, .012);--pinkA2: hsla(323, 100%, 51%, .032);--pinkA3: hsla(323, 98.9%, 47.3%, .067);--pinkA4: hsla(323, 99.9%, 44.3%, .102);--pinkA5: hsla(324, 99.9%, 42.3%, .153);--pinkA6: hsla(323, 99.5%, 39.6%, .224);--pinkA7: hsla(323, 99.7%, 38.5%, .322);--pinkA8: hsla(323, 99.5%, 37.7%, .444);--pinkA9: hsla(322, 99.7%, 39.3%, .75);--pinkA10: hsla(322, 100%, 39.1%, .808);--pinkA11: hsla(322, 99.8%, 39%, .887);--pinkA12: hsla(321, 99.8%, 10%, .961);--plum1: 292deg 90% 99.4%;--plum2: 300deg 100% 98.6%;--plum3: 299deg 71.2% 96.4%;--plum4: 299deg 62% 93.8%;--plum5: 298deg 56.1% 90.5%;--plum6: 296deg 51.3% 85.8%;--plum7: 295deg 48.2% 78.9%;--plum8: 292deg 47.7% 70.8%;--plum9: 292deg 45% 51%;--plum10: 292deg 50.2% 46.9%;--plum11: 292deg 60% 42.5%;--plum12: 291deg 66% 14%;--plumA1: hsla(280, 100%, 51%, .012);--plumA2: hsla(300, 100%, 51%, .028);--plumA3: hsla(300, 99%, 40.9%, .063);--plumA4: hsla(300, 99.9%, 38.5%, .102);--plumA5: hsla(298, 98.2%, 35.9%, .15);--plumA6: hsla(297, 99.6%, 33.7%, .216);--plumA7: hsla(295, 99.7%, 32.6%, .314);--plumA8: hsla(292, 99.6%, 32.4%, .432);--plumA9: hsla(292, 99.9%, 31%, .71);--plumA10: hsla(292, 99.9%, 30.8%, .765);--plumA11: hsla(292, 99.8%, 30.7%, .832);--plumA12: hsla(291, 99.9%, 9.7%, .953);--purple1: 280deg 65% 99.4%;--purple2: 276deg 100% 99%;--purple3: 276deg 83.1% 97%;--purple4: 275deg 76.4% 94.7%;--purple5: 275deg 70.8% 91.8%;--purple6: 274deg 65.4% 87.8%;--purple7: 273deg 61% 81.7%;--purple8: 272deg 60% 73.5%;--purple9: 272deg 51% 54%;--purple10: 272deg 46.8% 50.3%;--purple11: 272deg 50% 45.8%;--purple12: 272deg 66% 16%;--purpleA1: hsla(300, 94.3%, 34.6%, .012);--purpleA2: hsla(276, 100%, 51%, .02);--purpleA3: hsla(277, 99.6%, 46.5%, .055);--purpleA4: hsla(274, 97.9%, 44.3%, .095);--purpleA5: hsla(276, 98.6%, 42%, .142);--purpleA6: hsla(275, 100%, 39.2%, .2);--purpleA7: hsla(273, 99.2%, 38.2%, .295);--purpleA8: hsla(272, 99.7%, 37.6%, .424);--purpleA9: hsla(272, 99.6%, 34%, .695);--purpleA10: hsla(272, 99.7%, 32%, .73);--purpleA11: hsla(272, 99.8%, 29.7%, .773);--purpleA12: hsla(272, 99.2%, 11.3%, .946);--red1: 359deg 100% 99.4%;--red2: 359deg 100% 98.6%;--red3: 360deg 100% 96.8%;--red4: 360deg 97.9% 94.8%;--red5: 360deg 90.2% 91.9%;--red6: 360deg 81.7% 87.8%;--red7: 359deg 74.2% 81.7%;--red8: 359deg 69.5% 74.3%;--red9: 358deg 75% 59%;--red10: 358deg 69.4% 55.2%;--red11: 358deg 65% 48.7%;--red12: 354deg 50% 14.6%;--redA1: hsla(0, 100%, 51%, .012);--redA2: hsla(0, 100%, 51%, .032);--redA3: hsla(0, 100%, 50.2%, .063);--redA4: hsla(0, 100%, 50%, .102);--redA5: hsla(0, 99.9%, 47.5%, .153);--redA6: hsla(0, 99.5%, 44.9%, .224);--redA7: hsla(359, 99.7%, 42.7%, .318);--redA8: hsla(359, 99.6%, 41.1%, .436);--redA9: hsla(358, 99.9%, 42.9%, .718);--redA10: hsla(358, 99.9%, 41%, .761);--redA11: hsla(358, 99.8%, 38.3%, .832);--redA12: hsla(355, 99.3%, 7.9%, .926);--sage1: 155deg 30% 98.8%;--sage2: 150deg 16.7% 97.6%;--sage3: 151deg 10.6% 95.2%;--sage4: 151deg 8.8% 93%;--sage5: 151deg 7.8% 90.8%;--sage6: 152deg 7.2% 88.4%;--sage7: 153deg 6.7% 85.3%;--sage8: 154deg 6.1% 77.5%;--sage9: 155deg 3.5% 55.5%;--sage10: 154deg 2.8% 51.7%;--sage11: 155deg 3% 43%;--sage12: 155deg 24% 9%;--sageA1: hsla(150, 92.6%, 26.5%, .016);--sageA2: hsla(150, 87.7%, 16%, .028);--sageA3: hsla(160, 98.4%, 10.9%, .055);--sageA4: hsla(140, 92.3%, 8.5%, .075);--sageA5: hsla(160, 86%, 6.9%, .099);--sageA6: hsla(156, 95.1%, 8.2%, .126);--sageA7: hsla(156, 98.6%, 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new file mode 100644
index 0000000..5d85380
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diff --git a/public/lambert-ga/_astro/attributions.b36d46b4.css b/public/lambert-ga/_astro/attributions.b36d46b4.css
new file mode 100644
index 0000000..108c9ec
--- /dev/null
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new file mode 100644
index 0000000..9a2abde
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diff --git a/public/lambert-ga/_astro/team.b6b40ce8.css b/public/lambert-ga/_astro/team.b6b40ce8.css
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+.imgStyle:where(.astro-6SQSH2PF){width:1211px;height:576px;-o-object-fit:cover;object-fit:cover;border-radius:48px}.teamImg:where(.astro-6SQSH2PF) img:where(.astro-6SQSH2PF){display:block}.teamImg:where(.astro-6SQSH2PF) .gif:where(.astro-6SQSH2PF){display:none;transition:opacity .3s ease}.teamImg:where(.astro-6SQSH2PF):hover img:where(.astro-6SQSH2PF){display:none}.teamImg:where(.astro-6SQSH2PF):hover .gif:where(.astro-6SQSH2PF){display:block;opacity:1}.meetTheTeam:where(.astro-6SQSH2PF){padding-bottom:2rem}
diff --git a/public/measurement/index.html b/public/measurement/index.html
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+++ b/public/measurement/index.html
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+<!DOCTYPE html>
+<html lang="en">
+ <head>
+ <meta charset="UTF-8">
+ <meta name="viewport" content="width=device-width">
+ <link rel="icon" type="image/svg+xml" href="/favicon.svg">
+ <meta name="generator" content="Astro v2.10.15">
+ <link href="../components/fonts.css" rel="stylesheet">
+ <!-- <link
+ rel="stylesheet"
+ href="https://cdn.jsdelivr.net/npm/katex@0.16.9/dist/katex.min.css"
+ integrity="sha384-n8MVd4RsNIU0tAv4ct0nTaAbDJwPJzDEaqSD1odI+WdtXRGWt2kTvGFasHpSy3SV"
+ crossorigin="anonymous"
+ />
+ <link
+ rel="stylesheet"
+ href="https://cdn.jsdelivr.net/npm/katex@0.16.9/dist/katex.min.css"
+ integrity="sha384-n8MVd4RsNIU0tAv4ct0nTaAbDJwPJzDEaqSD1odI+WdtXRGWt2kTvGFasHpSy3SV"
+ crossorigin="anonymous"
+ /> -->
+ <title>Measurement</title>
+ <link rel="stylesheet" href="/lambert-ga/lambert-ga/_astro/RCAERCA.96298b81.css" />
+<link rel="stylesheet" href="/lambert-ga/lambert-ga/_astro/RCAOverview2022.5440303d.css" />
+<link rel="stylesheet" href="/lambert-ga/lambert-ga/_astro/contribution.8701276b.css" />
+<link rel="stylesheet" href="/lambert-ga/lambert-ga/_astro/contribution.ca893883.css" /></head>
+ <astro-island uid="15kb8U" prefix="r22" component-url="/lambert-ga/_astro/PageLayoutComp.a397ad4e.js" component-export="default" renderer-url="/lambert-ga/_astro/client.4f88357e.js" props="{"title":[0,"Measurement"],"header":[0,"https://static.igem.wiki/teams/4683/wiki/img-1116.jpg"],"file":[0,"/Users/ryandu/Desktop/iGEM/2023/wiki/src/pages/measurement.mdx"],"url":[0,"/lambert-ga/measurement"]}" ssr="" client="load" opts="{"name":"Layout","value":true}" await-children=""><body class="w-screen">https://static.igem.wiki/teams/4683/wiki/img-1116.jpg<div class="bg-cover absolute w-screen left-0 top-0 p-0 m-0 z-10 h-1/2" style="background:linear-gradient(to bottom, rgba(255, 255, 255, 0) 36.46%, #FFFFFF 100%), url(https://static.igem.wiki/teams/4683/wiki/img-1116.jpg);background-size:cover"></div><div class="bg-slate-100 fixed top-0 left-0 right-0 pt-8" style="margin-top:-40px"><div class="bottom-0 left-0 pb-1 mt-2 right-0 bg-yellow-400 origin-left" style="transform:scaleX(0) translateZ(0)"></div></div><div class="w-full flex justify-center items-center"><div class="flex justify-between m-5 z-50 max-w-6xl grow"><a href="https://2023.igem.wiki/lambert-ga/" class="font-serif z-30 text-white "><p class="bg-blackA-11 text-xl rounded-full p-2">Lambert-GA</p></a><div class="flex justify-end z-30"><nav aria-label="Main" data-orientation="horizontal" dir="ltr" class="relative "><div style="position:relative"><ul data-orientation="horizontal" class="flex flex-row rounded-full bg-blackA-11 p-2 space-x-2" dir="ltr"><div><li><button id="radix-:r22Rk:-trigger-radix-:r22Rbk:" data-state="closed" aria-expanded="false" aria-controls="radix-:r22Rk:-content-radix-:r22Rbk:" class="px-4 py-2 rounded-full hover:bg-whiteA-6 font-medium text-gray-3 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Team</button></li></div><div><li><button id="radix-:r22Rk:-trigger-radix-:r22Rjk:" data-state="closed" aria-expanded="false" aria-controls="radix-:r22Rk:-content-radix-:r22Rjk:" class="px-4 py-2 rounded-full hover:bg-whiteA-6 font-medium text-gray-3 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Project</button></li></div><div><li><button id="radix-:r22Rk:-trigger-radix-:r22Rrk:" data-state="closed" aria-expanded="false" aria-controls="radix-:r22Rk:-content-radix-:r22Rrk:" class="px-4 py-2 rounded-full hover:bg-whiteA-6 font-medium text-gray-3 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Wetlab</button></li></div><div><li><button id="radix-:r22Rk:-trigger-radix-:r22R13k:" data-state="closed" aria-expanded="false" aria-controls="radix-:r22Rk:-content-radix-:r22R13k:" class="px-4 py-2 rounded-full hover:bg-whiteA-6 font-medium text-gray-3 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Drylab</button></li></div><div><li><button id="radix-:r22Rk:-trigger-radix-:r22R1bk:" data-state="closed" aria-expanded="false" aria-controls="radix-:r22Rk:-content-radix-:r22R1bk:" class="px-4 py-2 rounded-full hover:bg-whiteA-6 font-medium text-gray-3 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Human Practices</button></li></div></ul></div></nav></div></div></div><div class="flex justify-center w-full items-center"><div class="w-full m-5 max-w-3xl "><p class="font-serif font-bold z-30 absolute top-1/2 -translate-y-1/2 lg:-translate-x-20 text-5xl sm:text-6xl md:text-7xl">Measurement</p><div class="absolute top-1/2 translate-y-20 h-32 lg:-translate-x-20 lg:border-l-2 border-gray-900 z-50"></div><div class="
+prose max-w-3xl
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+
+mt-[50vh] break-words grow sm:mt-[44vh] md:mt-[40vh]"><astro-slot>
+ <h1 id="micro-q-pro">Micro-Q Pro</h1>
+<h2 id="overviewbackground">Overview/Background</h2>
+<p>Traditional methods of diagnosing coronary artery disease (CAD) such as angiograms and echocardiograms are tedious and expensive, with costs ranging from $500 to $20,000. The length of time for diagnosis is prolonged by the fact it takes multiple days to receive test results for patients (Fogoros, 2023). To increase accessibility of CAD diagnostics, our team at Lambert iGEM developed a cost-effective biosensor that detects microRNA (miRNA) biomarkers correlated with CAD progression. Our biosensor utilizes rolling circle amplification (RCA) to correlate fluorescence intensity to specific miRNA concentrations, allowing doctors and clinicians to monitor CAD progression (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA</a>). However, existing fluorometers are bulky, immobile, and have prices upwards of $10,000, making point-of-care testing (POCT) impractical (Hixson et al., 2022).</p>
+<p>In 2022, our team developed Micro-Q: a ~$15 PCR-tube fluorometer capable of quantifying fluorescent samples in seconds with 97.01% accuracy (See Lambert iGEM Hardware, 2022). This affordable solution, paired with its user-friendly mobile app, increases the accessibility of POCT in underfunded labs. However, after testing Micro-Q in Thailand, a couple of limitations were revealed: 1) quantifying batches of samples is tedious; 2) tuning excitation spectra for diverse fluorophores is complicated. Ultimately, these issues make testing and experimentation inefficient.</p>
+<p>This year, we addressed these issues by developing Micro-Q Pro—a camera-based fluorescence viewer under $10 capable of quantifying up to 8 PCR tubes simultaneously across the full visible wavelength spectrum. Since this detection uses camera imaging rather than photodiodes, users can record fluorescence videos and create real-time curves by snipping images at certain time points with 95.37% accuracy. This open-sourced fluorometer enables accurate and rapid sample quantification for diverse applications.</p>
+<h2 id="fluorescence-background">Fluorescence Background</h2>
+<p>Fluorescence is a type of luminescence that occurs when particular substances absorb light at one wavelength and then emit light at a longer wavelength. When a fluorescent sample is exposed to a specific excitation wavelength, it absorbs the light energy, raising its molecules to higher energy states. As these excited molecules return to lower energy ground states, they release excess energy as fluorescent light (see Fig. 1).</p>
+<p>This fundamental process enables diverse applications for fluorescence, including bioimaging, medical diagnostics, forensics, and environmental monitoring. By tuning excitation light and detecting emission wavelengths, researchers can selectively visualize, quantify, and track fluorescent probes for many purposes.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/screen-shot-2023-08-31-at-9-19-08-am.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. The process of fluorescence excitation and emission from a sample.</div></div>
+<h2 id="design">Design</h2>
+<style>astro-island,astro-slot,astro-static-slot{display:contents}</style><script>(()=>{var e=async t=>{await(await t())()};(self.Astro||(self.Astro={})).load=e;window.dispatchEvent(new Event("astro:load"));})();;(()=>{var d;{let p={0:t=>u(t),1:t=>l(t),2:t=>new RegExp(t),3:t=>new Date(t),4:t=>new Map(l(t)),5:t=>new Set(l(t)),6:t=>BigInt(t),7:t=>new URL(t),8:t=>new Uint8Array(t),9:t=>new Uint16Array(t),10:t=>new Uint32Array(t)},h=t=>{let[e,n]=t;return e in p?p[e](n):void 0},l=t=>t.map(h),u=t=>typeof t!="object"||t===null?t:Object.fromEntries(Object.entries(t).map(([e,n])=>[e,h(n)]));customElements.get("astro-island")||customElements.define("astro-island",(d=class extends HTMLElement{constructor(){super(...arguments);this.hydrate=async()=>{var i;if(!this.hydrator||!this.isConnected)return;let e=(i=this.parentElement)==null?void 0:i.closest("astro-island[ssr]");if(e){e.addEventListener("astro:hydrate",this.hydrate,{once:!0});return}let n=this.querySelectorAll("astro-slot"),o={},a=this.querySelectorAll("template[data-astro-template]");for(let r of a){let s=r.closest(this.tagName);s!=null&&s.isSameNode(this)&&(o[r.getAttribute("data-astro-template")||"default"]=r.innerHTML,r.remove())}for(let r of n){let s=r.closest(this.tagName);s!=null&&s.isSameNode(this)&&(o[r.getAttribute("name")||"default"]=r.innerHTML)}let c;try{c=this.hasAttribute("props")?u(JSON.parse(this.getAttribute("props"))):{}}catch(r){let s=this.getAttribute("component-url")||"<unknown>",y=this.getAttribute("component-export");throw y&&(s+=` (export ${y})`),console.error(`[hydrate] Error parsing props for component ${s}`,this.getAttribute("props"),r),r}await this.hydrator(this)(this.Component,c,o,{client:this.getAttribute("client")}),this.removeAttribute("ssr"),this.dispatchEvent(new CustomEvent("astro:hydrate"))}}connectedCallback(){!this.hasAttribute("await-children")||this.firstChild?this.childrenConnectedCallback():new MutationObserver((e,n)=>{n.disconnect(),setTimeout(()=>this.childrenConnectedCallback(),0)}).observe(this,{childList:!0})}async childrenConnectedCallback(){let e=this.getAttribute("before-hydration-url");e&&await import(e),this.start()}start(){let e=JSON.parse(this.getAttribute("opts")),n=this.getAttribute("client");if(Astro[n]===void 0){window.addEventListener(`astro:${n}`,()=>this.start(),{once:!0});return}Astro[n](async()=>{let o=this.getAttribute("renderer-url"),[a,{default:c}]=await Promise.all([import(this.getAttribute("component-url")),o?import(o):()=>()=>{}]),i=this.getAttribute("component-export")||"default";if(!i.includes("."))this.Component=a[i];else{this.Component=a;for(let r of i.split("."))this.Component=this.Component[r]}return this.hydrator=c,this.hydrate},e,this)}attributeChangedCallback(){this.hydrate()}},d.observedAttributes=["props"],d))}})();</script><astro-island uid="1pEiJ9" prefix="r2" component-url="/lambert-ga/_astro/ImageCarousel.440b7446.js" component-export="default" renderer-url="/lambert-ga/_astro/client.4f88357e.js" props="{"caption":[0,"Figure 2. 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Different angles of Micro-Q Pro design</div></div></astro-island>
+<h3 id="parts-list">Parts List</h3>
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+<table><thead><tr><th>Part</th><th>Price</th></tr></thead><tbody><tr><td>3D-printed shell PLA (217.25g)</td><td>$2.33</td></tr><tr><td>5000K LED Strip (4 inches)</td><td>$0.68</td></tr><tr><td>Musou Black Paint (0.1 Fl Oz)</td><td>$0.58</td></tr><tr><td>ESP-32 Camera</td><td>$6.33</td></tr><tr><td><strong>Total:</strong></td><td><strong>$9.92</strong></td></tr></tbody></table>
+<p>All the codes, steps for assembly, and CAD files can be found in the open-source Github repository: <a href="https://github.com/Lambert-iGEM-2023/Micro-Q-Pro">Micro-Q Pro Files</a>.</p>
+<h3 id="light-source">Light Source</h3>
+<p>Last year, Micro-Q used a singular 405 nm laser for high-precision green fluorescent sample excitation, minimizing needed software processing. However, when presented to Kiatichai Faksri of Khon Kaen University in Thailand, he commended its single-tube quantification accuracy but noted difficulties switching excitation lasers and filters to quantify different materials.
+Commercial spectrophotometers utilize white light lasers, which cover the full visible spectrum, in conjunction with diffraction gratings to choose the optimal excitation wavelength for a given biological sample (Dondelinger, 2011). However, these lasers often exceed $3,000, as they combine an array of laser wavelengths into a single compressed laser. Additionally, the many moving parts make these systems prone to breaking, while increasing size and bulkiness. A cost-effective alternative is a 5000K LED emitting the full 350-750 nm visible spectrum (Liu et al., 2018). To test our LED, we excited green fluorescein dyes at 30°, 45°, and 60°, finding 45° optimal for fluorescence visualization with minimal glare (Fig. 3). Additionally, we coated the inside of Micro-Q Pro with Musou Black Paint, which absorbs 99% of visible light, to eliminate excess ambient light for clearer imaging (Chu, 2019).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/measurement/fig3-2.png" style="max-width:350" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. Fluorescein sample excited at A) 30° B) 45° C) 60°; 45° showed least glare and best fluorescence.</div></div>
+<h3 id="fluorescence-quantification">Fluorescence Quantification</h3>
+<p>The original Micro-Q design utilized a photoresistor (a light intensity sensor) behind an emission filter to quantify the fluorescence intensity of the sample. While this design worked relatively well for discerning between high fluorescence concentrations, it struggled to differentiate between lower ones. Its accuracy could significantly be improved by replacing the photoresistor with a photodiode (a more sensitive light intensity sensor), however this year, Lambert iGEM implemented a camera-based detection system to enable real-time visualizations and quantification of sample fluorescence. Additionally, with this quantification mechanism, more samples can be quantified in a fraction of the time. This camera-based detection enables users to automate fluorescence curves by quantifying fluorescence across video segments.</p>
+<p>To quantify fluorescence, the Micro-Q Pro ESP32 camera captures two images: 1) a blank image with just light; 2) a sample image with fluorescent tubes (Fig. 4). Then, the algorithm subtracts the blank pixels (image 1) from the sample image (image 2) to remove non-fluorescent features, and isolate the sample’s fluorescence intensity. The image is then preprocessed to improve quality before calculating RFU values. Preprocessing involves median blurring to reduce noise, inverting the image, and applying gamma correction. This compensates for the non-linear relationship between true and measured sample brightness (see Fig. 5).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/measurement/fig4.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Software pipeline of Micro-Q Pro ESP-32 Camera.</div></div>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/measurement/fig5-1.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 5. Tubes of fluorescein from 200µm 1.5µm a) before preprocessing b) after preprocessing.</div></div>
+<p>After preprocessing, Otsu’s thresholding masks out the remaining background noise, leaving only the foreground fluorescence signal. Individual binary masks are created for each PCR tube, and the average pixel brightness within each mask is calculated to determine the RFU values (Fig. 6). Since the masking algorithm will isolate the sample of fluorescence from excess light, users can quantify multiple samples without emission filters, unless a tube contains multiple fluorophores.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/measurement/fig6-1.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 6. Tubes of fluorescein from 200µm 1.5µm a) original b) after masking fluorescence.</div></div>
+<h3 id="3d-printed-shell">3D-Printed Shell:</h3>
+<p>The original Micro-Q device was initially designed to easily quantify individual PCR tubes. To enable multisample and multispectral quantification, Micro-Q Pro was redesigned as a 10.8 cm x 8.7cm x 8.3 cm 3D printed box with three key compartments. A sample chamber, an exchangeable emission filter, and a camera holster (see Fig. 7). The sample chamber was modified to hold a strip of 8 PCR tubes separated by dividers to minimize crosstalk between fluorescent signals, which could skew the data (Arrpe et al. 2017).</p>
+<p>Moreover, Micro-Q Pro uses a 45-degree angled white LED strip paired with the emission filter to detect fluorescence across all relevant wavelengths. The ESP-32 camera module is placed 7.8 cm away from the samples to ensure it can view all 8 tubes at once. These optimized elements work seamlessly together to enable robust, high-quality fluorescence imaging of multiple samples.</p>
+<astro-island uid="19HPwO" prefix="r7" component-url="/lambert-ga/_astro/ImageCarousel.440b7446.js" component-export="default" renderer-url="/lambert-ga/_astro/client.4f88357e.js" props="{"caption":[0,"Figure 7. Schematic diagram of the redesigned Micro-Q Pro device highlighting the sample chamber, emission filter compartment, and camera holster."],"images":[1,[[0,"https://static.igem.wiki/teams/4683/wiki/measurement/fig7-1.jpg"],[0,"https://static.igem.wiki/teams/4683/wiki/measurement/fig7-2.jpg"],[0,"https://static.igem.wiki/teams/4683/wiki/measurement/fig7-3.jpg"],[0,"https://static.igem.wiki/teams/4683/wiki/measurement/fig7-4.jpg"]]]}" ssr="" client="load" opts="{"name":"Image","value":true}" await-children=""><div class="flex w-full flex-col items-center justify-center p-1"><div class="carousel-root" tabindex="0"><div class="carousel carousel-slider" style="width:100%"><ul class="control-dots"><li class="dot selected" value="0" role="button" tabindex="0" aria-label="slide item 1"></li><li class="dot" value="1" role="button" tabindex="0" aria-label="slide item 2"></li><li class="dot" value="2" role="button" tabindex="0" aria-label="slide item 3"></li><li class="dot" value="3" role="button" tabindex="0" aria-label="slide item 4"></li></ul><button type="button" aria-label="previous slide / item" class="control-arrow control-prev control-disabled"></button><div class="slider-wrapper axis-horizontal"><ul class="slider animated" style="-webkit-transform:translate3d(0,0,0);-ms-transform:translate3d(0,0,0);-o-transform:translate3d(0,0,0);transform:translate3d(0,0,0);-webkit-transition-duration:350ms;-moz-transition-duration:350ms;-o-transition-duration:350ms;transition-duration:350ms;-ms-transition-duration:350ms"><li class="slide selected previous"><div class="slide"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/measurement/fig7-1.jpg"/></div></li><li class="slide"><div class="slide"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/measurement/fig7-2.jpg"/></div></li><li class="slide"><div class="slide"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/measurement/fig7-3.jpg"/></div></li><li class="slide"><div class="slide"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/measurement/fig7-4.jpg"/></div></li></ul></div><button type="button" aria-label="next slide / item" class="control-arrow control-next"></button><p class="carousel-status">1 of 4</p></div><div class="carousel"><div class="thumbs-wrapper axis-vertical"><button type="button" class="control-arrow control-prev control-disabled" aria-label="previous slide / item"></button><ul class="thumbs animated" style="-webkit-transform:translate3d(0,0,0);-moz-transform:translate3d(0,0,0);-ms-transform:translate3d(0,0,0);-o-transform:translate3d(0,0,0);transform:translate3d(0,0,0);-ms-transform:translate3d(0,0,0);-webkit-transition-duration:350ms;-moz-transition-duration:350ms;-ms-transition-duration:350ms;-o-transition-duration:350ms;transition-duration:350ms;-ms-transition-duration:350ms"><li class="thumb selected" aria-label="slide item 1" style="width:80px" role="button" tabindex="0"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/measurement/fig7-1.jpg"/></li><li class="thumb" aria-label="slide item 2" style="width:80px" role="button" tabindex="0"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/measurement/fig7-2.jpg"/></li><li class="thumb" aria-label="slide item 3" style="width:80px" role="button" tabindex="0"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/measurement/fig7-3.jpg"/></li><li class="thumb" aria-label="slide item 4" style="width:80px" role="button" tabindex="0"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/measurement/fig7-4.jpg"/></li></ul><button type="button" class="control-arrow control-next control-disabled" aria-label="next slide / item"></button></div></div></div><div class="text-center italic">Figure 7. Schematic diagram of the redesigned Micro-Q Pro device highlighting the sample chamber, emission filter compartment, and camera holster.</div></div></astro-island>
+<h2 id="results-and-analysis">Results and Analysis</h2>
+<h3 id="preliminary-testing">Preliminary Testing</h3>
+<p>To validate Micro-Q Pro’s accuracy, we quantified a range of fluorescein concentrations (0-200 μM) similar to GFP working ranges (de Jonge et al.). Triplicate PCR tubes of 8 were quantified for each of the concentrations below.</p>
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+<table><thead><tr><th>Tube</th><th>Fluorescein Concentration (μM)</th></tr></thead><tbody><tr><td>Tube 1</td><td>200</td></tr><tr><td>Tube 2</td><td>97</td></tr><tr><td>Tube 3</td><td>48</td></tr><tr><td>Tube 4</td><td>24.25</td></tr><tr><td>Tube 5</td><td>12.3</td></tr><tr><td>Tube 6</td><td>6.06</td></tr><tr><td>Tube 7</td><td>3.032</td></tr><tr><td>Tube 8</td><td>0</td></tr></tbody></table>
+<p>Initial comparisons of raw relative fluorescence values (RFU) from Micro-Q Pro, and a plate reader revealed a logarithmic relationship, rather than the expected linear correlation between fluorescence and concentration (Fig. 8)(Itagaki, 2000). After further investigation, we discovered this was due to the camera’s reduced sensitivity at higher concentrations, causing oversaturation. This means small perceived camera differences equate to larger fluorescence intensity differences, as proven by the plate reader. To account for this non-linearity, the data requires processing.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/measurement/fig8.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 8. Raw RFU values calculated from the original quantification algorithm).</div></div>
+<p>To linearize our data, we applied a power series regression on all the trials from the 3 triplicates. The curve below achieved the highest R2 value when linearized, so we chose to apply this formula to linearize any further results (See Fig. 9).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/measurement/fig9.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 9. Power series regression to linearize Micro-Q values to plate reader values.</div></div>
+<p>Applying the power series regression formula to the previous triplicate test, we get a linear relationship (See Fig. 10).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/measurement/fig10.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 10. Micro-Q Pro triplicate results after power series linearization.</div></div>
+<h3 id="final-results">Final Results</h3>
+<p>After confirming the linearization of the RFU values from Micro-Q pro, we reran triplicates of the same experiment, and compared the results to the plate reader. Since relative fluorescence units (RFU) are relative, we scaled the output of Micro-Q to match the scale of the output from the plate reader so that they can be compared (see Fig. 11).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/measurement/fig11.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 11. Measurements from plate reader and Micro-Q Pro from 0-200 μM. </div></div>
+<p>In order to have an accurate comparison of the data from the plate reader and Micro-Q, we added points at the origin to our data set, and calculated a slope from a linear regression for each measurement device, assuming a y-intercept of 0. The slopes of the data are ~72.1 and ~75.6 from Micro-Q Pro and the Plate reader, respectively, achieving a percent error of -4.629%</p>
+<h2 id="future-directions--conclusion">Future Directions & Conclusion</h2>
+<p>Micro-Q Pro demonstrates promising accuracy in quantifying green fluorescent samples, with no significant differences between Micro-Q Pro and plate reader outputs. This validates the fluorescence measurement capabilities of our hardware device. Although we primarily characterized performance with green fluorophores, preliminary data with red fluorescent materials is also encouraging.</p>
+<p>Future iterations could incorporate internal heating pads, enabling fully autonomous fluorescence curves creation during incubations. We also aim to develop an intuitive mobile app for live-streaming fluorescence data and make sample quantification as simple as a click of a button. Additionally, we will resume full characterization across additional spectra once we return to the lab.</p>
+<p>Micro-Q Pro retains the core functionalities of the original Micro-Q, while dramatically enhancing the ability to efficiently quantify multiple samples across diverse fluorescent spectra—all within a simple $10 device.</p>
+<p>As characterization of Micro-Q Pro continues on different fluorophores, Micro-Q Pro makes robust fluorescence quantification widely accessible regardless of financial limitations. Ultimately, this technology enables portable, efficient point-of-care diagnostic testing our miRNA biosensors and other fluorescence-based assays.</p>
+<h2 id="references">References</h2>
+<div class="pl-6 -indent-6"><p>Arppe, R., Carro-Temboury, M. R., Hempel, C., Vosch, T., & Just Sørensen, T.
+(2017, November 27). Investigating dye performance and crosstalk in
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+Fluorophores for the Violet (405 nm) Laser.
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+<div class="pl-6 -indent-6"><p>Chu, J. (2019, September 12). MIT engineers develop “blackest black†material
+to date. MIT News | Massachusetts Institute of Technology.
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+<a href="https://meridian.allenpress.com/bit/article/45/2/139/142090/Spectrophotometers">https://meridian.allenpress.com/bit/article/45/2/139/142090/Spectrophotometers</a></p></div>
+<div class="pl-6 -indent-6"><p>De Jonge, Jennifer, et al. “Salicylic Acid Interferes with GFP Fluorescence in
+Vivo.†Journal of Experimental Botany, vol. 68, no. 7, 1 Mar. 2017, pp.
+1689–1696, <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5441896">www.ncbi.nlm.nih.gov/pmc/articles/PMC5441896</a> /,
+<a href="https://doi.org/10.1093/jxb/erx031">https://doi.org/10.1093/jxb/erx031</a>. Accessed 17 Oct. 2022.</p></div>
+<div class="pl-6 -indent-6"><p>Hixson, J. L., & Ward, A. S. (2022, March 17). Hardware selection and
+performance of low-cost fluorometers. Sensors (Basel, Switzerland).
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+ <h1 id="erca--rca-models">eRCA & RCA Models</h1>
+<h2 id="rca-overview">RCA Overview</h2>
+<p>In 2022, Lambert iGEM created a mathematical model for rolling circle amplification (RCA) (see Fig. 1). RCA can be summarized by the steps below (see RCA):</p>
+<ul>
+<li>Hybridization: The target microRNA (miRNA) hybridizes to a linear DNA padlock probe, bringing the ends closer together (Graugnard et al., 2010).</li>
+<li>Ligation: SplintR ligase circularizes the padlock probe (Jin et al., 2016; Lohman et al., 2013).</li>
+<li>Amplification: The miRNA acts as a primer for phi29 DNA polymerase (Esteban et al., 1993) to amplify the padlock probe sequence, thus synthesizing the rolling circle product (RCP) made of interspaced repeats of the miRNA sequence and the reporting mechanism sequence.</li>
+<li>Quantification: Linear probes are short DNA sequences tagged with either a fluorophore or quencher molecule. When the linear probes bind to the RCP, they are in close proximity, allowing the quencher to absorb the light emitted by the fluorophore. This results in a quantifiable decrease in fluorescence.</li>
+</ul>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/model-fig1.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. Diagram of the reactions of the RCA pathway in MATLAB Simbiology software</div></div>
+<p>Last year, Lambert iGEM modeled the RCA to guide and validate our wet-lab results. To accomplish this task, we relied on the Mass Action Laws and Michaelis-Menten Kinetics to simulate each reaction in the pathway.</p>
+<p>This year, as part of our ongoing commitment to enhancing the RCA model, we conducted a scientific literature review to source estimated parameters such as degradation values for rolling circle product (RCP) and the nicking endonuclease Nb.BbvCI, which is utilized in exponential RCA (Shen et al., 2019). We sourced a number of ‘k’ constants associated with the array of reversible reactions in our system. We then used equilibrium equations to derive the specific value of these reverse reactions. This process has notably improved results in our model simulations resulting in higher accuracy.</p>
+<h2 id="development">Development</h2>
+<p>To assist in interpreting and validating RCA reactions, Lambert iGEM incorporated a deterministic ordinary differential equations (ODE) model to simulate the reaction and correlate fluorescence caused by the RCP-fluorophore-quencher (RFQ) to the initial miRNA concentration.</p>
+<h2 id="signaling-pathway">Signaling Pathway</h2>
+<p>The team used MATLAB’s SimBiology software to diagram each reaction using the Law of Mass Action and Michaelis-Menten equations. The Mass Action equations are used to diagram the collision of reactants in a solution, which are needed for most reactants in our reaction pathways, while Michaelis-Menten equations are needed to properly diagram catalytic enzyme kinetics (Park, 2022; Zi, 2012). The pathway begins with the introduction of miRNA into the system and ends with the RFQ (see Fig. 2).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/model-fig2.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 2. Diagram of the reactions of the Updated RCA pathway in MATLAB Simbiology software</div></div>
+<h2 id="biochemical-reactions">Biochemical Reactions</h2>
+<p>The model contains a total of 3 biochemical reactions (see Fig. 2).</p>
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+<table><thead><tr><th align="center">Reactions</th><th align="center">Description</th></tr></thead><tbody><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mi>R</mi><mi>F</mi><mo>+</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><munderover><mo stretchy="true" minsize="3.0em">⇌</mo><mpadded width="+0.6em" lspace="0.3em"><msub><mi>k</mi><mn>6</mn></msub></mpadded><mpadded width="+0.6em" lspace="0.3em"><msub><mi>k</mi><mn>5</mn></msub></mpadded></munderover><mi>R</mi><mi>F</mi><mi>Q</mi></mrow><annotation encoding="application/x-tex">RF + Q_{free} \xrightleftharpoons[{k_{6}}]{k_{5}} RFQ</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:0.7667em;vertical-align:-0.0833em"></span><span class="mord mathnormal" style="margin-right:0.13889em">RF</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">+</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:2.0103em;vertical-align:-0.8052em"></span><span class="mord"><span class="mord mathnormal">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:0em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel x-arrow"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.2051em"><span style="top:-3.435em"><span class="pstrut" style="height:2.716em"></span><span class="sizing reset-size6 size3 mtight x-arrow-pad"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3173em"><span style="top:-2.357em;margin-left:-0.0315em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mtight">5</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.143em"><span></span></span></span></span></span></span></span></span></span><span class="svg-align" style="top:-2.608em"><span class="pstrut" style="height:2.716em"></span><span class="stretchy" style="height:0.716em;min-width:1.75em"><span class="halfarrow-left" style="height:0.716em"><svg xmlns="http://www.w3.org/2000/svg" width="400em" height="0.716em" viewBox="0 0 400000 716" preserveAspectRatio="xMinYMin slice"><path d="M7 435c-4 4-6.3 8.7-7 14 0 5.3.7 9 2 11s5.3 5.3 12
+ 10c90.7 54 156 130 196 228 3.3 10.7 6.3 16.3 9 17 2 .7 5 1 9 1h5c10.7 0 16.7
+-2 18-6 2-2.7 1-9.7-3-21-32-87.3-82.7-157.7-152-211l-3-3h399907v-40H7zm93 0
+v40h399900v-40zM0 241v40h399900v-40zm0 0v40h399900v-40z"></path></svg></span><span class="halfarrow-right" style="height:0.716em"><svg xmlns="http://www.w3.org/2000/svg" width="400em" height="0.716em" viewBox="0 0 400000 716" preserveAspectRatio="xMaxYMin slice"><path d="M0 241v40h399993c4.7-4.7 7-9.3 7-14 0-9.3-3.7-15.3-11
+-18-92.7-56.7-159-133.7-199-231-3.3-9.3-6-14.7-8-16-2-1.3-7-2-15-2-10.7 0-16.7
+ 2-18 6-2 2.7-1 9.7 3 21 15.3 42 36.7 81.8 64 119.5 27.3 37.7 58 69.2 92 94.5z
+m0 0v40h399900v-40z m100 194v40h399900v-40zm0 0v40h399900v-40z"></path></svg></span></span></span><span style="top:-2.0109em"><span class="pstrut" style="height:2.716em"></span><span class="sizing reset-size6 size3 mtight x-arrow-pad"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3173em"><span style="top:-2.357em;margin-left:-0.0315em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mtight">6</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.143em"><span></span></span></span></span></span></span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.8052em"><span></span></span></span></span></span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:0.8778em;vertical-align:-0.1944em"></span><span class="mord mathnormal">RFQ</span></span></span></span></td><td align="center">RCP-fluorophore complexes (<strong>RF</strong>) bind to free quenchers (<strong>Q_free</strong>) to form RCP-quencher-fluorophore complexes (<strong>RFQ</strong>)</td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mi>M</mi><mo>+</mo><mi>P</mi><mover><mo stretchy="true" minsize="3.0em">→</mo><mpadded width="+0.6em" lspace="0.3em"><mi>k</mi></mpadded></mover><mi>M</mi><mi>P</mi></mrow><annotation encoding="application/x-tex">M + P \xrightarrow{k} MP</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:0.7667em;vertical-align:-0.0833em"></span><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">+</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.1191em;vertical-align:-0.011em"></span><span class="mord mathnormal" style="margin-right:0.13889em">P</span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel x-arrow"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.1081em"><span style="top:-3.322em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight x-arrow-pad"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.03148em">k</span></span></span></span><span class="svg-align" style="top:-2.689em"><span class="pstrut" style="height:2.7em"></span><span class="hide-tail" style="height:0.522em;min-width:1.469em"><svg xmlns="http://www.w3.org/2000/svg" width="400em" height="0.522em" viewBox="0 0 400000 522" preserveAspectRatio="xMaxYMin slice"><path d="M0 241v40h399891c-47.3 35.3-84 78-110 128
+-16.7 32-27.7 63.7-33 95 0 1.3-.2 2.7-.5 4-.3 1.3-.5 2.3-.5 3 0 7.3 6.7 11 20
+ 11 8 0 13.2-.8 15.5-2.5 2.3-1.7 4.2-5.5 5.5-11.5 2-13.3 5.7-27 11-41 14.7-44.7
+ 39-84.5 73-119.5s73.7-60.2 119-75.5c6-2 9-5.7 9-11s-3-9-9-11c-45.3-15.3-85
+-40.5-119-75.5s-58.3-74.8-73-119.5c-4.7-14-8.3-27.3-11-40-1.3-6.7-3.2-10.8-5.5
+-12.5-2.3-1.7-7.5-2.5-15.5-2.5-14 0-21 3.7-21 11 0 2 2 10.3 6 25 20.7 83.3 67
+ 151.7 139 205zm0 0v40h399900v-40z"></path></svg></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.011em"><span></span></span></span></span></span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:0.6833em"></span><span class="mord mathnormal" style="margin-right:0.13889em">MP</span></span></span></span></td><td align="center">Input miRNA (<strong>M</strong>) binds to the complementary arms of free padlock probes (<strong>P</strong>) to form miRNA-padlock probe complexes (<strong>MP</strong>)</td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mi>M</mi><mi>P</mi><mo>+</mo><mi>S</mi><mi>p</mi><mi>l</mi><mi>R</mi><mo>⇌</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi><mi>i</mi><mi>z</mi><mi>e</mi><mi>d</mi></mrow></msub></mrow><annotation encoding="application/x-tex">MP + SplR \rightleftharpoons MP_{circularized}</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:0.7667em;vertical-align:-0.0833em"></span><span class="mord mathnormal" style="margin-right:0.13889em">MP</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">+</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:0.8889em;vertical-align:-0.1944em"></span><span class="mord mathnormal" style="margin-right:0.00773em">SplR</span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">⇌</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:0.8333em;vertical-align:-0.15em"></span><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">ze</span><span class="mord mathnormal mtight">d</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span></span></span></span></td><td align="center">miRNA-padlock probe complexes (<strong>MP</strong>) are ligated by SplintR ligase (<strong>SplR</strong>) to circularized complexes (<strong>MP_circularized</strong>)</td></tr></tbody></table>
+<h2 id="initial-values-of-species">Initial Values of Species</h2>
+<p>In order to simulate our reactions, our team needed to input values to initialize each component in our model. The initial value of each species was obtained from wet lab protocols:</p>
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+<table><thead><tr><th align="center">Variable</th><th align="center">Species</th><th align="center">Initial Value</th><th align="center">Units</th></tr></thead><tbody><tr><td align="center">M</td><td align="center">miRNA</td><td align="center">0.5</td><td align="center">picomoles</td></tr><tr><td align="center">P</td><td align="center">padlock probes</td><td align="center">0.05</td><td align="center">picomoles</td></tr><tr><td align="center">SplR</td><td align="center">SplintR</td><td align="center">25</td><td align="center">molecules</td></tr><tr><td align="center">phi29_xt</td><td align="center">phi29XT DNA polymerase</td><td align="center">12.5</td><td align="center">molecules</td></tr><tr><td align="center">R_free</td><td align="center">free RCP</td><td align="center">0</td><td align="center">molecules</td></tr><tr><td align="center">F_free</td><td align="center">free fluorophores</td><td align="center">180664245</td><td align="center">molecules</td></tr><tr><td align="center">Q_free</td><td align="center">free quenchers</td><td align="center">361328490</td><td align="center">molecules</td></tr><tr><td align="center">RF</td><td align="center">bound RCP-fluorophores</td><td align="center">0</td><td align="center">picomoles</td></tr><tr><td align="center">RQ</td><td align="center">bound RCP-quenchers</td><td align="center">0</td><td align="center">picomoles</td></tr><tr><td align="center">RFQ</td><td align="center">bound RCP-fluorophores-quenchers</td><td align="center">0</td><td align="center">picomoles</td></tr></tbody></table>
+<h2 id="parameters">Parameters</h2>
+<p>Rate constants and degradation rates are required to input into our Mass Action Laws and Michaelis-Menten equations. These values were derived from literature or estimated if the values could not be found. The value of each parameter is shown below:</p>
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+<table><thead><tr><th align="center">Variable</th><th align="center">Reaction</th><th align="center">Estimated Values</th><th align="center">Units</th></tr></thead><tbody><tr><td align="center">Mdeg</td><td align="center">rate constant for miRNA degradation</td><td align="center">0.000019254</td><td align="center">1/second</td></tr><tr><td align="center">k_padlock</td><td align="center">rate constant for miRNA-padlock binding</td><td align="center">32000</td><td align="center">1/(moles*second)</td></tr><tr><td align="center">Vmcircular</td><td align="center">maximal rate of reaction needed for Michaelis-Menten enzyme kinetics for MP-SplintR binding</td><td align="center">0.008</td><td align="center">moles/second</td></tr><tr><td align="center">Kmcircular</td><td align="center">Michaelis-Menten constant for MP-SplintR binding</td><td align="center">1</td><td align="center">moles</td></tr><tr><td align="center">Vmphi29_xt</td><td align="center">maximal rate of reaction needed for Michaelis-Menten enzyme kinetics for MP_circular-phi29 binding</td><td align="center">824</td><td align="center">moles/second</td></tr><tr><td align="center">Kmphi29_xt</td><td align="center">Michaelis-Menten constant for MP_circular-phi29 binding</td><td align="center">242</td><td align="center">moles</td></tr><tr><td align="center">RCPdeg</td><td align="center">rate constant for free RCP degradation</td><td align="center">0</td><td align="center">1/second</td></tr><tr><td align="center">k1</td><td align="center">forward reaction rate constant for free RCP and free fluorophore binding</td><td align="center">2.2e12</td><td align="center">1/(moles*second)</td></tr><tr><td align="center">k2</td><td align="center">backward reaction rate constant for free RCP and free fluorophore binding</td><td align="center">12.4326576778</td><td align="center">1/second</td></tr><tr><td align="center">k3</td><td align="center">forward reaction rate constant for free RCP and free quencher binding</td><td align="center">2.2e12</td><td align="center">1/(moles*second)</td></tr><tr><td align="center">k4</td><td align="center">backward reaction rate constant for free RCP and free quencher binding</td><td align="center">12.4326576778</td><td align="center">1/second</td></tr><tr><td align="center">k5</td><td align="center">forward reaction rate constant for bound RF and free quencher binding</td><td align="center">1.34e12</td><td align="center">1/(moles*second)</td></tr><tr><td align="center">k6</td><td align="center">backward reaction rate constant for bound RF and free quencher binding</td><td align="center">7.57261876741</td><td align="center">1/second</td></tr><tr><td align="center">k7</td><td align="center">forward reaction rate constant for bound RQ and free fluorophore binding</td><td align="center">1e12</td><td align="center">1/(moles*second)</td></tr><tr><td align="center">k8</td><td align="center">backward reaction rate constant for bound RQ and free fluorophore binding</td><td align="center">5.65120803538</td><td align="center">1/second</td></tr></tbody></table>
+<h2 id="ordinary-differential-equations">Ordinary Differential Equations</h2>
+<p>As mentioned previously, Lambert iGEM incorporated the Mass Action Kinetic Laws and Michaelis-Menten Equations to represent the reactions for the RCA model.</p>
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+<table><thead><tr><th align="center">Ordinary Differential Equations</th></tr></thead><tbody><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>M</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mrow><mi>p</mi><mi>a</mi><mi>d</mi><mi>l</mi><mi>o</mi><mi>c</mi><mi>k</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>P</mi><mo stretchy="false">]</mo><mo>−</mo><msub><mi>M</mi><mrow><mi>d</mi><mi>e</mi><mi>g</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo></mrow><annotation encoding="application/x-tex">\frac{d[M]}{dt} = -(k_{padlock}[M][P] - M_{deg}[M])</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord">−</span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">oc</span><span class="mord mathnormal mtight" style="margin-right:0.03148em">k</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">P</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.109em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">e</span><span class="mord mathnormal mtight" style="margin-right:0.03588em">g</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="mclose">])</span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>P</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><msub><mi>k</mi><mrow><mi>p</mi><mi>a</mi><mi>d</mi><mi>l</mi><mi>o</mi><mi>c</mi><mi>k</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>P</mi><mo stretchy="false">]</mo></mrow><annotation encoding="application/x-tex">\frac{d[P]}{dt} = -k_{padlock}[M][P]</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord">−</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">oc</span><span class="mord mathnormal mtight" style="margin-right:0.03148em">k</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">P</span><span class="mclose">]</span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>S</mi><mi>p</mi><mi>l</mi><mi>R</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mfrac><mrow><msub><mi>V</mi><mrow><mi>m</mi><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><mi>P</mi><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mrow><mi>m</mi><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo>+</mo><mo stretchy="false">[</mo><mi>M</mi><mi>P</mi><mo stretchy="false">]</mo></mrow></mfrac></mrow><annotation encoding="application/x-tex">\frac{d[SplR]}{dt} = -\frac{V_{mcircular}[MP]}{K_{mcircular} + [MP]}</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.00773em">SplR</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.53em;vertical-align:-0.52em"></span><span class="mord">−</span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.07153em">K</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0715em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mbin mtight">+</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">MP</span><span class="mclose mtight">]</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.22222em">V</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.2222em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">MP</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.52em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>M</mi><mi>P</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><msub><mi>k</mi><mrow><mi>p</mi><mi>a</mi><mi>d</mi><mi>l</mi><mi>o</mi><mi>c</mi><mi>k</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>P</mi><mo stretchy="false">]</mo><mo>−</mo><mfrac><mrow><msub><mi>V</mi><mrow><mi>m</mi><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><mi>P</mi><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mrow><mi>m</mi><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo>+</mo><mo stretchy="false">[</mo><mi>M</mi><mi>P</mi><mo stretchy="false">]</mo></mrow></mfrac></mrow><annotation encoding="application/x-tex">\frac{d[MP]}{dt} = k_{padlock}[M][P] - \frac{V_{mcircular}[MP]}{K_{mcircular} + [MP]}</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">MP</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">oc</span><span class="mord mathnormal mtight" style="margin-right:0.03148em">k</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">P</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.53em;vertical-align:-0.52em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.07153em">K</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0715em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mbin mtight">+</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">MP</span><span class="mclose mtight">]</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.22222em">V</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.2222em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">MP</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.52em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mfrac><mrow><msub><mi>V</mi><mrow><mi>m</mi><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><mi>P</mi><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mrow><mi>m</mi><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo>+</mo><mo stretchy="false">[</mo><mi>M</mi><mi>P</mi><mo stretchy="false">]</mo></mrow></mfrac><mo>−</mo><mfrac><mrow><msub><mi>V</mi><mrow><mi>m</mi><mi>p</mi><mi>h</mi><mi>i</mi><mn>29</mn></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mrow><mi>m</mi><mi>p</mi><mi>h</mi><mi>i</mi><mn>29</mn></mrow></msub><mo>+</mo><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow></mfrac></mrow><annotation encoding="application/x-tex">\frac{d[MP_{circular}]}{dt} = \frac{V_{mcircular}[MP]}{K_{mcircular} + [MP]} - \frac{V_{mphi29}[MP_{circular}]}{K_{mphi29} + [MP_{circular}]}</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.53em;vertical-align:-0.52em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.07153em">K</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0715em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mbin mtight">+</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">MP</span><span class="mclose mtight">]</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.22222em">V</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.2222em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">MP</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.52em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.5862em;vertical-align:-0.5481em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.0381em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.07153em">K</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0715em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">hi</span><span class="mord mtight">29</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2901em"><span></span></span></span></span></span></span><span class="mbin mtight">+</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.5131em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.22222em">V</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.2222em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">hi</span><span class="mord mtight">29</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2901em"><span></span></span></span></span></span></span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.5481em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>p</mi><mi>h</mi><mi>i</mi><mn>2</mn><msub><mn>9</mn><mi>x</mi></msub><mi>t</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mfrac><mrow><msub><mi>V</mi><mrow><mi>m</mi><mi>p</mi><mi>h</mi><mi>i</mi><mn>2</mn><msub><mn>9</mn><mi>x</mi></msub><mi>t</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mrow><mi>m</mi><mi>p</mi><mi>h</mi><mi>i</mi><mn>2</mn><msub><mn>9</mn><mi>x</mi></msub><mi>t</mi></mrow></msub><mo>+</mo><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow></mfrac></mrow><annotation encoding="application/x-tex">\frac{d[phi29_xt]}{dt} = - \frac{V_{mphi29_xt}[MP_{circular}]}{K_{mphi29_xt} + [MP_{circular}]}</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">hi</span><span class="mord mtight">2</span><span class="mord mtight"><span class="mord mtight">9</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.1645em"><span style="top:-2.357em;margin-left:0em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mathnormal mtight">x</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.143em"><span></span></span></span></span></span></span><span class="mord mathnormal mtight">t</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.5917em;vertical-align:-0.5509em"></span><span class="mord">−</span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.0409em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.07153em">K</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0715em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">hi</span><span class="mord mtight">2</span><span class="mord mtight"><span class="mord mtight">9</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.2306em"><span style="top:-2.3em;margin-left:0em;margin-right:0.1em"><span class="pstrut" style="height:2.5em"></span><span class="mord mathnormal mtight">x</span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2em"><span></span></span></span></span></span></span><span class="mord mathnormal mtight">t</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2941em"><span></span></span></span></span></span></span><span class="mbin mtight">+</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.5159em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.22222em">V</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.2222em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">hi</span><span class="mord mtight">2</span><span class="mord mtight"><span class="mord mtight">9</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.2306em"><span style="top:-2.3em;margin-left:0em;margin-right:0.1em"><span class="pstrut" style="height:2.5em"></span><span class="mord mathnormal mtight">x</span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2em"><span></span></span></span></span></span></span><span class="mord mathnormal mtight">t</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2941em"><span></span></span></span></span></span></span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.5509em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mfrac><mrow><msub><mi>V</mi><mrow><mi>m</mi><mi>p</mi><mi>h</mi><mi>i</mi><mn>2</mn><msub><mn>9</mn><mi>x</mi></msub><mi>t</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mrow><mi>m</mi><mi>p</mi><mi>h</mi><mi>i</mi><mn>2</mn><msub><mn>9</mn><mi>x</mi></msub><mi>t</mi></mrow></msub><mo>+</mo><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow></mfrac><mo>−</mo><mo stretchy="false">(</mo><mi>R</mi><mi>C</mi><msub><mi>P</mi><mrow><mi>d</mi><mi>e</mi><mi>g</mi></mrow></msub><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">)</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>1</mn></msub><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><msub><mi>F</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>2</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>3</mn></msub><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>4</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo></mrow><annotation encoding="application/x-tex">\frac{d[R_{free}]}{dt} = \frac{V_{mphi29_xt}[MP_{circular}]}{K_{mphi29_xt} + [MP_{circular}]} -(RCP_{deg}[R_{free}]) - (k_1[R_{free}][F_{free}] - k_2[RF]) - (k_3[R_{free}][Q_{free}] - k_4[RQ])</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.3831em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.0381em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.5131em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0077em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2901em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.5917em;vertical-align:-0.5509em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.0409em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.07153em">K</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0715em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">hi</span><span class="mord mtight">2</span><span class="mord mtight"><span class="mord mtight">9</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.2306em"><span style="top:-2.3em;margin-left:0em;margin-right:0.1em"><span class="pstrut" style="height:2.5em"></span><span class="mord mathnormal mtight">x</span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2em"><span></span></span></span></span></span></span><span class="mord mathnormal mtight">t</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2941em"><span></span></span></span></span></span></span><span class="mbin mtight">+</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.5159em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.22222em">V</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.2222em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">hi</span><span class="mord mtight">2</span><span class="mord mtight"><span class="mord mtight">9</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.2306em"><span style="top:-2.3em;margin-left:0em;margin-right:0.1em"><span class="pstrut" style="height:2.5em"></span><span class="mord mathnormal mtight">x</span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2em"><span></span></span></span></span></span></span><span class="mord mathnormal mtight">t</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2941em"><span></span></span></span></span></span></span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.5509em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord mathnormal" style="margin-right:0.07153em">RC</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">e</span><span class="mord mathnormal mtight" style="margin-right:0.03588em">g</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0077em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">])</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">1</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0077em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">F</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">2</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">RF</span><span class="mclose">])</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">3</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0077em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:0em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">4</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RQ</span><span class="mclose">])</span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><msub><mi>F</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>1</mn></msub><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><msub><mi>F</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>2</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>7</mn></msub><mo stretchy="false">[</mo><msub><mi>F</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>R</mi><mi>Q</mi><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>8</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo></mrow><annotation encoding="application/x-tex">\frac{d[F_{free}]}{dt} = -(k_1[R_{free}][F_{free}] - k_2[RF]) - (k_7[F_{free}][RQ] - k_8[RFQ])</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.3831em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.0381em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.5131em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">F</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2901em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord">−</span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">1</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0077em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">F</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">2</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">RF</span><span class="mclose">])</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">7</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">F</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal">RQ</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">8</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RFQ</span><span class="mclose">])</span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>3</mn></msub><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>2</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>5</mn></msub><mo stretchy="false">[</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>6</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo></mrow><annotation encoding="application/x-tex">\frac{d[Q_{free}]}{dt} = -(k_3[R_{free}][Q_{free}] - k_2[RQ]) - (k_5[Q_{free}][RF] - k_6[RFQ])</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.3831em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.0381em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.5131em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mtight"><span class="mord mathnormal mtight">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:0em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2901em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord">−</span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">3</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0077em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:0em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">2</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RQ</span><span class="mclose">])</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">5</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:0em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">RF</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">6</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RFQ</span><span class="mclose">])</span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>1</mn></msub><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><msub><mi>F</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>2</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>5</mn></msub><mo stretchy="false">[</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>6</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo></mrow><annotation encoding="application/x-tex">\frac{d[RF]}{dt} = -(k_1[R_{free}][F_{free}] - k_2[RF]) - (k_5[Q_{free}][RF] - k_6[RFQ])</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">RF</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord">−</span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">1</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0077em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">F</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">2</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">RF</span><span class="mclose">])</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">5</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:0em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">RF</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">6</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RFQ</span><span class="mclose">])</span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>R</mi><mi>Q</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>3</mn></msub><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>4</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>7</mn></msub><mo stretchy="false">[</mo><msub><mi>F</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>R</mi><mi>Q</mi><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>8</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo></mrow><annotation encoding="application/x-tex">\frac{d[RQ]}{dt} = -(k_3[R_{free}][Q_{free}] - k_4[RQ]) - (k_7[F_{free}][RQ] - k_8[RFQ])</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight">RQ</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord">−</span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">3</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0077em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:0em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">4</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RQ</span><span class="mclose">])</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">7</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">F</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal">RQ</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">8</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RFQ</span><span class="mclose">])</span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mi>Q</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>7</mn></msub><mo stretchy="false">[</mo><msub><mi>F</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>R</mi><mi>Q</mi><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>8</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo><mo>+</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>5</mn></msub><mo stretchy="false">[</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>6</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo></mrow><annotation encoding="application/x-tex">\frac{d[RFQ]}{dt} = -(k_7[F_{free}][RQ] - k_8[RFQ]) + (k_5[Q_{free}][RF] - k_6[RFQ])</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight">RFQ</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord">−</span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">7</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">F</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal">RQ</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">8</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RFQ</span><span class="mclose">])</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">+</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">5</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:0em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">RF</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">6</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RFQ</span><span class="mclose">])</span></span></span></span></td></tr></tbody></table>
+<h2 id="results">Results</h2>
+<p>Lambert iGEM’s 2022 RCA protocol requires samples to be incubated at a very specific temperature for eight hours. However, phi29-XT DNA polymerase, an optimized enzyme with improved thermostability and sensitivity, shortens this to a two-hour amplification time (New England Biolabs). Therefore, we simulated the updated RCA model containing reverse reaction values and updated parameters, such as phi29-XT. The comparison of the activity of both the original RCA (see Fig. 3) and revised RCA (see Fig. 4) models clearly demonstrates the larger quantity of DNA phi29-XT produces .</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/model-fig3.png
+" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. Relationship between miRNA concentrations ranging from 0 to 25 picomoles and RFU based on simultations completed with the original RCA Model</div></div>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/model-fig4.png
+" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Relationship between miRNA concentrations ranging from 0 to 25 picomoles and RFU based on simultations completed with the updated RCA Model</div></div>
+<h2 id="erca-overview">eRCA Overview</h2>
+<p>This year, we also investigated exponential rolling circle amplification (eRCA), an adaptation of RCA that produces exponentially greater fluorescence per unit of miRNA, therefore improving the accuracy and sensitivity of the reaction. The eRCA process includes the addition of the nicking endonuclease Nb.BbvCI to cleave the continuously produced RCP at specifically placed nicking sites. This allows the miRNA sequence to be cut out of the RCP, bind to another padlock probe, and begin the amplification process again.</p>
+<h2 id="development-1">Development</h2>
+<p>In order to validate our wetlab experimentation, Lambert iGEM incorporated a deterministic ordinary differential equations (ODE) model to simulate the eRCA reaction (see Fig.4). Our model converted several reactions in the eRCA system into ODEs and created a loop that updates miRNA values to ensure the reaction continues until the initial amount of padlock probes are depleted. Lambert iGEM also reached out to Dr. Mark Styczynski from the Georgia Institute of Technology to aid in the identification of parameters and the steps for creating a model that functions exponentially rather than linearly.</p>
+<h2 id="signaling-pathway-1">Signaling Pathway</h2>
+<p>The team used MATLAB’s SimBiology software to diagram each reaction using the Law of Mass Action and Michaelis-Menten equations. The Mass Action equations are used to diagram the collision of reactants in a solution, which is needed for most reactants in our reaction pathways, while Michaelis-Menten equations are needed to properly diagram catalytic enzyme kinetics (Park, 2022; Zi, 2012). The pathway begins with the introduction of miRNA into the system and ends with the RFQ (see Fig. 5).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/model-fig5.png
+" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 5. Diagram of the reactions of the eRCA pathway in MATLAB Simbiology software</div></div>
+<h2 id="biochemical-reactions-1">Biochemical Reactions</h2>
+<p>The model contains a total of 3 biochemical reactions (see Fig. 5).</p>
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+<table><thead><tr><th align="center">Reactions</th><th align="center">Description</th></tr></thead><tbody><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mi>R</mi><mi>F</mi><mo>+</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><munderover><mo stretchy="true" minsize="3.0em">⇌</mo><mpadded width="+0.6em" lspace="0.3em"><msub><mi>k</mi><mn>6</mn></msub></mpadded><mpadded width="+0.6em" lspace="0.3em"><msub><mi>k</mi><mn>5</mn></msub></mpadded></munderover><mi>R</mi><mi>F</mi><mi>Q</mi></mrow><annotation encoding="application/x-tex">RF + Q_{free} \xrightleftharpoons[{k_{6}}]{k_{5}} RFQ</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:0.7667em;vertical-align:-0.0833em"></span><span class="mord mathnormal" style="margin-right:0.13889em">RF</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">+</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:2.0103em;vertical-align:-0.8052em"></span><span class="mord"><span class="mord mathnormal">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:0em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel x-arrow"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.2051em"><span style="top:-3.435em"><span class="pstrut" style="height:2.716em"></span><span class="sizing reset-size6 size3 mtight x-arrow-pad"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3173em"><span style="top:-2.357em;margin-left:-0.0315em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mtight">5</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.143em"><span></span></span></span></span></span></span></span></span></span><span class="svg-align" style="top:-2.608em"><span class="pstrut" style="height:2.716em"></span><span class="stretchy" style="height:0.716em;min-width:1.75em"><span class="halfarrow-left" style="height:0.716em"><svg xmlns="http://www.w3.org/2000/svg" width="400em" height="0.716em" viewBox="0 0 400000 716" preserveAspectRatio="xMinYMin slice"><path d="M7 435c-4 4-6.3 8.7-7 14 0 5.3.7 9 2 11s5.3 5.3 12
+ 10c90.7 54 156 130 196 228 3.3 10.7 6.3 16.3 9 17 2 .7 5 1 9 1h5c10.7 0 16.7
+-2 18-6 2-2.7 1-9.7-3-21-32-87.3-82.7-157.7-152-211l-3-3h399907v-40H7zm93 0
+v40h399900v-40zM0 241v40h399900v-40zm0 0v40h399900v-40z"></path></svg></span><span class="halfarrow-right" style="height:0.716em"><svg xmlns="http://www.w3.org/2000/svg" width="400em" height="0.716em" viewBox="0 0 400000 716" preserveAspectRatio="xMaxYMin slice"><path d="M0 241v40h399993c4.7-4.7 7-9.3 7-14 0-9.3-3.7-15.3-11
+-18-92.7-56.7-159-133.7-199-231-3.3-9.3-6-14.7-8-16-2-1.3-7-2-15-2-10.7 0-16.7
+ 2-18 6-2 2.7-1 9.7 3 21 15.3 42 36.7 81.8 64 119.5 27.3 37.7 58 69.2 92 94.5z
+m0 0v40h399900v-40z m100 194v40h399900v-40zm0 0v40h399900v-40z"></path></svg></span></span></span><span style="top:-2.0109em"><span class="pstrut" style="height:2.716em"></span><span class="sizing reset-size6 size3 mtight x-arrow-pad"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3173em"><span style="top:-2.357em;margin-left:-0.0315em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mtight">6</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.143em"><span></span></span></span></span></span></span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.8052em"><span></span></span></span></span></span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:0.8778em;vertical-align:-0.1944em"></span><span class="mord mathnormal">RFQ</span></span></span></span></td><td align="center">RCP-fluorophore complexes (<strong>RF</strong>) bind to free quenchers (<strong>Q_free</strong>) to form RCP-quencher-fluorophore complexes (<strong>RFQ</strong>)</td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mi>M</mi><mo>+</mo><mi>P</mi><mover><mo stretchy="true" minsize="3.0em">→</mo><mpadded width="+0.6em" lspace="0.3em"><mi>k</mi></mpadded></mover><mi>M</mi><mi>P</mi></mrow><annotation encoding="application/x-tex">M + P \xrightarrow{k} MP</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:0.7667em;vertical-align:-0.0833em"></span><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">+</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.1191em;vertical-align:-0.011em"></span><span class="mord mathnormal" style="margin-right:0.13889em">P</span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel x-arrow"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.1081em"><span style="top:-3.322em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight x-arrow-pad"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.03148em">k</span></span></span></span><span class="svg-align" style="top:-2.689em"><span class="pstrut" style="height:2.7em"></span><span class="hide-tail" style="height:0.522em;min-width:1.469em"><svg xmlns="http://www.w3.org/2000/svg" width="400em" height="0.522em" viewBox="0 0 400000 522" preserveAspectRatio="xMaxYMin slice"><path d="M0 241v40h399891c-47.3 35.3-84 78-110 128
+-16.7 32-27.7 63.7-33 95 0 1.3-.2 2.7-.5 4-.3 1.3-.5 2.3-.5 3 0 7.3 6.7 11 20
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+-40.5-119-75.5s-58.3-74.8-73-119.5c-4.7-14-8.3-27.3-11-40-1.3-6.7-3.2-10.8-5.5
+-12.5-2.3-1.7-7.5-2.5-15.5-2.5-14 0-21 3.7-21 11 0 2 2 10.3 6 25 20.7 83.3 67
+ 151.7 139 205zm0 0v40h399900v-40z"></path></svg></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.011em"><span></span></span></span></span></span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:0.6833em"></span><span class="mord mathnormal" style="margin-right:0.13889em">MP</span></span></span></span></td><td align="center">Input miRNA (<strong>M</strong>) binds to the complementary arms of free padlock probes (<strong>P</strong>) to form miRNA-padlock probe complexes (<strong>MP</strong>)</td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mi>M</mi><mi>P</mi><mo>+</mo><mi>S</mi><mi>p</mi><mi>l</mi><mi>R</mi><mo>⇌</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi><mi>i</mi><mi>z</mi><mi>e</mi><mi>d</mi></mrow></msub></mrow><annotation encoding="application/x-tex">MP + SplR \rightleftharpoons MP_{circularized}</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:0.7667em;vertical-align:-0.0833em"></span><span class="mord mathnormal" style="margin-right:0.13889em">MP</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">+</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:0.8889em;vertical-align:-0.1944em"></span><span class="mord mathnormal" style="margin-right:0.00773em">SplR</span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">⇌</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:0.8333em;vertical-align:-0.15em"></span><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">ze</span><span class="mord mathnormal mtight">d</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span></span></span></span></td><td align="center">miRNA-padlock probe complexes (<strong>MP</strong>) are ligated by SplintR ligase (<strong>SplR</strong>) to circularized complexes (<strong>MP_circularized</strong>)</td></tr></tbody></table>
+<h2 id="initial-values-of-species-1">Initial Values of Species</h2>
+<p>In order to simulate our reactions, our team needed to input values to initialize each component in our model. The initial value of each species was obtained from wet lab protocols:</p>
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+<table><thead><tr><th align="center">Variable</th><th align="center">Species</th><th align="center">Initial Value</th><th align="center">Units</th></tr></thead><tbody><tr><td align="center">M</td><td align="center">miRNA</td><td align="center">0.5</td><td align="center">picomoles</td></tr><tr><td align="center">M_1</td><td align="center">miRNA</td><td align="center">0</td><td align="center">picomoles</td></tr><tr><td align="center">M_2</td><td align="center">miRNA</td><td align="center">0</td><td align="center">picomoles</td></tr><tr><td align="center">P</td><td align="center">padlock probes</td><td align="center">0.05</td><td align="center">picomoles</td></tr><tr><td align="center">SplR</td><td align="center">SplintR Ligase</td><td align="center">25</td><td align="center">molecules</td></tr><tr><td align="center">phi29</td><td align="center">phi29 DNA polymerase</td><td align="center">12.5</td><td align="center">molecules</td></tr><tr><td align="center">Nb.BbvCI</td><td align="center">phi29 DNA polymerase</td><td align="center">12500</td><td align="center">molecules</td></tr><tr><td align="center">R_free</td><td align="center">free RCP</td><td align="center">0</td><td align="center">molecules</td></tr><tr><td align="center">F_free</td><td align="center">free fluorophores</td><td align="center">180664245</td><td align="center">molecules</td></tr><tr><td align="center">Q_free</td><td align="center">free quenchers</td><td align="center">361328490</td><td align="center">molecules</td></tr><tr><td align="center">RF</td><td align="center">bound RCP-fluorophores</td><td align="center">0</td><td align="center">picomoles</td></tr><tr><td align="center">RQ</td><td align="center">bound RCP-quenchers</td><td align="center">0</td><td align="center">picomoles</td></tr><tr><td align="center">RFQ</td><td align="center">bound RCP-fluorophores-quenchers</td><td align="center">0</td><td align="center">picomoles</td></tr></tbody></table>
+<h2 id="parameters-1">Parameters</h2>
+<p>Rate constants and degradation rates are required to input into our Mass Action Laws and Michaelis-Menten equations. These values were derived from literature or estimated if the values could not be found. The value of each parameter is shown below:</p>
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+<table><thead><tr><th align="center">Variable</th><th align="center">Reaction</th><th align="center">Estimated Values</th><th align="center">Units</th></tr></thead><tbody><tr><td align="center">Mdeg</td><td align="center">rate constant for miRNA degradation</td><td align="center">0.000019254</td><td align="center">1/second</td></tr><tr><td align="center">M_1deg</td><td align="center">rate constant for miRNA degradation</td><td align="center">0.000019254</td><td align="center">1/second</td></tr><tr><td align="center">M_2deg</td><td align="center">rate constant for miRNA degradation</td><td align="center">0.000019254</td><td align="center">1/second</td></tr><tr><td align="center">k_padlock</td><td align="center">rate constant for miRNA-padlock binding</td><td align="center">32000</td><td align="center">1/(moles*second)</td></tr><tr><td align="center">Vmcircular</td><td align="center">maximal rate of reaction needed for Michaelis-Menten enzyme kinetics for MP-SplintR binding</td><td align="center">0.008</td><td align="center">moles/second</td></tr><tr><td align="center">Kmcircular</td><td align="center">Michaelis-Menten constant for MP-SplintR binding</td><td align="center">1</td><td align="center">moles</td></tr><tr><td align="center">Vmphi29</td><td align="center">maximal rate of reaction needed for Michaelis-Menten enzyme kinetics for MP_circular-phi29xt binding</td><td align="center">824</td><td align="center">moles/second</td></tr><tr><td align="center">Kmphi29</td><td align="center">Michaelis-Menten constant for MP_circular-phi29 binding</td><td align="center">31</td><td align="center">moles</td></tr><tr><td align="center">VmNb.BbvCI</td><td align="center">maximal rate of reaction needed for Michaelis-Menten enzyme kinetics for MP_circular-Nb.BbvCI binding</td><td align="center">112</td><td align="center">moles/second</td></tr><tr><td align="center">RCPdeg</td><td align="center">rate constant for free RCP degradation</td><td align="center">0</td><td align="center">1/second</td></tr><tr><td align="center">k1</td><td align="center">forward reaction rate constant for free RCP and free fluorophore binding</td><td align="center">2.2e12</td><td align="center">1/(moles*second)</td></tr><tr><td align="center">k2</td><td align="center">backward reaction rate constant for free RCP and free fluorophore binding</td><td align="center">12.4326576778</td><td align="center">1/second</td></tr><tr><td align="center">k3</td><td align="center">forward reaction rate constant for free RCP and free quencher binding</td><td align="center">2.2e12</td><td align="center">1/(moles*second)</td></tr><tr><td align="center">k4</td><td align="center">backward reaction rate constant for free RCP and free quencher binding</td><td align="center">12.4326576778</td><td align="center">1/second</td></tr><tr><td align="center">k5</td><td align="center">forward reaction rate constant for bound RF and free quencher binding</td><td align="center">1.34e12</td><td align="center">1/(moles*second)</td></tr><tr><td align="center">k6</td><td align="center">backward reaction rate constant for bound RF and free quencher binding</td><td align="center">7.57261876741</td><td align="center">1/second</td></tr><tr><td align="center">k7</td><td align="center">forward reaction rate constant for bound RQ and free fluorophore binding</td><td align="center">1e12</td><td align="center">1/(moles*second)</td></tr><tr><td align="center">k8</td><td align="center">backward reaction rate constant for bound RQ and free fluorophore binding</td><td align="center">5.65120803538</td><td align="center">1/second</td></tr></tbody></table>
+<h2 id="ordinary-differential-equations-1">Ordinary Differential Equations</h2>
+<p>As mentioned previously, Lambert iGEM incorporated the Mass Action Kinetic Laws and Michaelis-Menten
+Equations to represent the reactions for the eRCA model.</p>
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+<table><thead><tr><th align="center">Ordinary Differential Equations</th></tr></thead><tbody><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>M</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mrow><mi>p</mi><mi>a</mi><mi>d</mi><mi>l</mi><mi>o</mi><mi>c</mi><mi>k</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>P</mi><mo stretchy="false">]</mo><mo>−</mo><msub><mi>M</mi><mrow><mi>d</mi><mi>e</mi><mi>g</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo></mrow><annotation encoding="application/x-tex">\frac{d[M]}{dt} = -(k_{padlock}[M][P] - M_{deg}[M])</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord">−</span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">oc</span><span class="mord mathnormal mtight" style="margin-right:0.03148em">k</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">P</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.109em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">e</span><span class="mord mathnormal mtight" style="margin-right:0.03588em">g</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="mclose">])</span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>P</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><msub><mi>k</mi><mrow><mi>p</mi><mi>a</mi><mi>d</mi><mi>l</mi><mi>o</mi><mi>c</mi><mi>k</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>P</mi><mo stretchy="false">]</mo></mrow><annotation encoding="application/x-tex">\frac{d[P]}{dt} = -k_{padlock}[M][P]</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord">−</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">oc</span><span class="mord mathnormal mtight" style="margin-right:0.03148em">k</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">P</span><span class="mclose">]</span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>S</mi><mi>p</mi><mi>l</mi><mi>R</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mfrac><mrow><msub><mi>V</mi><mrow><mi>m</mi><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><mi>P</mi><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mrow><mi>m</mi><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo>+</mo><mo stretchy="false">[</mo><mi>M</mi><mi>P</mi><mo stretchy="false">]</mo></mrow></mfrac></mrow><annotation encoding="application/x-tex">\frac{d[SplR]}{dt} = -\frac{V_{mcircular}[MP]}{K_{mcircular} + [MP]}</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.00773em">SplR</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.53em;vertical-align:-0.52em"></span><span class="mord">−</span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.07153em">K</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0715em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mbin mtight">+</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">MP</span><span class="mclose mtight">]</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.22222em">V</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.2222em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">MP</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.52em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>M</mi><mi>P</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><msub><mi>k</mi><mrow><mi>p</mi><mi>a</mi><mi>d</mi><mi>l</mi><mi>o</mi><mi>c</mi><mi>k</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>P</mi><mo stretchy="false">]</mo><mo>−</mo><mfrac><mrow><msub><mi>V</mi><mrow><mi>m</mi><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><mi>P</mi><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mrow><mi>m</mi><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo>+</mo><mo stretchy="false">[</mo><mi>M</mi><mi>P</mi><mo stretchy="false">]</mo></mrow></mfrac></mrow><annotation encoding="application/x-tex">\frac{d[MP]}{dt} = k_{padlock}[M][P] - \frac{V_{mcircular}[MP]}{K_{mcircular} + [MP]}</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">MP</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">oc</span><span class="mord mathnormal mtight" style="margin-right:0.03148em">k</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">P</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.53em;vertical-align:-0.52em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.07153em">K</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0715em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mbin mtight">+</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">MP</span><span class="mclose mtight">]</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.22222em">V</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.2222em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">MP</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.52em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mfrac><mrow><msub><mi>V</mi><mrow><mi>m</mi><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><mi>P</mi><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mrow><mi>m</mi><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo>+</mo><mo stretchy="false">[</mo><mi>M</mi><mi>P</mi><mo stretchy="false">]</mo></mrow></mfrac><mo>−</mo><mfrac><mrow><msub><mi>V</mi><mrow><mi>m</mi><mi>p</mi><mi>h</mi><mi>i</mi><mn>29</mn></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mrow><mi>m</mi><mi>p</mi><mi>h</mi><mi>i</mi><mn>29</mn></mrow></msub><mo>+</mo><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow></mfrac></mrow><annotation encoding="application/x-tex">\frac{d[MP_{circular}]}{dt} = \frac{V_{mcircular}[MP]}{K_{mcircular} + [MP]} - \frac{V_{mphi29}[MP_{circular}]}{K_{mphi29} + [MP_{circular}]}</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.53em;vertical-align:-0.52em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.07153em">K</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0715em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mbin mtight">+</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">MP</span><span class="mclose mtight">]</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.22222em">V</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.2222em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">MP</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.52em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.5862em;vertical-align:-0.5481em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.0381em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.07153em">K</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0715em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">hi</span><span class="mord mtight">29</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2901em"><span></span></span></span></span></span></span><span class="mbin mtight">+</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.5131em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.22222em">V</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.2222em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">hi</span><span class="mord mtight">29</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2901em"><span></span></span></span></span></span></span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.5481em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>p</mi><mi>h</mi><mi>i</mi><mn>29</mn><mi>x</mi><mi>t</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mfrac><mrow><msub><mi>V</mi><mrow><mi>m</mi><mi>p</mi><mi>h</mi><mi>i</mi><mn>2</mn><msub><mn>9</mn><mi>x</mi></msub><mi>t</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mrow><mi>m</mi><mi>p</mi><mi>h</mi><mi>i</mi><mn>2</mn><msub><mn>9</mn><mi>x</mi></msub><mi>t</mi></mrow></msub><mo>+</mo><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow></mfrac></mrow><annotation encoding="application/x-tex">\frac{d[phi29xt]}{dt} = - \frac{V_{mphi29_xt}[MP_{circular}]}{K_{mphi29_xt} + [MP_{circular}]}</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">hi</span><span class="mord mtight">29</span><span class="mord mathnormal mtight">x</span><span class="mord mathnormal mtight">t</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.5917em;vertical-align:-0.5509em"></span><span class="mord">−</span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.0409em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.07153em">K</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0715em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">hi</span><span class="mord mtight">2</span><span class="mord mtight"><span class="mord mtight">9</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.2306em"><span style="top:-2.3em;margin-left:0em;margin-right:0.1em"><span class="pstrut" style="height:2.5em"></span><span class="mord mathnormal mtight">x</span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2em"><span></span></span></span></span></span></span><span class="mord mathnormal mtight">t</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2941em"><span></span></span></span></span></span></span><span class="mbin mtight">+</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.5159em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.22222em">V</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.2222em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">hi</span><span class="mord mtight">2</span><span class="mord mtight"><span class="mord mtight">9</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.2306em"><span style="top:-2.3em;margin-left:0em;margin-right:0.1em"><span class="pstrut" style="height:2.5em"></span><span class="mord mathnormal mtight">x</span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2em"><span></span></span></span></span></span></span><span class="mord mathnormal mtight">t</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2941em"><span></span></span></span></span></span></span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.5509em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>N</mi><mi>b</mi><mi mathvariant="normal">.</mi><mi>B</mi><mi>b</mi><mi>v</mi><mi>C</mi><mi>I</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mfrac><mrow><msub><mi>V</mi><mrow><mi>m</mi><mi>N</mi><mi>b</mi><mi mathvariant="normal">.</mi><mi>B</mi><mi>b</mi><mi>v</mi><mi>C</mi><mi>I</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mrow><mi>m</mi><mi>N</mi><mi>b</mi><mi mathvariant="normal">.</mi><mi>B</mi><mi>b</mi><mi>v</mi><mi>C</mi><mi>I</mi></mrow></msub><mo>+</mo><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow></mfrac></mrow><annotation encoding="application/x-tex">\frac{d[Nb.BbvCI]}{dt} = - \frac{V_{mNb.BbvCI}[MP_{circular}]}{K_{mNb.BbvCI} + [MP_{circular}]}</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">N</span><span class="mord mathnormal mtight">b</span><span class="mord mtight">.</span><span class="mord mathnormal mtight" style="margin-right:0.05017em">B</span><span class="mord mathnormal mtight">b</span><span class="mord mathnormal mtight" style="margin-right:0.03588em">v</span><span class="mord mathnormal mtight" style="margin-right:0.07153em">C</span><span class="mord mathnormal mtight" style="margin-right:0.07847em">I</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.53em;vertical-align:-0.52em"></span><span class="mord">−</span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.07153em">K</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0715em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">N</span><span class="mord mathnormal mtight">b</span><span class="mord mtight">.</span><span class="mord mathnormal mtight" style="margin-right:0.05017em">B</span><span class="mord mathnormal mtight">b</span><span class="mord mathnormal mtight" style="margin-right:0.03588em">v</span><span class="mord mathnormal mtight" style="margin-right:0.07153em">C</span><span class="mord mathnormal mtight" style="margin-right:0.07847em">I</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mbin mtight">+</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.22222em">V</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.2222em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">N</span><span class="mord mathnormal mtight">b</span><span class="mord mtight">.</span><span class="mord mathnormal mtight" style="margin-right:0.05017em">B</span><span class="mord mathnormal mtight">b</span><span class="mord mathnormal mtight" style="margin-right:0.03588em">v</span><span class="mord mathnormal mtight" style="margin-right:0.07153em">C</span><span class="mord mathnormal mtight" style="margin-right:0.07847em">I</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.52em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mfrac><mrow><msub><mi>V</mi><mrow><mi>m</mi><mi>p</mi><mi>h</mi><mi>i</mi><mn>2</mn><msub><mn>9</mn><mi>x</mi></msub><mi>t</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mrow><mi>m</mi><mi>p</mi><mi>h</mi><mi>i</mi><mn>2</mn><msub><mn>9</mn><mi>x</mi></msub><mi>t</mi></mrow></msub><mo>+</mo><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow></mfrac><mo>−</mo><mo stretchy="false">(</mo><mi>R</mi><mi>C</mi><msub><mi>P</mi><mrow><mi>d</mi><mi>e</mi><mi>g</mi></mrow></msub><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">)</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>1</mn></msub><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><msub><mi>F</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>2</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>3</mn></msub><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>4</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo></mrow><annotation encoding="application/x-tex">\frac{d[R_{free}]}{dt} = \frac{V_{mphi29_xt}[MP_{circular}]}{K_{mphi29_xt} + [MP_{circular}]} -(RCP_{deg}[R_{free}]) - (k_1[R_{free}][F_{free}] - k_2[RF]) - (k_3[R_{free}][Q_{free}] - k_4[RQ])</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.3831em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.0381em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.5131em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0077em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2901em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.5917em;vertical-align:-0.5509em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.0409em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.07153em">K</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0715em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">hi</span><span class="mord mtight">2</span><span class="mord mtight"><span class="mord mtight">9</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.2306em"><span style="top:-2.3em;margin-left:0em;margin-right:0.1em"><span class="pstrut" style="height:2.5em"></span><span class="mord mathnormal mtight">x</span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2em"><span></span></span></span></span></span></span><span class="mord mathnormal mtight">t</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2941em"><span></span></span></span></span></span></span><span class="mbin mtight">+</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.5159em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.22222em">V</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.2222em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight">p</span><span class="mord mathnormal mtight">hi</span><span class="mord mtight">2</span><span class="mord mtight"><span class="mord mtight">9</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.2306em"><span style="top:-2.3em;margin-left:0em;margin-right:0.1em"><span class="pstrut" style="height:2.5em"></span><span class="mord mathnormal mtight">x</span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2em"><span></span></span></span></span></span></span><span class="mord mathnormal mtight">t</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2941em"><span></span></span></span></span></span></span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.5509em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord mathnormal" style="margin-right:0.07153em">RC</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">e</span><span class="mord mathnormal mtight" style="margin-right:0.03588em">g</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0077em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">])</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">1</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0077em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">F</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">2</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">RF</span><span class="mclose">])</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">3</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0077em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:0em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">4</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RQ</span><span class="mclose">])</span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><msub><mi>M</mi><mn>1</mn></msub><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mfrac><mrow><msub><mi>V</mi><mrow><msub><mi>m</mi><mi>x</mi></msub><mi>t</mi></mrow></msub><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><msub><mi>K</mi><mrow><mi>m</mi><mi>N</mi><mi>b</mi><mi mathvariant="normal">.</mi><mi>B</mi><mi>b</mi><mi>v</mi><mi>C</mi><mi>I</mi></mrow></msub><mo>+</mo><mo stretchy="false">[</mo><mi>M</mi><msub><mi>P</mi><mrow><mi>c</mi><mi>i</mi><mi>r</mi><mi>c</mi><mi>u</mi><mi>l</mi><mi>a</mi><mi>r</mi></mrow></msub><mo stretchy="false">]</mo></mrow></mfrac><mo>−</mo><mo stretchy="false">(</mo><msub><mi>M</mi><mrow><mn>1</mn><mi>d</mi><mi>e</mi><mi>g</mi></mrow></msub><mo stretchy="false">[</mo><msub><mi>M</mi><mn>1</mn></msub><mo stretchy="false">]</mo><mo stretchy="false">)</mo></mrow><annotation encoding="application/x-tex">\frac{d[M_{1}]}{dt} = \frac{V_{m_xt}[MP_{circular}]}{K_{mNb.BbvCI} + [MP_{circular}]} -(M_{1deg}[M_{1}])</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3173em"><span style="top:-2.357em;margin-left:-0.109em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mtight">1</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.143em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.5551em;vertical-align:-0.52em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.0351em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.07153em">K</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.0715em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">N</span><span class="mord mathnormal mtight">b</span><span class="mord mtight">.</span><span class="mord mathnormal mtight" style="margin-right:0.05017em">B</span><span class="mord mathnormal mtight">b</span><span class="mord mathnormal mtight" style="margin-right:0.03588em">v</span><span class="mord mathnormal mtight" style="margin-right:0.07153em">C</span><span class="mord mathnormal mtight" style="margin-right:0.07847em">I</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mbin mtight">+</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.5101em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.22222em">V</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.2963em"><span style="top:-2.357em;margin-left:-0.2222em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mtight"><span class="mord mathnormal mtight">m</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.2306em"><span style="top:-2.3em;margin-left:0em;margin-right:0.1em"><span class="pstrut" style="height:2.5em"></span><span class="mord mathnormal mtight">x</span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2em"><span></span></span></span></span></span></span><span class="mord mathnormal mtight">t</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2859em"><span></span></span></span></span></span></span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.10903em">M</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">P</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">c</span><span class="mord mathnormal mtight">i</span><span class="mord mathnormal mtight">rc</span><span class="mord mathnormal mtight">u</span><span class="mord mathnormal mtight" style="margin-right:0.01968em">l</span><span class="mord mathnormal mtight">a</span><span class="mord mathnormal mtight" style="margin-right:0.02778em">r</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.1512em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.52em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.109em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight">1</span><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">e</span><span class="mord mathnormal mtight" style="margin-right:0.03588em">g</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.10903em">M</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.109em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight">1</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mclose">])</span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><msub><mi>F</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>1</mn></msub><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><msub><mi>F</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>2</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>7</mn></msub><mo stretchy="false">[</mo><msub><mi>F</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>R</mi><mi>Q</mi><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>8</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo></mrow><annotation encoding="application/x-tex">\frac{d[F_{free}]}{dt} = -(k_1[R_{free}][F_{free}] - k_2[RF]) - (k_7[F_{free}][RQ] - k_8[RFQ])</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.3831em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.0381em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.5131em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.13889em">F</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:-0.1389em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2901em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord">−</span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">1</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0077em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">F</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">2</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">RF</span><span class="mclose">])</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">7</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">F</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal">RQ</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">8</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RFQ</span><span class="mclose">])</span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>3</mn></msub><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>2</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>5</mn></msub><mo stretchy="false">[</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>6</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo></mrow><annotation encoding="application/x-tex">\frac{d[Q_{free}]}{dt} = -(k_3[R_{free}][Q_{free}] - k_2[RQ]) - (k_5[Q_{free}][RF] - k_6[RFQ])</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.3831em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.0381em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.5131em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mtight"><span class="mord mathnormal mtight">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3448em"><span style="top:-2.3488em;margin-left:0em;margin-right:0.0714em"><span class="pstrut" style="height:2.5em"></span><span class="sizing reset-size3 size1 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2901em"><span></span></span></span></span></span></span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord">−</span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">3</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0077em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:0em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">2</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RQ</span><span class="mclose">])</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">5</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:0em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">RF</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">6</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RFQ</span><span class="mclose">])</span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>1</mn></msub><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><msub><mi>F</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>2</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>5</mn></msub><mo stretchy="false">[</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>6</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo></mrow><annotation encoding="application/x-tex">\frac{d[RF]}{dt} = -(k_1[R_{free}][F_{free}] - k_2[RF]) - (k_5[Q_{free}][RF] - k_6[RFQ])</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight" style="margin-right:0.13889em">RF</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord">−</span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">1</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0077em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">F</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">2</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">RF</span><span class="mclose">])</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">5</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:0em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">RF</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">6</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RFQ</span><span class="mclose">])</span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>R</mi><mi>Q</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>3</mn></msub><mo stretchy="false">[</mo><msub><mi>R</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>4</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>7</mn></msub><mo stretchy="false">[</mo><msub><mi>F</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>R</mi><mi>Q</mi><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>8</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo></mrow><annotation encoding="application/x-tex">\frac{d[RQ]}{dt} = -(k_3[R_{free}][Q_{free}] - k_4[RQ]) - (k_7[F_{free}][RQ] - k_8[RFQ])</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight">RQ</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord">−</span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">3</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.00773em">R</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.0077em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:0em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">4</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RQ</span><span class="mclose">])</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">7</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">F</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal">RQ</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">8</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RFQ</span><span class="mclose">])</span></span></span></span></td></tr><tr><td align="center"><span class="katex"><span class="katex-mathml"><math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mrow><mfrac><mrow><mi>d</mi><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mi>Q</mi><mo stretchy="false">]</mo></mrow><mrow><mi>d</mi><mi>t</mi></mrow></mfrac><mo>=</mo><mo>−</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>7</mn></msub><mo stretchy="false">[</mo><msub><mi>F</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>R</mi><mi>Q</mi><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>8</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo><mo>+</mo><mo stretchy="false">(</mo><msub><mi>k</mi><mn>5</mn></msub><mo stretchy="false">[</mo><msub><mi>Q</mi><mrow><mi>f</mi><mi>r</mi><mi>e</mi><mi>e</mi></mrow></msub><mo stretchy="false">]</mo><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mo stretchy="false">]</mo><mo>−</mo><msub><mi>k</mi><mn>6</mn></msub><mo stretchy="false">[</mo><mi>R</mi><mi>F</mi><mi>Q</mi><mo stretchy="false">]</mo><mo stretchy="false">)</mo></mrow><annotation encoding="application/x-tex">\frac{d[RFQ]}{dt} = -(k_7[F_{free}][RQ] - k_8[RFQ]) + (k_5[Q_{free}][RF] - k_6[RFQ])</annotation></semantics></math></span><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.355em;vertical-align:-0.345em"></span><span class="mord"><span class="mopen nulldelimiter"></span><span class="mfrac"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:1.01em"><span style="top:-2.655em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mord mathnormal mtight">t</span></span></span></span><span style="top:-3.23em"><span class="pstrut" style="height:3em"></span><span class="frac-line" style="border-bottom-width:0.04em"></span></span><span style="top:-3.485em"><span class="pstrut" style="height:3em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight">d</span><span class="mopen mtight">[</span><span class="mord mathnormal mtight">RFQ</span><span class="mclose mtight">]</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.345em"><span></span></span></span></span></span><span class="mclose nulldelimiter"></span></span><span class="mspace" style="margin-right:0.2778em"></span><span class="mrel">=</span><span class="mspace" style="margin-right:0.2778em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mord">−</span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">7</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.13889em">F</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:-0.1389em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal">RQ</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">8</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RFQ</span><span class="mclose">])</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">+</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1.0361em;vertical-align:-0.2861em"></span><span class="mopen">(</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">5</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord"><span class="mord mathnormal">Q</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3361em"><span style="top:-2.55em;margin-left:0em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mathnormal mtight" style="margin-right:0.10764em">f</span><span class="mord mathnormal mtight">ree</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2861em"><span></span></span></span></span></span></span><span class="mclose">]</span><span class="mopen">[</span><span class="mord mathnormal" style="margin-right:0.13889em">RF</span><span class="mclose">]</span><span class="mspace" style="margin-right:0.2222em"></span><span class="mbin">−</span><span class="mspace" style="margin-right:0.2222em"></span></span><span class="base"><span class="strut" style="height:1em;vertical-align:-0.25em"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.03148em">k</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em"><span style="top:-2.55em;margin-left:-0.0315em;margin-right:0.05em"><span class="pstrut" style="height:2.7em"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight">6</span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em"><span></span></span></span></span></span></span><span class="mopen">[</span><span class="mord mathnormal">RFQ</span><span class="mclose">])</span></span></span></span></td></tr></tbody></table>
+<h2 id="results-1">Results</h2>
+<p>Our eRCA model provides an accurate mathematical representation of the reaction. According to the model, eRCA produces an exponentially larger decrease in fluorescence than RCA. The trend of decreasing fluorescence is much clearer in the eRCA model than in the RCA model. This makes it more reliable to derive accurate miRNA levels from the RFU values obtained from the eRCA model (See Fig. 6).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/model-fig6.png
+" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 6. Relationship between miRNA concentrations ranging from 0 to 0.49 picomoles and RFU based on simultations completed with the eRCA Model</div></div>
+<p>The model simulation has produced a graph that clearly illustrates an exponential relationship between RFU and miRNA concentration (see Fig. 6). This outcome serves to corroborate the notion that eRCA, which is based on the premise of an exponential increase in RCP concentration, can significantly enhance the accuracy of wetlab experiments (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: eRCA</a>). The observed exponential relationship further reinforces the concept that as RCP concentration increases exponentially, the margin of error in miRNA concentration detection decreases. Notably, the simulation results for miRNA concentrations ranging from 0 to 0.49 picomolar above provide compelling evidence of this decrease in error (see Fig. 6). In this range, the points on the graph are distinctly separated, leading to a remarkable reduction in measurement error indicating the effect of the model on wetlab accuracy.</p>
+<h2 id="assumptions">Assumptions</h2>
+<p>Our RCA and eRCA ODE models assume all rate constants remain constant due to a lack of external environmental factors. Thus, this model replicates only the miRNA-padlock probe binding process to simulate RCP production and RQF. The model also assumes the total quantity of padlock probes, SplintR ligase, phi29 DNA polymerase, fluorophores, and quenchers is known initially and remains constant over time. Finally, DNA polymerase activity is expected to remain the same regardless of the primer, miRNA, or dNTP concentration.</p>
+<h1 id="capillary-tube-rca-models">Capillary Tube RCA Models</h1>
+<h2 id="capillary-rca-overview">Capillary RCA Overview:</h2>
+<p>Lambert iGEM designed capillary rolling circle amplification (cpRCA), a frugal, capillary tube-based diagnostic for coronary artery disease (CAD). cpRCA works as an extension of exponential rolling circle amplification (eRCA) or rolling circle amplification (RCA) to serve as a time-effective diagnostic tool.</p>
+<p>To corroborate the results from cpRCA, we created an updated RCA model that utilized the SYBRsafe-based reporting mechanism. The model used the biochemical equations from the RCA model while modifying specific parameters to fit the capillary tube setting, enabling us to map the initial miRNA concentration to the final amount of SYBRsafe fluorescence (See Fig. 7).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/model-fig7.png
+" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 7. Diagram of the reactions of the cpRCA pathway in MATLAB Simbiology software</div></div>
+<h2 id="results-2">Results</h2>
+<p>The capillary model offers a precise simulation of the cpRCA pathway. When initialized with a 0.00166 picomoles of miRNA—a value determined through our wet lab experiments—the model accurately predicted a count of approximately 47 molecules for the R_Sybr complex, which represents the RCP-SYBR™ Safe interaction (See Fig. 8). This outcome substantiates the validity of our cpRCA protocols, as the simulated count of 47 molecules closely aligns with the expected value of around 50. Not only will this count likely reduce overlap that can negatively affect the miRNA count, but has been validated with our wetlab results, having a cpRCA reaction work at 50 molecules of miRNA (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: outputs</a>).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/model-fig8.png
+" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 8. Simulation results of RCP-SYBRâ„¢ Safe complexes over 4 hours</div></div>
+<h2 id="dna-and-mirna-diffusion">DNA and miRNA Diffusion</h2>
+<p>To validate the functionality of cpRCA, we created a model to examine the diffusion of microRNA (miRNA) and rolling circle product (RCP) within the capillary tube which is crucial for the imaging process and formation of fluorescent dots indicating miRNA concentration. This model enabled us to simulate and analyze the diffusion and concentration of miRNA and RCP under different experimental conditions, providing insights into the correlation between diffusion patterns and fluorescent dot formation. Additionally, the model utilized various parameters and optimized the accuracy of the simulation by studying tube dimensions, reaction times, and the impact of miRNA/RCP concentrations on diffusion and fluorescent signals.</p>
+<h2 id="development-and-results">Development and Results</h2>
+<p>In order to simulate the diffusion of the miRNA and RCP within the capillary tube we utilized a random walk model in conjunction with the Stokes-Einstein law of diffusion.</p>
+<p>As illustrated in Figure 9, these computational simulations provide insights into the dynamic behavior of miRNA and RCP within the capillary tube. The results confirm that significant diffusion of RCP is unlikely, and there is minimal overlap between the RCP and miRNA species. This validates the imaging step in the cpRCA pathway that relies on fluorescent dots for accurate detection and quantification of miRNA.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/dnadiff.png
+" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 9. 20 minute simulation of miRNA and DNA positions inside a capillary tube</div></div>
+<h2 id="limitations">Limitations</h2>
+<p>The model uses the input parameters to provide a simulation of the diffusion tailored towards cpRCA. However, the model has many limitations and may not be an accurate representation of the diffusion of the species. The Stokes-Einstein equation’s assumptions pose limitations when applied to DNA and miRNA diffusion in a capillary tube. Specifically, DNA and miRNA are not spherical and can interact with other particles, violating the equation’s premises. Additionally, the capillary’s confined geometry can lead to wall interactions affecting diffusion, and biological fluids like blood are often non-Newtonian, altering viscosity dynamically. Consequently, while our results provide initial insights, they should be considered approximations that require refinements to account for these biological complexities.</p>
+<h3 id="references">References</h3>
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+<div class="pl-6 -indent-6"><p>Park, C. (2022). Visual interpretation of the meaning of kcat/km in enzyme
+kinetics. Journal of Chemical Education, 99(7), 2556–2562.
+<a href="https://doi.org/10.1021/acs.jchemed.1c01268">https://doi.org/10.1021/acs.jchemed.1c01268</a></p></div>
+<div class="pl-6 -indent-6"><p>Zi, Z. (2012). A tutorial on mathematical modeling of Biological Signaling
+Pathways. Methods in Molecular Biology, 41–51.
+<a href="https://doi.org/10.1007/978-1-61779-833-7_3">https://doi.org/10.1007/978-1-61779-833-7_3</a></p></div>
+<div class="pl-6 -indent-6"><p>Shen, B. W., Doyle, L., Bradley, P., Heiter, D. F., Lunnen, K. D., Wilson, G.
+G., & Stoddard, B. L. (2019, January 10). Structure, subunit organization and
+behavior of the asymmetric type IIT restriction endonuclease bbvci. Nucleic
+acids research. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326814/">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326814/</a></p></div>
+<div class="pl-6 -indent-6"><p>Allen, M. P., Debye, P., Einstein, A., Hanley, H. J. M., Hoheisel, C., Ishii,
+Y., Mazo, R. M., & Mazza, M. G. (2020, July 11). Molecular size and shape
+effects: Rotational diffusion and the stokes-einstein-debye relation. Journal
+of Molecular Liquids.
+<a href="https://www.sciencedirect.com/science/article/abs/pii/S0167732220316937">https://www.sciencedirect.com/science/article/abs/pii/S0167732220316937</a></p></div>
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+ <h1 id="rca-lab-notebook">RCA Lab Notebook</h1>
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+<h1 id="phi29-dna-polymerase-protein-purification-lab-notebook">phi29 DNA Polymerase Protein Purification Lab Notebook</h1>
+<div class="w-full grow flex my-4"><iframe src="https://static.igem.wiki/teams/4683/wiki/notebooks/phi29-dna-polymerase-protein-purification-lab-notebook-compressed.pdf" height="1000px" width="120%"></iframe></div>
+<h1 id="capillary-rca-lab-notebook">Capillary RCA Lab Notebook</h1>
+<div class="w-full grow flex my-4"><iframe src="https://static.igem.wiki/teams/4683/wiki/notebooks/capillary-rca-lab-notebook-compressed.pdf" height="1000px" width="120%"></iframe></div>
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style="transform:scaleX(0) translateZ(0)"></div></div><div class="w-full flex justify-center items-center"><div class="flex justify-between m-5 z-50 max-w-6xl grow"><a href="https://2023.igem.wiki/lambert-ga/" class="font-serif z-30 text-white "><p class="bg-blackA-11 text-xl rounded-full p-2">Lambert-GA</p></a><div class="flex justify-end z-30"><nav aria-label="Main" data-orientation="horizontal" dir="ltr" class="relative "><div style="position:relative"><ul data-orientation="horizontal" class="flex flex-row rounded-full bg-blackA-11 p-2 space-x-2" dir="ltr"><div><li><button id="radix-:r1Rk:-trigger-radix-:r1Rbk:" data-state="closed" aria-expanded="false" aria-controls="radix-:r1Rk:-content-radix-:r1Rbk:" class="px-4 py-2 rounded-full hover:bg-whiteA-6 font-medium text-gray-3 focus:outline-none focus-visible:ring focus-visible:ring-purple-500 focus-visible:ring-opacity-75" data-radix-collection-item="">Team</button></li></div><div><li><button id="radix-:r1Rk:-trigger-radix-:r1Rjk:" 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+mt-[50vh] break-words grow sm:mt-[44vh] md:mt-[40vh]"><astro-slot>
+ <h1 id="parts-library">Parts Library</h1>
+<p>Lambert iGEM 2023 worked with 44 parts in total, contributing 28 to the registry this year.</p>
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+<table><thead><tr><th>Workstream</th><th>Biobrick number</th><th>Part Name</th><th>Designer</th><th>Description</th><th>Status</th><th>Length (bp)</th></tr></thead><tbody><tr><td>Exponential Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683000"></a><a href="http://parts.igem.org/Part:BBa_K4683000">BBa_K4683000</a></td><td>Lettuce Complement</td><td>Natalie Tan, Manasvi Gupta, Vandana Vijay Kumar, Kate Sharer</td><td>Basic, DNA</td><td>New, Utilized</td><td>99</td></tr><tr><td>Exponential Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_M31961">BBa_M31961</a></td><td>BbvCI restriction enzyme site</td><td>Rebecca Adams</td><td>Basic, DNA</td><td>Existing, Utilized</td><td>7</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683003">BBa_K4683</a><a href="http://parts.igem.org/Part:BBa_K4683003">00</a>3</td><td>hsa-miR-1-3p 1SNV</td><td>Natalie Tan, Manasvi Gupta, Vandana Vijay Kumar, Kate Sharer</td><td>Basic, DNA</td><td>New, Utilized</td><td>22</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683004">BBa_K4683004</a></td><td>hsa-miR-1-3p 3 SNV</td><td>Natalie Tan, Manasvi Gupta, Vandana Vijay Kumar, Kate Sharer</td><td>Basic, DNA</td><td>New, Utilized</td><td>22</td></tr><tr><td>Exponential Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683002">BBa_K4683002</a></td><td>hsa-miR-1-3p eRCA Padlock Probe</td><td>Natalie Tan, Manasvi Gupta, Vandana Vijay Kumar, Kate Sharer</td><td>Composite</td><td>New, Utilized</td><td>135</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683005">BBa_K4683005</a></td><td>hsa-miR-20b</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, RNA</td><td>New, Utilized</td><td>18</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683006">BBa_K4683006</a></td><td>3’ arm for hsa-miR-20b</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, DNA</td><td>New, Utilized</td><td>8</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683007">BBa_K4683007</a></td><td>5’ arm for hsa-miR-20b</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, DNA</td><td>New, Utilized</td><td>10</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683008">BBa_K4683008</a></td><td>hsa-miR-20b RCA Padlock Probe</td><td>Sahana Narayanan, Kate Sharer</td><td>Composite</td><td>New, Utilized</td><td>43</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683009">BBa_K4683009</a></td><td>hsa-mir-150-5p</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, RNA</td><td>New, Utilized</td><td>22</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683010">BBa_K4683010</a></td><td>3’ arm for hsa-mir-150-5p</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, DNA</td><td>New, Utilized</td><td>6</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683011">BBa_K4683011</a></td><td>5’ arm for hsa-mir-150-5p</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, DNA</td><td>New, Utilized</td><td>6</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683012">BBa_K4683012</a></td><td>hsa-mir-150-5p RCA Padlock Probe</td><td>Sahana Narayanan, Kate Sharer</td><td>Composite</td><td>New, Utilized</td><td>53</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683013">BBa_K4683013</a></td><td>hsa-mir-122-5p</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, RNA</td><td>New, Utilized</td><td>20</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683014">BBa_K4683014</a></td><td>3’ arm for hsa-mir-122-5p</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, DNA</td><td>New, Utilized</td><td>11</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683015">BBa_K4683015</a></td><td>5’ arm for hsa-mir-122-5p</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, DNA</td><td>New, Utilized</td><td>9</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683016">BBa_K4683016</a></td><td>hsa-mir-122-5p RCA Padlock Probe</td><td>Sahana Narayanan, Kate Sharer</td><td>Composite</td><td>New, Utilized</td><td>45</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683017">BBa_K4683017</a></td><td>hsa-mir-30c-5p</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, RNA</td><td>New, Utilized</td><td>18</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683018">BBa_K4683018</a></td><td>3’ arm for hsa-mir-30c-5p</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, DNA</td><td>New, Utilized</td><td>6</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683019">BBa_K4683019</a></td><td>5’ arm for hsa-mir-30c-5p</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, DNA</td><td>New, Utilized</td><td>10</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683020">BBa_K4683020</a></td><td>hsa-mir-30c-5p RCA Padlock Probe</td><td>Sahana Narayanan, Kate Sharer</td><td>Composite</td><td>New, Utilized</td><td>45</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683021">BBa_K4683021</a></td><td>hsa-mir-328-3p</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, RNA</td><td>New, Utilized</td><td>18</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683022">BBa_K4683022</a></td><td>3’ arm for hsa-mir-328-3p</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, DNA</td><td>New, Utilized</td><td>10</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683023">BBa_K4683023</a></td><td>5’ arm for hsa-mir-328-3p</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, DNA</td><td>New, Utilized</td><td>8</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683024">BBa_K4683024</a></td><td>hsa-mir-328-3p RCA Padlock Probe</td><td>Sahana Narayanan, Kate Sharer</td><td>Composite</td><td>New, Utilized</td><td>43</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683025">BBa_K4683025</a></td><td>hsa-mir-146a</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, RNA</td><td>New, Utilized</td><td>21</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683026">BBa_K4683026</a></td><td>3’ arm for hsa-mir-146a</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, DNA</td><td>New, Utilized</td><td>11</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683027">BBa_K4683027</a></td><td>5’ arm for hsa-mir-146a</td><td>Sahana Narayanan, Kate Sharer</td><td>Basic, DNA</td><td>New, Utilized</td><td>10</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4683028">BBa_K4683028</a></td><td>hsa-mir-146a RCA Padlock Probe</td><td>Sahana Narayanan, Kate Sharer</td><td>Composite</td><td>New, Utilized</td><td>46</td></tr><tr><td>Rolling Circle, eRCA</td><td><a href="http://parts.igem.org/Part:BBa_K4245006">BBa_K4245006</a></td><td>hsa-miR-1-3p</td><td>Akshaya Poonepalle, Michelle Jing, Shivaek Venkateswaran, Daeun Lee, Sahana Ram Narayanan, Richard Jiang, Sishnukeshav Balamurali, Christina Yi, Varnica Basavaraj, Manaswi Gorle, Janet Standeven</td><td>Basic, RNA</td><td>Existing, Utilized</td><td>22</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4245009">BBa_K4245009</a></td><td>hsa-mir-133a-3p</td><td>Akshaya Poonepalle, Michelle Jing, Shivaek Venkateswaran, Daeun Lee, Sahana Ram Narayanan, Richard Jiang, Sishnukeshav Balamurali, Christina Yi, Varnica Basavaraj, Manaswi Gorle, Janet Standeven</td><td>Basic, RNA</td><td>Existing, Utilized</td><td>22</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4245100">BBa_K4245100</a></td><td>3’ arm for hsa-miR-1-3p</td><td>Akshaya Poonepalle, Michelle Jing, Shivaek Venkateswaran, Daeun Lee, Sahana Ram Narayanan, Richard Jiang, Sishnukeshav Balamurali, Christina Yi, Varnica Basavaraj, Manaswi Gorle, Janet Standeven</td><td>Basic, DNA</td><td>Existing, Utilized</td><td>11</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4245009"></a><a href="http://parts.igem.org/Part:BBa_K4245009">BBa_K4245009</a></td><td>hsa-mir-133a-3p</td><td>Akshaya Poonepalle, Michelle Jing, Shivaek Venkateswaran, Daeun Lee, Sahana Ram Narayanan, Richard Jiang, Sishnukeshav Balamurali, Christina Yi, Varnica Basavaraj, Manaswi Gorle, Janet Standeven</td><td>Basic, RNA</td><td>Existing, Utilized</td><td>22</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4245107">BBa_K4245107</a></td><td>5’ arm for hsa-miR-1-3p</td><td>Akshaya Poonepalle, Michelle Jing, Shivaek Venkateswaran, Daeun Lee, Sahana Ram Narayanan, Richard Jiang, Sishnukeshav Balamurali, Christina Yi, Varnica Basavaraj, Manaswi Gorle, Janet Standeven</td><td>Basic, DNA</td><td>Existing, Utilized</td><td>11</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4245130">BBa_K4245130</a></td><td>6-carboxyl-fluorescein (FAM) Labeled DNA Probe</td><td>Fulin Zhou, Baoxin Li, and Jiyuan Maa</td><td>Basic, Reporter</td><td>Existing, Utilized</td><td>12</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4245131">BBa_K4245131</a></td><td>Middle sequence</td><td>Fulin Zhou, Baoxin Li, and Jiyuan Maa</td><td>Basic, DNA</td><td>Existing, Utilized</td><td>25</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4245132">BBa_K4245132</a></td><td>Black Hole Quencher 1 (BHQ1) DNA Probe</td><td>Fulin Zhou, Baoxin Li, and Jiyuan Maa</td><td>Basic, Reporter</td><td>Existing, Utilized</td><td>12</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4245133">BBa_K4245133</a></td><td>Lettuce Aptamer</td><td>Bria S VarnBuhler, Jared Moon, Sourav Kumar Dey, Jiahui Wu, Samie R Jaffrey</td><td>Basic, Reporter</td><td>Existing, Utilized</td><td>99</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4245134">BBa_K4245134</a></td><td>Modified Lettuce Left</td><td>Bria S VarnBuhler, Jared Moon, Sourav Kumar Dey, Jiahui Wu, Samie R Jaffrey, Akshaya Poonepalle, Shivaek Venkateswaran, Varnica Basavaraj, Manaswi Gorle, Sahana Ram Narayanan, Daeun Lee, Janet Standeven</td><td>Basic, Reporter</td><td>Existing, Utilized</td><td>43</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4245135">BBa_K4245135</a></td><td>Lettuce Right</td><td>Bria S VarnBuhler, Jared Moon, Sourav Kumar Dey, Jiahui Wu, Samie R Jaffrey, Akshaya Poonepalle, Shivaek Venkateswaran, Varnica Basavaraj, Manaswi Gorle, Sahana Ram Narayanan, Daeun Lee, Janet Standeven</td><td>Basic, Reporter</td><td>Existing, Utilized</td><td>26</td></tr><tr><td>Rolling Circle</td><td><a href="http://parts.igem.org/Part:BBa_K4245200">BBa_K4245200</a></td><td>hsa-miR-1-3p RCA Padlock Probe</td><td>Akshaya Poonepalle, Shivaek Venkateswaran, Varnica Basavaraj, Manaswi Gorle, Sahana Ram Narayanan, Janet Standeven</td><td>Composite</td><td>Existing, Utilized</td><td>47</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>
+ </astro-slot></div></div></div></body></astro-island>
+ <footer class="relative bg-[#292524] pt-8 pb-6 w-screen"><div class="w-full max-w-5xl mx-16 mt-8 container mx-auto px-4 py-10"><div class="flex flex-wrap text-left lg:text-left"><div class="w-full lg:w-6/12 px-4"><h1 class="text-4xl text-white font-serif font-light">Lambert-GA</h1><br/><h3 class="text-xs w-3/4 mt-0 mb-2 font-sans text-white">© <span id="get-current-year">2023</span> - Content on this site is licensed under a<a href="https://creativecommons.org/licenses/by/4.0/" class="underline hover:text-gray-300" target="_blank">Creative Commons Attribution 4.0 International license.</a><br/><br/>The repository used to create this website is available at<a href="https://gitlab.igem.org/2023/lambert-ga" class="underline hover:text-gray-300" target="_blank">https://gitlab.igem.org/2023/lambert-ga</a></h3></div><div class="w-full lg:w-5/12 px-4"><div class="flex flex-wrap items-top mb-6"><div class="w-full lg:w-4/12 px-4 ml-auto"><ul class="w-full list-unstyled"><li class="flex"><svg width="25" height="25" viewBox="0 0 25 25" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M18.7498 2.0835H15.6248C14.2435 2.0835 12.9187 2.63223 11.942 3.60898C10.9652 4.58573 10.4165 5.91049 10.4165 7.29183V10.4168H7.2915V14.5835H10.4165V22.9168H14.5832V14.5835H17.7082L18.7498 10.4168H14.5832V7.29183C14.5832 7.01556 14.6929 6.75061 14.8883 6.55526C15.0836 6.35991 15.3486 6.25016 15.6248 6.25016H18.7498V2.0835Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">Lambert iGEM</a></li><li class="flex"><svg width="25" height="28" viewBox="0 0 25 28" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M22.9168 4.52889C22.9168 4.52889 22.1877 6.90661 20.8335 8.37853C22.5002 19.701 11.0418 27.9664 2.0835 21.5126C4.37516 21.6258 6.66683 20.8332 8.3335 19.2481C3.12516 17.5497 0.520996 10.8695 3.12516 5.66114C5.41683 8.60498 8.9585 10.3033 12.5002 10.1901C11.5627 5.43469 16.6668 2.7173 19.7918 5.88759C20.9377 5.88759 22.9168 4.52889 22.9168 4.52889Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">@lamberths_iGEM</a></li><li class="flex "><svg width="25" height="25" viewBox="0 0 25 25" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M17.7085 2.21484H7.29183C4.41535 2.21484 2.0835 4.49911 2.0835 7.31688V17.521C2.0835 20.3387 4.41535 22.623 7.29183 22.623H17.7085C20.585 22.623 22.9168 20.3387 22.9168 17.521V7.31688C22.9168 4.49911 20.585 2.21484 17.7085 2.21484Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path><path d="M16.6667 11.7759C16.7952 12.6251 16.6472 13.4924 16.2435 14.2545C15.8399 15.0165 15.2012 15.6345 14.4184 16.0205C13.6355 16.4064 12.7484 16.5408 11.8831 16.4044C11.0179 16.268 10.2185 15.8678 9.59881 15.2608C8.9791 14.6537 8.57057 13.8707 8.43134 13.0231C8.29211 12.1754 8.42926 11.3064 8.82328 10.5395C9.2173 9.77269 9.84813 9.14706 10.626 8.75165C11.404 8.35625 12.2893 8.21119 13.1563 8.33712C14.0406 8.46558 14.8592 8.86923 15.4914 9.48846C16.1235 10.1077 16.5356 10.9096 16.6667 11.7759Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">@lambertigem</a></li><li class="flex"><svg width="25" height="24" viewBox="0 0 25 24" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M12.5002 18.2922C10.1043 18.2922 5.8335 18.1028 4.06266 17.7241C3.3335 17.5347 2.81266 17.0612 2.60433 16.3983C2.29183 15.3566 2.0835 13.1786 2.0835 11.6635C2.0835 10.1483 2.29183 7.97027 2.60433 6.92861C2.81266 6.26573 3.3335 5.79224 4.06266 5.60285C5.8335 5.22406 10.1043 5.03467 12.5002 5.03467C14.896 5.03467 19.1668 5.22406 20.9377 5.60285C21.6668 5.79224 22.1877 6.26573 22.396 6.92861C22.7085 7.97027 22.9168 10.1483 22.9168 11.6635C22.9168 13.1786 22.7085 15.3566 22.396 16.3983C22.1877 17.0612 21.6668 17.5347 20.9377 17.7241C19.1668 18.1028 14.896 18.2922 12.5002 18.2922Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path><path d="M10.4165 14.5046L15.6248 11.6637L10.4165 8.82275V14.5046Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">Lambert iGEM</a></li></ul></div></div></div></div></div></footer>
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\ No newline at end of file
diff --git a/public/protein-purification/index.html b/public/protein-purification/index.html
new file mode 100644
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--- /dev/null
+++ b/public/protein-purification/index.html
@@ -0,0 +1,74 @@
+<!DOCTYPE html>
+<html lang="en">
+ <head>
+ <meta charset="UTF-8">
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+ <title>Protein Purification</title>
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+ <h2 id="overview">Overview</h2>
+<p>Lambert iGEM utilized padlock probes with rolling circle amplification (RCA) <a href="https://2022.igem.wiki/lambert-ga/rca">see RCA: Overview</a> to detect various microRNAs (miRNAs) that are upregulated in correlation with coronary artery disease (CAD) (Fichtlscherer et al., 2010). The RCA reaction utilizes phi29 DNA polymerase to amplify the padlock probe sequence (Jonstrup et al., 2006 & Fang et al., 2021). However, a single RCA reaction is $8.98, with phi29 DNA polymerase making up 56% of the cost. Moreover, multiple reactions need to be performed for a more thorough diagnosis, restricting its use to privileged communities, and making it unaffordable for most demographics. To increase the preventative aspect of healthcare and decrease the cost of the overall assay, we produced phi29 DNA polymerase through protein purification.</p>
+<h2 id="protein-purification">Protein Purification</h2>
+<p>Protein purification is commonly used to isolate proteins from a complex mixture (Labrou, 2014). Specifically, immobilized metal affinity chromatography (IMAC) is a protein purification technique used to purify recombinant proteins fused to six consecutive histidine tags (his-tags). His-tags are short DNA sequences inserted at an end terminal of the DNA encoding protein in a recombinant plasmid (see Fig. 1).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/frame-7.png" style="max-width:[250px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. Recombinant plasmid containing DNA encoding protein fused with His-tags.</div></div>
+<p>To purify phi29 DNA polymerase, we utilized an existing plasmid from Addgene that produces the protein attached to six His-tags (see Fig. 1). We isolated the plasmid inside DH5α through miniprep then transformed the plasmid into BL21 for protein expression and cultured it in Luria Broth (LB) growth medium. The resulting cells in the solution were then lysed and filtered through a column that contained positively-charged nickel affinity resin, prompting a bond with the negatively-charged His-tags and polymerase (see Fig. 2) (Corbin & Falke, 2013). To remove the other parts of the lysate and elute the proteins from the columns, we added the His-Wash and His-Elution buffers, respectively (see Fig. 2).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/protein-purification-figure-2.png" style="max-width:500px" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 2. phi29 DNA purification through the use of nickel column and the chelation bond of the His-tag and nickel affinity resin.</div></div>
+<p>We then performed a Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), a technique commonly used for protein separation to measure the protein’s size to ensure only the correct protein has been purified.</p>
+<h2 id="design">Design</h2>
+<p>To produce phi29 DNA polymerase, Lambert iGEM initially used TAS Taipei part, BBa_K3352009 (see Fig. 3), with pet-11a-ABLE (see Fig. 4), an ampicillin-resistant and IPTG-inducible 6xHis-TEV-ABLE backbone from Addgene (Addgene: pet11a-ABLE, 2023). However, when we attempted to purify the pet-11a-ABLE backbone, we obtained repetitive impure results. Therefore, our team acquired pGS-21a his6-GST-WTPhi29DNAP, a plasmid that produces an affinity-tagged phi29 DNA polymerase (see Fig. 5), allowing us to bypass restriction digest and purify the protein (PGS-21A His6-GST-WTPHI29DNAP (plasmid #166863)).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/screen-shot-2023-10-09-at-4-49-01-pm.png" style="max-width:500" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. Construct of pET T7 Promoter with Modified UTR, Extended RBS, phi29 DNA polymerase, and T7 Terminator (BBa_K3352009)</div></div>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/screen-shot-2023-10-09-at-4-10-35-pm.png" style="max-width:500px" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Full Sequence Map of pet11a-ABLE from SnapGene</div></div>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/screen-shot-2023-10-09-at-4-12-14-pm.png" style="max-width:[250px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 5. Full Sequence Map of pGS-21a his6-GST-WTPhi29DNAP from SnapGene</div></div>
+<h2 id="results">Results</h2>
+<p>After running our purified phi29 DNA polymerase from immobilized metal affinity chromatography (IMAC) on the Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE)<a href="https://2023.igem.wiki/lambert-ga/experiments/">see Experiments: Protein Purification</a>, Lambert iGEM saw bands around 50kD ~ 75kD. When we compared this band to the commercial phi29 DNA polymerase from New England Biolabs, it indicated that the protein was purified as the bands appeared in the same area (see Fig. 6).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/frame-10-1.png" style="max-width:800" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 6. SDS-PAGE with purified phi29 DNA polymerase, displaying bands at 50kD ~ 75kD</div></div>
+<p>Our team tested the efficiency of the purified phi29 DNA polymerase in rolling circle amplification (RCA) by comparing the rolling circle products (RCP) of the commercial and purified phi29 DNA polymerase. We added the same volume of both to RCA reactions and ran the rolling circle products on a gel. We saw fluorescent bands of DNA very close to the wells, indicating that both the RCA assay utilizing commercial and purified enzyme reactions were successful (see Fig. 7).</p>
+<p>If we buy phi29 DNA polymerase from New England Biolabs, it will cost $251.00 for 1,250 units. However, after we utilized the NEBExpress Ni Spin Columns ($10 per prep to purify the enzyme), we were able to produce 200ul in one prep, or approximately 2,000 units. Because, it can be assumed that the unit between the commercially bought enzyme and our purified enzyme is similar, if commercially bought, this 200ul amount would cost around $400. Therefore, we can determine that our purified protein is significantly more affordable than the commercially bought protein.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/hardware/screenshot-2023-10-12-at-10-37-15-am.png" style="max-width:800" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 7. Gel comparing the rolling circle product (RCP) between the team’s purified phi29 DNA polymerase and that commercially available phi29</div></div>
+<p>In the future, we plan to more accurately quantify the activity of the purified units of phi29 DNA polymerase. Additionally, we hope to test the accuracy of our enzyme by quantifying the RCP using linear DNA probes (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: outputs</a>) to validate its use of point-of-care testing.</p>
+<h2 id="references">References</h2>
+<div class="pl-6 -indent-6"> Falke, J. J., & Corbin, J. A. (2013). Affinity Tags for Protein Purification. Encyclopedia of Biological Chemistry. Retrieved June 10, 2023, from <a href="https://doi.org/10.1016/B978-0-12-378630-2.00173-0">https://doi.org/10.1016/B978-0-12-378630-2.00173-0</a> </div>
+<div class="pl-6 -indent-6"> Fichtlscherer, S., De Rosa, S., Fox, H., Schwietz, T., Fischer, A., Liebetrau, C., … & Dimmeler, S. (2010). Circulating microRNAs in patients with coronary artery disease. Circulation research, 107(5), 677-684. <a href="https://doi.org/10.1161/circresaha.109.215566">https://doi.org/10.1161/circresaha.109.215566</a> </div>
+<div class="pl-6 -indent-6">Jonstrup, S. P., Koch, J., & Kjems, J. (2006). A microRNA detection system based on padlock probes and rolling circle amplification. RNA (New York, N.Y.), 12(9), 1747–1752. <a href="https://doi.org/10.1261/rna.110706">https://doi.org/10.1261/rna.110706</a> </div>
+<div class="pl-6 -indent-6"> Labrou, N. E. (2014). Protein purification: An overview. PubMed. <a href="https://pubmed.ncbi.nlm.nih.gov/24648062/">https://pubmed.ncbi.nlm.nih.gov/24648062/</a> </div>
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+ <astro-island uid="2cRQI8" prefix="r62" component-url="/lambert-ga/_astro/TabReact.a7adacc1.js" component-export="default" renderer-url="/lambert-ga/_astro/client.4f88357e.js" props="{}" ssr="" client="load" opts="{"name":"TabReact","value":true}" await-children=""><div dir="ltr" data-orientation="horizontal" class="w-full "><div role="tablist" aria-orientation="horizontal" class=" h-[54px] bg-neutral-50 rounded-[33px] shadow border border-gray-6 flex flex-shrink mb-10" tabindex="-1" data-orientation="horizontal" style="outline:none"><button type="button" role="tab" aria-selected="true" aria-controls="radix-:r62R0:-content-a" data-state="active" id="radix-:r62R0:-trigger-a" class="grow text-center text-gray-11 font-sans text-lg font-medium hover:bg-gray-3 rounded-full m-1 data-[state=active]:bg-gray-4 data-[state=active]:text-gray-12" tabindex="-1" data-orientation="horizontal" data-radix-collection-item="">2022 Overview</button><button type="button" role="tab" aria-selected="false" aria-controls="radix-:r62R0:-content-b" data-state="inactive" id="radix-:r62R0:-trigger-b" class="grow text-center text-gray-11 font-sans text-lg font-medium hover:bg-gray-3 rounded-full m-1 data-[state=active]:bg-gray-4 data-[state=active]:text-gray-12" tabindex="-1" data-orientation="horizontal" data-radix-collection-item="">Outputs</button><button type="button" role="tab" aria-selected="false" aria-controls="radix-:r62R0:-content-c" data-state="inactive" id="radix-:r62R0:-trigger-c" class="grow text-center text-gray-11 font-sans text-lg font-medium hover:bg-gray-3 rounded-full m-1 data-[state=active]:bg-gray-4 data-[state=active]:text-gray-12" tabindex="-1" data-orientation="horizontal" data-radix-collection-item="">Optimization</button><button type="button" role="tab" aria-selected="false" aria-controls="radix-:r62R0:-content-d" data-state="inactive" id="radix-:r62R0:-trigger-d" class="grow text-center text-gray-11 font-sans text-lg font-medium hover:bg-gray-3 rounded-full m-1 data-[state=active]:bg-gray-4 data-[state=active]:text-gray-12" tabindex="-1" data-orientation="horizontal" data-radix-collection-item="">eRCA</button><button type="button" role="tab" aria-selected="false" aria-controls="radix-:r62R0:-content-e" data-state="inactive" id="radix-:r62R0:-trigger-e" class="grow text-center text-gray-11 font-sans text-lg font-medium hover:bg-gray-3 rounded-full m-1 data-[state=active]:bg-gray-4 data-[state=active]:text-gray-12" tabindex="-1" data-orientation="horizontal" data-radix-collection-item="">Inclusivity</button></div><div data-state="active" data-orientation="horizontal" role="tabpanel" aria-labelledby="radix-:r62R0:-trigger-a" id="radix-:r62R0:-content-a" tabindex="0" style="animation-duration:0s"><astro-slot name="a"><div><h1 id="overview">Overview</h1>
+<h2 id="problem">Problem</h2>
+<p>Heart disease has been one of the most prevalent diseases in the United States, with 1 in 4 deaths caused by cardiovascular disease (Centers for Disease Control, 2022). According to the Centers for Disease Control and Prevention, heart disease has contributed to over 25% of all deaths in Georgia, Lambert iGEM’s home region, in 2018 - that is 5% greater than the overwhelmingly large national statistic. Historically, the southern region has had issues concerning obesity, high blood pressure, smoking, and diabetes, all of which are exacerbated by a family history of coronary artery disease (CAD). These problems only increase with the “coincidental†ethnic and racial minorities present within these low-income populations (Hames and Greenlund). However, with a process that considers both affordability and accuracy, we can proliferate the means of diagnosing CAD. This starts with inclusive initiatives targeting these overlooked demographics.</p>
+<h2 id="methodology">Methodology</h2>
+<p>Last year, Lambert iGEM created CADlock, an accessible point-of-care diagnostic tool to both detect and quantify microRNA (miRNA) by utilizing rolling circle amplification (RCA). The rolling circle product (RCP) is a continuous transcription of the team’s designed padlock probe, which consists of many interspaced repeats of the reporting mechanism and target miRNA’s complementary sequence (Shi et al., 2012; Song et al., 2010; Ouyang et al., 2019; Ye et al., 2019; Zheng et al., 2016). We identified two miRNAs found to be upregulated to CAD, hsa-miR-1-3p (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245006">BBa_K4245006</a>) and hsa-miR-133a-3p (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245009">BBa_K4245009</a>), to serve as an early indication of the disease (Kaur et al., 2020).</p>
+<p>Initially, we considered Reverse Transcription Quantification PCR (RT-qPCR) which was appealing due to its regularity with current screening, especially with the COVID-19 tests. However, through conversations with experts and further analysis of protocols, we identified the dangers of this method due to the high rate of false positive mismatches and determined that RCA held the most advantages. Not only does RCA eliminate the use of primers–which RT-qPCR requires–but it has no data concerning any mismatch or inconsistent results from our literature studies (Shi et al., 2012; Song et al., 2010; Ouyang et al., 2019; Ye et al., 2019; Zheng et al., 2016).</p>
+<p>Moreover, diagnostic tools such as echocardiograms and angiograms are expensive – costing upwards of $1000 – and not readily available at most primary care facilities, preventing a large portion of the population from receiving effective screening (Hansing C. E.). However, our RCA assay costs approximately $9, which is less than 1% of most common diagnostic procedures.</p>
+<h2 id="padlock-design">Padlock Design</h2>
+<p>A padlock probe is a single-stranded DNA sequence (30-150 nucleotides in length) designed to hybridize to a specific RNA/DNA target– in this case, our target miRNA (Jonstrup et al., 2006). The ends or “arms†of the probe are complementary to the target sequence, which allows the miRNA to bind antiparallel to the padlock arms (see Fig. 1). We designed these ends to have similar annealing temperatures to maximize their binding efficiency with the miRNA. In between the ends is the reporter sequence, which can be utilized to quantify the initial miRNA concentration (see Fig. 1). Furthermore, we added a phosphate group modification to the 5’ end of the padlock sequence (Jonstrup et al., 2006).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-1-21.png" style="max-width:[250px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. Padlock probe formation and arrangement with miRNA</div></div>
+<p>The following steps are for generating a padlock probe by hand. Users will need a software tool such as SnapGene that displays melting temperatures of sequences.</p>
+<ol>
+<li>Paste target sequence in software of choice.</li>
+<li>Take the reverse complementary sequence of the target.</li>
+<li>Split the sequence in half. Use the first half of the sequence as the end of the padlock probe and the second half as the beginning of the probe.</li>
+<li>Insert the desired reporter sequence in between the two halves of the sequence.</li>
+<li>Calculate the annealing temperature of both halves and move nucleotides one at a time from one end of a half to the end of the other half until the difference between the annealing temperatures of the two arms is minimal.</li>
+</ol>
+<p>Last year, Lambert iGEM developed Probebuilder: a novel, intuitive, and user-friendly program for designing padlock probes. After validating the hsa-miR-451a padlock probe generated by Probebuilder, the team has continued to use the software to design padlocks for RCA reactions (see <a href="https://2022.igem.wiki/lambert-ga/software/">Lambert iGEM Wiki Software, 2022</a>).</p>
+<p>Probebuilder’s source code is available on the 2022 <a href="https://gitlab.igem.org/2022/software-tools/lambert-ga/">Gitlab repository</a></p>
+<h2 id="process">Process</h2>
+<p>The RCA process relies on the continuous replication of padlock probes to produce RCP that can be quantified and correlated to the target miRNA concentration. There are three main steps in the RCA process detailed by Jonstrup et al. in their 2006 paper: hybridization, ligation, and amplification. By designing the padlock probe to detect a specific miRNA target, the miRNA and padlock probe arms form a DNA-RNA hybridization (see Fig. 2a, 2b). The arms are then in closer proximity, and the enzyme SplintR ligase (which only ligates near RNA-DNA hybridization) utilizes a phosphate modification on the 5’ arm and ATP to circularize the padlock probe (Avantor Staff) (see Fig. 2c). After ligation, phi29 DNA polymerase uses the miRNA hybridized to the padlock arms as a primer to initiate and perform amplification (see Fig. 2d, 2e). The resultant rolling circle product (RCP) contains interspaced repeats of the middle sequence of the padlock probe (see Fig. 2f). This sequence of the RCP is complementary to the reporters (linear probes or lettuce) used to correlate fluorescence to varying concentrations of the target miRNA.</p>
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RCA process: (a, b) hybridization, (c) ligation, (d, e) amplification, (f) rolling circle product</div></div></astro-island>
+<h1 id="2022-results">2022 Results</h1>
+<p>In 2022, we conducted multiple rounds of experimentation to generate quantifiable results with our product. First, we conducted initial experiments to assess how well our hsa-miR-1-3p biosensor (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245200">BBa_K4245200</a>) performs in a controlled laboratory setting by evaluating the effectiveness of rolling circle amplification (RCA) and various reporter mechanisms. All results in Figures 4-11 and13 were quantified in a plate reader at exciation wavelength of 480 nm and emission intensity at 518 nm.</p>
+<ol>
+<li>
+<p>To quantify the rolling circle product (RCP), we first used the fluorescence dye SYBRâ„¢ Safe dye which fluoresces when bound to ssDNA. The results were inconsistent, so we tested the potential rolling circle product (RCP) through gel electrophoresis. The gel showed a band near the wells, indicating a long strand of DNA was produced, likely our RCP (see Fig. 3). <div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-updated-figure-10.png" style="max-width:[250px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. Gel electrophoresis of RCA reactions, depicting bands greater than 1 kB; 2% agarose gel ran for 1 hour at 48V</div></div></p>
+</li>
+<li>
+<p>Through a literature review, we found a lettuce aptamer (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245133">BBa_K4245133</a>) and tested it with simulated RCP (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245131">BBa_K4245131</a>). This showed an increase in fluorescence compared to the controls (see Fig. 4), indicating that the lettuce aptamer is able to fluoresce when bound to the simulated RCP (VarnBuhler et al., 2022). <div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-18.png" style="max-width:[250px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Lettuce aptamer with simulated RCP.</div></div></p>
+</li>
+<li>
+<p>We performed a series of experiments of the split Lettuce aptamer (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245134">BBa_K4245134</a>; <a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245135">BBa_K4245135</a>) with actual RCP. The results display a significant increase in fluorescence with RCP and split lettuce as compared to the controls, indicating that the lettuce aptamer was able to successfully bind to the RCP and induce fluorescence of DFHBI-1T (see Fig. 5). <div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rcp-lettuce.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 5. Graph of fluorescence from left to right: 1) water + dye control, 2) split lettuce + dye control, 3) split lettuce + RCP + dye, 4) split lettuce + padlock&miRNA control + dye. </div></div></p>
+</li>
+<li>
+<p>After consultation with other experts on linear DNA probes, we decided to test these with simulated RCP (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245131">BBa_K4245131</a>) to ensure linear probes were an effective and characterizable means of quantifying miRNA (Zhou et al., 2015). The FAM dye tagged probe (<a href="%5BBBa_K4245131%5D(http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245130)">BBa_K4245130</a>) and BHQ-1 quencher tagged probe (<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K4245132">BBa_K4245132</a>)) are complementary to our RCP. When the linear DNA probes are bound to the RCP, the fluorescence produced by the FAM Dye is quenched by the BHQ-1 quencher which significantly reduces fluorescent intensity (Zhou et al., 2015). As shown in Figure 6, linear DNA probes with simulated RCP have significantly less fluorescence than just the FAM dye, indicating the mechanism would be efficient with actual RCP. <div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-11.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 6. Linear DNA probes with simulated RCP.</div></div></p>
+</li>
+<li>
+<p>We further characterized the linear DNA probes with various simulated RCP concentrations. Figure 7 below shows a negative logarithmic correlation between the complement concentrations and the relative fluorescence units, a parallel relationship to our ODE model (see Fig. 8). <div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-12.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 7. Linear DNA probes with various concentrations of simulated RCP.</div></div> <div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-13.png
+" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 8. Deterministic ODE Model Simulation of RFU output dependent on concentration of linear DNA probe complement concentration.</div></div></p>
+</li>
+<li>
+<p>After we determined the vitality of using linear DNA probes with simulated RCP, we tested these probes with actual RCP. As shown in Figure 9 below, there is a statistically significant decrease in the fluorescence output of the probes with RCP as compared to that of just the FAM-tagged probe. This confirms that we produced our desired RCP.</p>
+</li>
+</ol>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-14.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 9. Fluorescent readout of RCP for miRNA-1-3p and miRNA-133a-3p using linear DNA probes.</div></div>
+<ol start="7">
+<li>We further characterized linear DNA probes to quantify the relationship between various miRNA concentrations and fluorescence. There is a negative logarithmic correlation between the miRNA concentrations and the RFU (see Fig. 10), a parallel relationship to our ODE model (see Fig. 11), validating that RCA coupled with linear probes are an effective means of quantifying miRNA.</li>
+</ol>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-15.png
+" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 10. Characterization curve for showing a negative logarithmic relationship between RFU from linear DNA probes and various miRNA concentrations.</div></div>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-16.png
+" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 11. Deterministic ODE Model Simulation of RFU output dependent on miRNA concentration</div></div>
+<h2 id="serum-testing">Serum Testing</h2>
+<p>To assess the feasibility of our biosensors in real-world applications, Lambert iGEM experimented with pooled human serum. Upon meeting with Dr. Charles Searles from the Emory University School of Medicine, we tested the ability of our RCA sensors to detect miRNAs in blood serum by spiking pooled human serum with hsa-miR-1-3p and RNase inhibitors (to prevent native RNases from degrading the added miRNA).
+The gel electrophoresis results show clear bands close to the wells (see Fig. 12), indicating our biosensors were able to produce a long strand, likely the RCP.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/proof-of-concept-figure-9.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 12. Agarose gel with RCP from RCA reactions and negative control reactions that had no added miRNA from serum treated with 10 nM miRNA; 2% agarose gel ran for 1 hour at 48V</div></div>
+<p>To experimentally validate that the long DNA strand that was produced is our desired RCP, we utilized linear DNA probes. As shown in Figure 13, there is a statistically significant decrease in the fluorescent output of the linear DNA probes with RCP as compared to that of just FAM tagged probes, validating the success of the RCA reaction with hsa-miR-1-3p in spiked serum.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rca-figure-17-updated.png
+" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 13. Linear DNA probes fluorescence output from RCA with serum extracted miRNA-1-3p.</div></div></div></astro-slot></div><div data-state="inactive" data-orientation="horizontal" role="tabpanel" aria-labelledby="radix-:r62R0:-trigger-b" hidden="" id="radix-:r62R0:-content-b" tabindex="0"></div><div data-state="inactive" data-orientation="horizontal" role="tabpanel" aria-labelledby="radix-:r62R0:-trigger-c" hidden="" id="radix-:r62R0:-content-c" tabindex="0"></div><div data-state="inactive" data-orientation="horizontal" role="tabpanel" aria-labelledby="radix-:r62R0:-trigger-d" hidden="" id="radix-:r62R0:-content-d" tabindex="0"></div><div data-state="inactive" data-orientation="horizontal" role="tabpanel" aria-labelledby="radix-:r62R0:-trigger-e" hidden="" id="radix-:r62R0:-content-e" tabindex="0"></div></div><template data-astro-template="b"><div><h1 id="linear-dna-probes-vs-lettuce">Linear DNA Probes vs Lettuce</h1>
+<p>Lettuce DNA aptamer and linear DNA probes were both tested as reporters for rolling circle amplification (RCA) with the hsa-miR-1-3p padlock probe (<a href="http://parts.igem.org/Part:BBa_K4245200">BBa_K4245200</a>); however, only linear probes were used for the successful characterization of miR-1 (<a href="http://parts.igem.org/Part:BBa_K4245006">BBa_K4245006</a>) in the 2022 season (see <a href="https://2022.igem.wiki/lambert-ga/rca">Lambert iGEM Wiki RCA, 2022</a>). Dr. Adam Silverman from Northwestern University and Dr. Mark Styzynski from the Georgia Institute of Technology both suggested utilizing an on-state reporter to make results more accurate and comprehensible; measuring gain of signal reduces fluorescent noise caused by the reaction and has less limitations compared to repressible systems. Linear DNA probes are an off-state reporter, therefore they produce an indirect relationship between microRNA (miRNA) concentration and fluorescence output. Utilizing an on-state reporter such as Lettuce would result in a direct relationship, in which a higher concentration of fluorescence will correspond to a higher concentration of miRNA.</p>
+<h2 id="linear-dna-probes">Linear DNA Probes</h2>
+<p>Fluorophore and quencher-tagged linear DNA probes were used to quantify the presence and concentration of target miRNA in samples. Each probe contains part of the complement to the middle sequence (<a href="http://parts.igem.org/Part:BBa_K4245131">BBa_K4245131</a>) of the rolling circle product (RCP): One is tagged with the fluorophore dye FAM (<a href="http://parts.igem.org/Part:BBa_K4245130">BBa_K4245130</a>), and the other is tagged with the quencher molecule BHQ1 (<a href="http://parts.igem.org/Part:BBa_K4245132">BBa_K4245132</a>). The fluorescent signal from the fluorophore is shut off by the quencher as a result of a fluorescence resonance energy transfer (FRET) reaction (Zhou et al., 2015). The reaction is distance-dependent; when the quencher and fluorophore-tagged linear probes bind to the RCP, they are in close proximity (see Fig. 1), allowing non-radiative energy to be transferred from the excited fluorophore to the quencher (Sekar & Periasamy, 2003). The decrease in fluorescence in the solution can be correlated with a specific concentration of miRNA through characterization.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/frame-2.jpeg" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. Linear DNA probes binding to RCP, leading to diminished fluorescence</div></div>
+<p>Similar to last year, we characterized and quantified RCP through the linear probes reporting mechanism (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Linear DNA Probes with RCP</a>). There is a negative logarithmic correlation between the miRNA concentrations and the relative fluorescence units (RFU) (see Fig. 2). Changes in fluorescence were measured using a plate reader in experimentation: the emission spectrum of FAM is 480 nm in wavelength, while the excitation spectrum is 528 nm in wavelength (Zhou et al., 2015).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/linearprobes.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 2. Characterization curve showing a negative logarithmic relationship between miR-1 concentrations and RFU from linear DNA probes </div></div>
+<h2 id="lettuce">Lettuce</h2>
+<p>Lettuce is a fluorescent DNA aptamer that binds with the dye DFHBI-1T within its secondary structure, thus causing the dye to fluoresce (see Fig. 3) (VarnBuhler et al., 2022). The split Lettuce design includes two halves of the Lettuce aptamer and their flanking sequences. After the RCA reaction, we add the split Lettuce sequences and the DFHBI-1T dye to the RCP. The left flanking sequence (<a href="http://parts.igem.org/Part:BBa_K4245134">BBa_K4245134</a>) will bind to the first half of the middle sequence of the RCP, and the right flanking sequence (<a href="http://parts.igem.org/Part:BBa_K4245135">BBa_K4245135</a>) will bind to the second half of the middle sequence. Once together, the dye is able to bind and produce fluorescence, therefore an increase in miRNA concentration should correlate with an increase in fluorescence.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-9.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. Split Lettuce aptamer with DFHBI-1T binding to RCP</div></div>
+<p>We characterized and quantified RCP through the Lettuce reporting mechanism (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Lettuce with RCP</a>). There is a positive logarithmic correlation between the miRNA concentrations and the relative fluorescence units (RFU) (see Fig. 4). Changes in fluorescence were measured using a plate reader in experimentation: the emission spectrum of DFHBI-1T is 480 nm in wavelength, while the excitation spectrum is 528 nm in wavelength (VarnBuhler et al., 2022).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/lettuce.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Characterization curve showing a positive logarithmic relationship between miR-1 concentrations and RFU from split Lettuce aptamer</div></div>
+<h2 id="comparison">Comparison</h2>
+<p>Both reporter mechanisms resulted in significant SEM overlap between the lower miRNA concentrations, making accurate differentiation of miRNAs difficult. However, there was no indication that Lettuce outperformed linear DNA probes. Therefore, we continued to conduct further experiments with linear probes. In the future, we hope to find another on-state reporter that would make reading RCA results more comprehensible and accurate. Such reporters include molecular beacons and other DNA aptamers.</p>
+<h1 id="capillary">Capillary</h1>
+<p>To create a more accessible miRNA detection system, Lambert iGEM also adopted another output approach: capillary-rolling circle amplification (cpRCA). This method offers faster reaction times and eliminates the need for expensive and specialized equipment like plate readers and fluorometers, making it suitable for rapid point-of-care testing of miRNAs (Hixson & Ward, 2021). Unlike our current reporter mechanisms, cpRCA amplifies miRNA to produce RCP within a glass capillary tube. The small diameter of the capillary tube (0.1mm) enables diffusion of small polymers (such as miRNA), while restricting diffusion of larger polymers (such as the RCP). Due to this principle (see <a href="https://2023.igem.wiki/lambert-ga/model/">Modeling</a>), the miRNA molecules are relatively spaced apart within the tube (see Fig. 5a). As the RCP is being synthesized from the miRNA molecules, the large DNA strands are unable to diffuse, creating spaced apart, isolated regions of ssDNA (see Fig. 5b). When the DNA fluorescent dye SYBR™ Safe is present in the reaction solution, it binds to the synthesized RCP to create regions of high fluorescence, or fluorescent “dots†(see Fig. 5c), a phenomenon that has been explored with PCR in capillary tubes (Choi et al., 2018). We can directly quantify the number of miRNA molecules by counting the number of dots, which can be done through use of a phone camera and counting algorithm, such as Open Computer Vision Library (OpenCV) (Abid et al, 2021).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-12-1.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 5. (a) miRNA and padlock probe diffusion in capillary tube. (b) Isolated regions of RCP within the capillary tube. (c) SYBRâ„¢ Safe fluorescence with RCP
+</div></div>
+<p>After discussion with Dr. Charles Searles from the Emory University School of Medicine, we determined that the upper limit of clinically relevant miRNA concentrations in patients with CAD is around 40 pM. We initially ran cpRCA with the miR-1 padlock probe (<a href="http://parts.igem.org/Part:BBa_K4245200">BBa_K4245200</a>) and 40.8 pM of miR-1 (<a href="http://parts.igem.org/Part:BBa_K4245006">BBa_K4245006</a>) but were not able to visualize any dots of miRNA within the capillary tube (see Fig. 6) (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Capillary RCA</a>). This is likely because the higher concentration of miRNA resulted in a significant overlap of fluorescent regions within the capillary tube, leading to inaccurate quantification through cpRCA.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/screenshot-2023-10-11-at-3-22-41-am.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 6. Visualization of cpRCA with 40.8 pM of miRNA
+</div></div>
+<p>To ensure the amplified products were countable, we diluted the sample down to 1.66 fM, or approximately 50 molecules of miRNA. To validate that cpRCA would still be successful at this lower concentration, we ran a gel electrophoresis on the RCP from the cpRCA reaction after 4 hours of amplification (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: blueGelâ„¢ with RCP</a>) using both 40pM and 1.66fM of miRNA to observe the difference. By analyzing the results on the gel, we concluded that RCP was likely produced with both concentrations as the gel exhibited fluorescent bands of DNA very close to the wells, while no band was expressed in the control (see Fig. 7). As a result, we can infer that cpRCA was successful after 4 hours of amplification and at only 50 molecules of miRNA, which is more efficient than the 8 hour amplification of traditional RCA, and increases the sensitivity of the assay.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/screenshot-2023-10-11-at-3-21-24-am.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 7. Lane 1: ladder, Lanes 2-3: cpRCA with 1.66 fM miR-1, Lanes 4-5: cpRCA with 40.8 pM miR-1, Lanes 6-7: control; 2% agarose gel ran for 1 hour at 48V</div></div>
+<p>In the future, we plan to conduct further experimentation with lower miRNA concentrations to better visualize the fluorescent regions in the capillary tube, as well as characterize the full range of relevant miRNA concentrations for CAD.</p></div></template><template data-astro-template="c"><div><h1 id="specificity">Specificity</h1>
+<p>While Lambert iGEM has been utilizing rolling circle amplification (RCA) to detect a single isolated microRNA (miRNA), human blood serum contains a total of 204 detectable miRNAs (Wang et al., 2012). Research conducted by Jonstrup et al. in 2006 found that the padlock probe ligates on a perfectly matching RNA template, distinguishing between differences in the target and other sequences. To test whether padlock probes would be able to detect specific miRNA, and therefore be applicable for serum testing, we ran RCA using the hsa-miR-1-3p padlock (<a href="http://parts.igem.org/Part:BBa_K4245200">BBa_K4245200</a>) in the presence of four different miRNA sequences (see Fig. 1). The first is the original miR-1 sequence (<a href="http://parts.igem.org/Part:BBa_K4245006">BBa_K4245006</a>), which is expected to hybridize to the padlock and result in the greatest fluorescence decrease. Two sequences with differing single nucleotide variants (SNVs) found from the National Library of Medicine microRNA 1-1 database were utilized to determine the specificity of RCA: one with a single SNV (<a href="http://parts.igem.org/Part:BBa_K4683003">BBa_K4683003</a>) and one with three SNVs (<a href="http://parts.igem.org/Part:BBa_K4683004">BBa_K4683004</a>). hsa-miR-133a-3p (<a href="http://parts.igem.org/Part:BBa_K4245009">BBa_K4245009</a>) was also included to ensure the padlock would not ligate to any miRNA.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/frame-1.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. Comparison of sequences used to test specificity of hsa-miR-1-3p RCA padlock: 1 bp SNV in the seed region, 3 SNVs outside of seed, and miR-133a-3p</div></div>
+<p>We ran the reactions and control on a gel electrophoresis; only the well with 40.8 pM of miR-1 showed visible bands of DNA near the top of the wells, which is likely our RCP (see Fig. 2) (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: blueGelâ„¢ with RCP</a>).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/specificity-gel.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 2. Gel results: RCA with A: miR-1, B: 1 SNV, C: 3 SNVs, D: miR-133a; 2% agarose gel ran for 1 hour at 48V</div></div>
+<p>We then tested the RCP with linear DNA probes and quantified the resultant fluorescence in a plate reader at an emission wavelength of 480 nm and an excitation wavelength of 528 nm (see Fig. 3) (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Linear DNA Probes with RCP</a>. The RCA reaction utilizing the miR-1 padlock probe with miR-1 exhibited significantly less fluorescence than the other miRNAs. Since linear DNA probes produce a negative correlation between fluorescence and miRNA concentration, this result, along with the gel, indicates that RCA is specific to single nucleotide differences.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/specificty-graph.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. Comparison of RCA with miR-1, 1SNV, 3SNVs, and 133a fluorescence output using linear DNA probes.</div></div>
+<h1 id="emory-testing">Emory Testing</h1>
+<p>Dr. Charles Searles is a cardiologist specialist working with biomarkers regarding coronary artery disease (CAD) and an associate professor of medicine at Emory Healthcare Hospital. Last year, he provided us with hsa-miR-451a (<a href="http://parts.igem.org/Part:BBa_K4245012">BBa_K4245012</a>) - which is unrelated to the pathophysiology of CAD - as a control for our proof of concept experimentation and also offered to test our biosensor in his lab. This year, we continued to communicate with him in order to test whether our biosensor could be practical and applicable as a diagnostic tool. This was done with the help of Kimberly Ann Rooney, a lab technician at the Searles Cardiovascular Lab led by Dr. Charles Searles, who ran the RCA reactions utilizing our protocol and miR-1 padlock probe (<a href="http://parts.igem.org/Part:BBa_K4245200">BBa_K4245200</a>), and their enzymes (see Fig. 4) (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: SYBRâ„¢ Safe with RCP</a>). They ran RCA on 40.8 pM of miR-1 (<a href="http://parts.igem.org/Part:BBa_K4245006">BBa_K4245006</a>) with SYBRâ„¢ Safe dye, which fluoresces when bound to ssDNA, as the output. Resultant fluoresence was quantified in a plate reader at exciation wavelength of 480 nm and emission intensity at 528 nm. As shown in Figure 4, there was a significant increase in fluorescence in the RCA reaction as compared to that of the controls, therefore validating the application of our biosensor in other labs.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/emory.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Triplicate of RCA with SYBRâ„¢ Safe output done by independent hands</div></div>
+<p>After running our assay twice, Ms. Rooney gave us feedback regarding our protocol such as reducing the number of variables as controls and performing serial dilutions for more precise concentrations of miRNA. Ultimately, she told us that our biosensor lacked practicality and applicability for point-of-care testing due to its long reaction time, which is approximately 15-16 hours‒ more than double the time needed for quantification through qRT-PCR. Subsequently, the next step we took to optimize RCA was to reduce the time required to run the reaction.</p>
+<h1 id="time-optimization">Time Optimization</h1>
+<h2 id="ligation-time">Ligation Time</h2>
+<p>Lambert iGEM’s 2022 RCA protocol (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: RCA 2022 protocol</a>) requires samples to be incubated in the thermocycler at 37°C for two hours. However, the properties and usage of SplintR Ligase show that the reaction is successful with a 15-minute ligation time (Avantor Staff). Therefore, we ran RCA utilizing the miR-1 padlock probe (<a href="http://parts.igem.org/Part:BBa_K4245200">BBa_K4245200</a>) with four different ligation times with miR-1 (<a href="http://parts.igem.org/Part:BBa_K4245006">BBa_K4245006</a>): 15 minutes, 30 minutes, one hour, and two hours. After amplification, the reactions and controls were run on a gel; the bright bands near the top of the well showed that DNA product was produced for all reactions except for 15 minutes (see Fig. 5). Moving forward, we implemented a 30-minute ligation time (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Optimized RCA protocol</a>).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/screenshot-2023-10-11-at-4-32-50-pm.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 5. 1: 15-minute ligation, 2: 30-minute ligation, 3: 1-hour ligation, 4: 2-hour ligation, A: control (no enzymes); 2% agarose gel ran for 1 hour at 48V</div></div>
+<h2 id="amplification-time">Amplification Time</h2>
+<p>Lambert iGEM’s 2022 RCA protocol (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: RCA 2022 protocol</a>) requires samples to be incubated in the thermocycler at 37°C for eight hours for amplification. To reduce this time, we ran RCA with two different concentrations of miR-1. After hybridization and ligation, we incubated the reactions in the plate reader at 37°C with 4uL SYBR™ Safe (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: SYBR™ Safe with RCP</a>). The reactions were run overnight and the subsequent fluorescence was quantified in a plate reader (excitation wavelength 480 nm; emission wavelength: 528 nm): 30-minute intervals (see Fig. 6). Over time, the two RCA reactions increased in fluorescence, with no SEM overlap observed between the starting time and 5-hour mark. This suggests that RCP can be produced optimally starting at 5 hours. The significant increase in fluorescence between the RCA reactions and controls shows that SYBR™ Safe can determine the presence of RCP; however, the lack of difference between the 40.8 pM and .41 pM of miR-1 fluorescence indicates that the dye is not sensitive enough to differentiate between miRNA concentrations. Therefore, we did not continue to utilize SYBR™ Safe as a reporter.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/specificty-graph.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 6. RCA amplification optimization reaction with 40.8 pM and 0.41 pM of miRNA. No SEM overlap between starting point and 5-hour incubation time. SEM overlaps between 40. pM and .41 pM show that miRNA concentrations cannot be differentiated by SYBRâ„¢ Safe.</div></div>
+<h2 id="phi29-xt">Phi29-XT</h2>
+<p>Lambert iGEM’s RCA protocol (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Optimized RCA protocol</a>) utilizes Phi29 DNA polymerase to perform amplification, resulting in an 5 hour amplification time. Phi29- XT DNA polymerase is an optimized enzyme with improved thermostability and sensitivity, which could shorten this time down to 2 hours (Biolabs). We ran RCA following the protocol for Phi29-XT on the New England Biolabs website (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Amplification with Phi29- XT</a>). The reactions and controls were run on a gel; no visible bands could be seen on the gel, indicating that RCP was not produced and therefore the reaction was not successful. As a result, we did not pursue utilizing Phi29-XT for further RCA reactions (see Fig. 7).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/phi-xt.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 7. A: control (no enzymes), X: RCA with phi29-XT DNA polymerase; 2% agarose gel ran for 1 hour at 48V</div></div>
+<p>Overall, we were able to reduce the RCA workflow from around 15 hours to 7 (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Optimized RCA Protocol</a>).</p></div></template><template data-astro-template="d"><div><h1 id="exponential-rolling-circle-amplification-erca">Exponential Rolling Circle Amplification (eRCA)</h1>
+<p>Although the data from testing rolling circle amplification (RCA) with the target microRNA (miRNA) indicates high sensitivity — detecting lower limits of 2 pM — it lacks the ability to clearly differentiate between various concentrations of miRNA. Throughout multiple iterations of testing, Lambert iGEM has consistently found a significant overlap in error bars for the fluorescent output, especially among lower concentrations of miRNA. Despite taking extra steps to decrease potential error (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: Optimization</a>), we still see a large margin of error. After further discussions with Dr. Mark Styczinski from the Georgia Institute of Technology, we concluded that this overlap in error bars is likely caused by the similarity of fluorescent outputs ‒ due to the minute scale of and minimal difference between concentrations ‒ rather than experimental variation. Exponential RCA (eRCA) is an adaptation of RCA that produces exponentially greater fluorescence per unit of miRNA (Liu et al., 2013; Li et al., 2017); therefore, it has the potential to increase the distinction between lower miRNA concentrations and subsequently decrease the margin of error.</p>
+<h2 id="rca-vs-erca">RCA vs. eRCA</h2>
+<p>There are two major differences between eRCA and RCA: the padlock probe design and the amplification process. The RCA padlock probe consists of two parts: the end or “arms†of the padlock probe and the middle sequence (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: 2022 Overview</a>). The arms are the reverse complement of the target miRNA, while the middle sequence corresponds to the reporter mechanism (Jonstrup et al., 2006). The three main steps of RCA are hybridization, ligation, and amplification (Jonstrup et al., 2006) (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: 2022 Overview</a>). The target miRNA first hybridizes to the padlock probe, bringing the two ends of the probe together. Then, the two ends are ligated by SplintR ligase. The bound miRNA serves as a primer for Phi29 DNA polymerase, initiating the amplification of the padlock probe. The result is a long repeating strand of DNA, known as the rolling circle product (RCP), used to quantify the initial concentration of the miRNA (Jonstrup et al., 2006).
+In eRCA, the padlock probes consist of an additional part between the arms and the reporter sequence: the complement of the endonuclease binding site (see Fig. 1) (Li et al., 2017). When the RCP is synthesized during amplification, the nicking endonuclease Nb.BbvCI will recognize this site and cleave the outer strand of DNA, or the RCP, while it is still bound to the padlock probe (Biolabs). This ensures that the padlock probe remains intact while releasing individual strands of the synthesized miRNA and reporter sequence. The produced miRNA can then bind to the padlock probe and initiate the eRCA process again, essentially creating an endless loop of amplification until the reagents are consumed or the reaction is deactivated (see Fig. 2) (Liu et al., 2013; Li et al., 2017). The exponential increase in output can increase the differences in fluorescence emission between miRNA concentrations, subsequently decreasing the margin of error and accuracy of our overall biosensor.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/group-11.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. Padlock probe design for eRCA</div></div>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/erca-overall-diagram.jpeg" style="max-width:700" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 2. eRCA process utilizing Nb.BbvCI</div></div>
+<p>Lettuce is a fluorescent DNA aptamer that binds with and induces the fluorescences of the DFHBI-1T dye (VarnBuhler et al., 2022). Initially, we considered producing whole Lettuce aptamers through the RCA reaction. However, Dr. Mark Styczsinski and Megan McSweeney from the Georgia Institute of Technology advised us against this as the secondary structures of the aptamers would likely interfere with each other on a single DNA strand. As a result, we chose to pursue other reporter mechanisms with the traditional RCA assay (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: Outputs</a>). Contrary to RCA, eRCA produces multiple isolated strands of the reporter sequence, offering the potential to create whole Lettuce aptamers without the risk of secondary structure interference (Liu et al., 2013; Li et al., 2017).
+The hsa-miR-1-3p eRCA padlock probe (<a href="http://parts.igem.org/Part:BBa_K4683002">BBa_K4683002</a>) consists of the following parts: 3’ arm for miR-1 (<a href="http://parts.igem.org/Part:BBa_K4245100">BBa_K4245100</a>), 5’ arm for miR-1 (<a href="http://parts.igem.org/Part:BBa_K4245107">BBa_K4245107</a>), Lettuce aptamer complement (<a href="http://parts.igem.org/Part:BBa_K4683000">BBa_K4683000</a>), and Nb.BbvCI binding sites (<a href="http://parts.igem.org/Part:BBa_M31961">BBa_M31961</a>). As the concentration of the target miRNA increases, the padlock probe produces more miRNAs and Lettuce aptamers, resulting in an exponential increase in fluorescence output in the presence of DFHBI-1T.</p>
+<h2 id="results">Results</h2>
+<p>We performed eRCA with 40.8 pM of miR-1 (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: eRCA Protocol</a>) then ran the reaction on a gel electrophoresis (see Fig. 3) (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: blueGelâ„¢ with RCP</a>). Since eRCA produces multiple shorter strands of DNA (~25 nucleotides), we do not expect to see any bands on the gel. Therefore, the gel in Figure 3 indicates that eRCA was likely successful.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rcagel.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes); 2% agarose gel ran for 1 hour at 48V</div></div>
+<p>We then characterized and quantified the RCP from the eRCA reaction through the Lettuce reporting mechanism (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: eRCA Readout</a>). Resultant fluoresence was quantified in a plate reader at exciation wavelength of 480 nm and emission intensity at 528 nm. The triplicate of eRCA with 40.8 pM of miR-1 exhibits significantly more fluorescence than that of the negative control (no enzyme), indicating that the eRCA reaction was successful (see Fig. 4).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/erca-graph.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Fluorescent readout of RCP for eRCA with 40.8 pM miR-1 using Lettuce aptamer</div></div>
+<p>In the future, we plan to test eRCA with the entire range of clinically relevant miRNA concentrations for coronary artery disease (CAD), as well as validate the applicability of eRCA with spiked serum samples. If the system proves to be more accurate than our current RCA biosensor, we will communicate with Dr. Charles Searles from the Emory University School of Medicine to test our biosensors in actual patient serum.</p></div></template><template data-astro-template="e"><div><h1 id="overview">Overview</h1>
+<p>One of Lambert iGEM’s major goals in 2023 was to increase the inclusivity of our biosensors for all demographics. Through literature review, we found that healthcare settings tend to emphasize responsive care for men, resulting in lower patient retention among women, especially during coronary artery disease (CAD) diagnosis (Johns Hopkins, 2018). We validated this disparity with Dr. Mindy B. Gentry, a cardiologist in Georgia, and Dr. DeLisa Fairweather, a director of research at the Department of Cardiovascular Diseases at Mayo Clinic. To further address this issue, we specifically targeted microRNAs (miRNAs) correlated to CAD in women (see Inclusivity: Estrogen). Discussion with these experts confirmed the use of hsa-miR-20b (<a href="http://parts.igem.org/Part:BBa_K4683005">BBa_K4683005</a>) as a target for our rolling circle amplification (RCA) assay. Moreover, we extended the correlation of miRNAs to CAD through specific racial groups, utilizing CADmir to identify hsa-miR-328-3p (<a href="http://parts.igem.org/Part:BBa_K4683021">BBa_K4683021</a>) and hsa-miR-146a (<a href="http://parts.igem.org/Part:BBa_K4683025">BBa_K4683025</a>). Further collaboration with Dr. Charles Searles, a professor of medicine at Emory Healthcare Hospital, enabled us to utilize hsa-miR-150-5p (<a href="http://parts.igem.org/Part:BBa_K4683009">BBa_K4683009</a>), hsa-miR-122-5p (<a href="http://parts.igem.org/Part:BBa_K4683013">BBa_K4683013</a>), and hsa-miR-30c-5p (<a href="http://parts.igem.org/Part:BBa_K4683017">BBa_K4683017</a>) as they correlate with CAD in African American populations. Therefore, we can incorporate these miRNAs into our research to further accommodate an inclusive approach for understanding and detecting CAD.</p>
+<h2 id="estrogen-mirna">Estrogen miRNA</h2>
+<p>Women after menopause are more likely to develop heart disease due to a decrease in estrogen hormone levels (Brandt, 2013). Studies conducted by Pare et al. in 2022 found that estrogen directly suppresses CAD through the Estrogen Receptor α (ERα) pathway; when estrogen is present in the bloodstream, it activates the ERα pathway, subsequently activating the production of CAD suppressors. Post-menopausal women have lower levels of estrogen within their bloodstream, resulting in the inhibition of CAD suppressors and therefore increasing patient susceptibility to CAD (Brandt, 2013; Pare et al., 2022).
+The concentration of hsa-miR-20b is positively correlated with that of estrogen (Pérez-Cremades et al., 2018). Like the estrogen molecule, miR-20b activates the ERα pathway, regulating the production of CAD suppressors (Pérez-Cremades et al., 2018). Dr. Fairweather validated the use of this miRNA to detect the risk of CAD in post-menopausal women (see Inclusivity: Estrogen).</p>
+<h2 id="other-mirnas">Other miRNAs</h2>
+<p>We conducted a comprehensive literature review using CADmir to efficiently identify relevant miRNAs (see Fig. 1) (see <a href="https://2023.igem.wiki/lambert-ga/software/">Software: CADmir</a>). In just 4 minutes and 12 seconds, we uncovered three miRNAs and a selection of related articles.</p>
+<p>One of these miRNAs, hsa-mir-328-3p, was found to be correlated with cardiovascular disease, specifically atherosclerosis. Therefore, it serves as a valuable predictive tool for early detection of heart disease, with a specific focus on the African American population (Iwańczyk, 2023). Similarly, hsa-mir-146a is a miRNA associated with the regulation of inflammation and CAD, predominantly linked to the Latin American population (Iwańczyk, 2023). These findings are of particular relevance to our team after considering the demographic makeup of Georgia. Notably, the African American and Latin American population have experienced significant growth, as demonstrated by the statistic: “the Black (non-Hispanic) population had the most growth increasing by 518,670 from 2.9 million in 2010 to 3.5 million in 2021†(Georgia Population by Year, County, Race, & More, 2023).</p>
+<p>To further complement our findings, we consulted with Dr. Searles, who recommended three additional miRNAs correlated with CAD in African American populations: hsa-mir-150-5p, hsa-mir-122-5p, and hsa-mir-30c-5p.</p>
+<h1 id="gel-experimentation">Gel Experimentation</h1>
+<p>We performed RCA on miR-20b, miR-328, miR-146a using our respective padlock probe designs (<a href="http://parts.igem.org/Part:BBa_K4683008">BBa_K4683008</a>; <a href="http://parts.igem.org/Part:BBa_K4683024">BBa_K4683024</a>; <a href="http://parts.igem.org/Part:BBa_K4683028">BBa_K4683028</a>) and ran the subsequent rolling circle product (RCP) on a 1% agarose gel (see Fig. 2). The gel exhibited a fluorescent band of DNA very close to the well, indicating that a long strand of DNA greater than 1 kB - our RCP - was produced. Therefore, we can validate that our reaction was successful. In the future, we plan to expand our collection of biosensors to detect more miRNAs related to CAD and other demographics.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/igem-estrogen-label.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 13. RCP from RCA reaction with miR-20b, miR-328,miR-146; 2% agarose gel ran for 1 hour at 48V </div></div></div></template></astro-island>
+<div class="py-6"></div>
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+<a href="https://doi.org/10.1039/c4ra14467h">https://doi.org/10.1039/c4ra14467h</a></p></div>
+ </astro-slot></div></div></div></body></astro-island>
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+ <h1 id="rca-outputs-results">RCA Outputs Results</h1>
+<h2 id="linear-dna-probes-vs-lettuce">Linear DNA Probes vs Lettuce</h2>
+<h3 id="linear-dna-probes">Linear DNA Probes</h3>
+<p>Fluorophore (<a href="http://parts.igem.org/Part:BBa_K4245130">BBa_K4245130</a>) and quencher-tagged linear DNA probes (<a href="http://parts.igem.org/Part:BBa_K4245132">BBa_K4245132</a>) were used to quantify the presence and concentration of target microRNA (miRNA) in samples (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: outputs</a>). The decrease in fluorescence in the solution can be correlated with a specific concentration of miRNA through characterization. Similar to last year, we characterized and quantified rolling circle product (RCP) through the linear probes reporting mechanism (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Linear DNA Probes with RCP</a>). Resultant fluoresence was quantified in plate reader at exciation wavelength of 480 nm and emission intensity at 528 nm. There is a negative logarithmic correlation between the miRNA concentrations and the relative fluorescence units (RFU) (see Fig. 1).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src=" https://static.igem.wiki/teams/4683/wiki/linearprobes.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. Characterization curve showing a negative logarithmic relationship between miR-1 concentrations and RFU from linear DNA probes </div></div>
+<h3 id="lettuce">Lettuce</h3>
+<p>Lettuce is a fluorescent DNA aptamer that binds with the dye DFHBI-1T within its secondary structure, thus causing the dye to fluoresce (VarnBuhler et al., 2022). After the rolling circle amplification (RCA) reaction, the apatamer is able to bind to the RCP and produce fluorescence; therefore, an increase in miRNA concentration should correlate with an increase in fluorescence (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: outputs</a>). We characterized and quantified RCP through the Lettuce reporting mechanism (<a href="http://parts.igem.org/Part:BBa_K4245134">BBa_K4245134</a>; <a href="http://parts.igem.org/Part:BBa_K4245135">BBa_K4245135</a>) (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Lettuce with RCP</a>). Resultant fluoresence was quantified in plate reader at exciation wavelength of 480 nm and emission intensity at 528 nm. There is a positive logarithmic correlation between the miRNA concentrations and the relative fluorescence units (RFU) (see Fig. 2).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/lettuce.png " alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 2. Characterization curve showing a positive logarithmic relationship between miR-1 concentrations and RFU from split Lettuce aptamer</div></div>
+<h3 id="comparison">Comparison</h3>
+<p>Both reporter mechanisms resulted in significant SEM overlap between the lower miRNA concentrations, making accurate differentiation of miRNAs difficult. However, there was no indication that Lettuce outperformed linear DNA probes. Therefore, we continued to conduct further experiments with linear probes. In the future, we hope to find another on-state reporter that would make reading RCA results more comprehensible and accurate. Such reporters include molecular beacons and other DNA aptamers.</p>
+<h1 id="rca-optimization-results">RCA Optimization Results</h1>
+<h2 id="specificity">Specificity</h2>
+<p>We ran RCA using the hsa-miR-1-3p padlock (<a href="http://parts.igem.org/Part:BBa_K4245200">BBa_K4245200</a>) in the presence of four different miRNA sequences (see Fig. 1). The first is the original miR-1 sequence (<a href="http://parts.igem.org/Part:BBa_K4245006">BBa_K4245006</a>), which is expected to hybridize to the padlock and result in the greatest fluorescence decrease. Two sequences with differing single nucleotide variants (SNVs) found from the National Library of Medicine microRNA 1-1 database were utilized to determine the specificity of RCA: one with a single SNV (<a href="http://parts.igem.org/Part:BBa_K4683003">BBa_K4683003</a>) and one with three SNVs (<a href="http://parts.igem.org/Part:BBa_K4683004">BBa_K4683004</a>). hsa-miR-133a-3p (<a href="http://parts.igem.org/Part:BBa_K4245009">BBa_K4245009</a>) was also included to ensure the padlock would not ligate to any miRNA.</p>
+<p>We ran the reactions and control on a gel electrophoresis; only the well with 40.8 pM of miR-1 showed visible bands of DNA near the top of the wells, which is likely our RCP (see Fig. 3) (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: blueGelâ„¢ with RCP</a>).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/specificity-gel.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. Gel results: RCA with A: miR-1, B: 1 SNV, C: 3 SNVs, D: miR-133a; 2% agarose gel ran for 1 hour at 48V</div></div>
+<p>We then tested the RCP with linear DNA probes and quantified the resultant fluorescence in a plate reader at an emission wavelength of 480 nm and an excitation wavelength of 528 nm (see Fig. 4) (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Linear DNA Probes with RCP</a>. The RCA reaction utilizing the miR-1 padlock probe with miR-1 exhibited significantly less fluorescence than the other miRNAs. Since linear DNA probes produce a negative correlation between fluorescence and miRNA concentration, this result, along with the gel, indicates that RCA is specific to single nucleotide differences.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/specificty-graph.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Comparison of RCA with miR-1, 1SNV, 3SNVs, and 133a fluorescence output using linear DNA probes.</div></div>
+<h2 id="emory-testing">Emory Testing</h2>
+<p>In 2023, Lambert iGEM continued communication with Dr. Charles Searles of the Emory University in order to test whether our biosensor could be practical and applicable as a diagnostic tool. Researchers in the Searles Cardiovascular Lab ran RCA on 40.8 pM of miR-1 with SYBRâ„¢ Safe dye (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: SYBRâ„¢ Safe with RCP</a>), which fluoresces when bound to ssDNA, as the output. As shown in Figure 5, there was a significant increase in fluorescence in the RCA reaction as compared to that of the controls, therefore validating the application of our biosensor in other labs.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/emory.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 5. Triplicate of RCA with SYBRâ„¢ Safe output done by independent hands</div></div>
+<h2 id="ligation-time">Ligation Time</h2>
+<p>Lambert iGEM’s 2022 RCA protocol (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: RCA 2022 protocol</a>) requires samples to be incubated in the thermocycler at 37°C for two hours. However, the properties and usage of SplintR Ligase show that the reaction is successful with a 15-minute ligation time (Avantor Staff). Therefore, we ran RCA utilizing the miR-1 padlock probe (<a href="http://parts.igem.org/Part:BBa_K4245200">BBa_K4245200</a>) with four different ligation times with miR-1 (<a href="http://parts.igem.org/Part:BBa_K4245006">BBa_K4245006</a>): 15 minutes, 30 minutes, one hour, and two hours. After amplification, the reactions and controls were run on a gel; the bright bands near the top of the well showed that DNA product was produced for all reactions except for 15 minutes (see Fig. 6). Moving forward, we implemented a 30-minute ligation time (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Optimized RCA protocol</a>).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/screenshot-2023-10-11-at-4-32-50-pm.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 6. 1: 15-minute ligation, 2: 30-minute ligation, 3: 1-hour ligation, 4: 2-hour ligation, A: control (no enzymes); 2% agarose gel ran for 1 hour at 48V</div></div>
+<h2 id="amplification-time">Amplification Time</h2>
+<p>Lambert iGEM’s 2022 RCA protocol (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: RCA 2022 protocol</a>) requires samples to be incubated in the thermocycler at 37°C for eight hours for amplification. To reduce this time, we ran RCA with two different concentrations of miR-1. After hybridization and ligation, we incubated the reactions in the plate reader at 37°C with 4uL SYBR™ Safe (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: SYBR™ Safe with RCP</a>). The reactions were run overnight and the subsequent fluorescence was quantified in a plate reader (excitation wavelength 480 nm; emission wavelength: 528 nm): 30-minute intervals (see Fig. 7). Over time, the two RCA reactions increased in fluorescence, with no SEM overlap observed between the starting time and 5-hour mark. This suggests that RCP can be produced optimally starting at 5 hours. The significant increase in fluorescence between the RCA reactions and controls shows that SYBR™ Safe can determine the presence of RCP; however, the lack of difference between the 40.8 pM and .41 pM of miR-1 fluorescence indicates that the dye is not sensitive enough to differentiate between miRNA concentrations. Therefore, we did not continue to utilize SYBR™ Safe as a reporter.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/specificty-graph.png" style="max-width:[400px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 7. RCA amplification optimization reaction with 40.8 pM and 0.41 pM of miRNA. No SEM overlap between starting point and 5-hour incubation time. SEM overlaps between 40. pM and .41 pM show that miRNA concentrations cannot be differentiated by SYBRâ„¢ Safe.</div></div>
+<h2 id="phi29-xt">Phi29-XT</h2>
+<p>Lambert iGEM’s RCA protocol (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Optimized RCA protocol</a>) utilizes Phi29 DNA polymerase to perform amplification, resulting in an 5 hour amplification time. Phi29- XT DNA polymerase is an optimized enzyme with improved thermostability and sensitivity, which could shorten this time down to 2 hours (Biolabs). We ran RCA following the protocol for Phi29-XT on the New England Biolabs website (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Amplification with Phi29- XT</a>). The reactions and controls were run on a gel; no visible bands could be seen on the gel, indicating that RCP was not produced and therefore the reaction was not successful. As a result, we did not pursue utilizing Phi29-XT for further RCA reactions (see Fig. 8).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/phi-xt.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 8. A: control (no enzymes), X: RCA with phi29-XT DNA polymerase; 2% agarose gel ran for 1 hour at 48V</div></div>
+<h1 id="exponential-rca-erca-results">Exponential RCA (eRCA) Results</h1>
+<p>Although the data from testing rolling circle amplification (RCA) with the target microRNA (miRNA) indicates high sensitivity — detecting lower limits of 2 pM — it lacks the ability to clearly differentiate between various concentrations of miRNA. Exponential RCA (eRCA) is an adaptation of RCA that produces exponentially greater fluorescence per unit of miRNA (Liu et al., 2013; Li et al., 2017); therefore, it has the potential to increase the distinction between lower miRNA concentrations and subsequently increase the margin of error (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: eRCA</a>).</p>
+<h2 id="rca-vs-erca">RCA vs. eRCA</h2>
+<p>We performed eRCA with 40.8 pM of miR-1 (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: eRCA Protocol</a>) then ran the reaction on a gel electrophoresis (see Fig. 9) (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: blueGelâ„¢ with RCP</a>). Since eRCA produces multiple shorter strands of DNA (~25 nucleotides), we do not expect to see any bands on the 2% agarose gel. Therefore, the gel in Figure 9 indicates that eRCA was likely successful.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/screen-shot-2023-10-11-at-4-41-35-pm.png" style="max-width:[250px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 9. A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes)</div></div>
+<p>We then characterized and quantified the RCP from the eRCA reaction through the Lettuce reporting mechanism (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: eRCA Readout</a>). The triplicate of eRCA with 40.8 pM of miR-1 significantly exhibits more fluorescence than that of the negative control (no enzyme), indicating that the eRCA reaction was successful (see Fig. 10).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/rca-optimization/rcagel.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 10. A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes); 2% agarose gel ran for 1 hour at 48V</div></div>
+<p>In the future, we plan to test eRCA with the entire range of clinically relevant miRNA concentrations for coronary artery disease (CAD), as well as validate the applicability of eRCA with spiked serum samples. If the system proves to be more accurate than our current RCA biosensor, we will communicate with Dr. Charles Searles from the Emory University School of Medicine to test our biosensors in actual patient serum.</p>
+<h1 id="capillary-rca-results">Capillary RCA Results</h1>
+<p>To create a more accessible miRNA detection system, Lambert iGEM also adopted another output approach: capillary-rolling circle amplification (cpRCA). This method offers faster reaction times and eliminates the need for expensive and specialized equipment like plate readers and fluorometers, making it suitable for rapid point-of-care testing of miRNAs (Hixson & Ward, 2021) (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: outputs</a>).</p>
+<p>After discussion with Dr. Charles Searles from the Emory University School of Medicine, we determined that the upper limit of clinically relevant miRNA concentrations in patients with CAD is around 40 pM. We initially ran cpRCA with the miR-1 padlock probe (<a href="http://parts.igem.org/Part:BBa_K4245200">BBa_K4245200</a>) and 40.8 pM of miR-1 (<a href="http://parts.igem.org/Part:BBa_K4245006">BBa_K4245006</a>) but were not able to visualize any dots of miRNA within the capillary tube (see Fig. 11) (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: Capillary RCA</a>). This is likely because the higher concentration of miRNA resulted in a significant overlap of fluorescent regions within the capillary tube, leading to inaccurate quantification through cpRCA.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/screenshot-2023-10-11-at-3-22-41-am.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 11. Visualization of cpRCA with 40.8 pM of miRNA
+</div></div>
+<p>To ensure the amplified products were countable, we diluted the sample down to 1.66 fM, or approximately 50 molecules of miRNA. To validate that cpRCA would still be successful at this lower concentration, we ran a gel electrophoresis on the RCP from the cpRCA reaction after 4 hours of amplification (see <a href="https://2023.igem.wiki/lambert-ga/experiments/">Experiments: blueGelâ„¢ with RCP</a>) using both 40pM and 1.66fM of miRNA to observe the difference. By analyzing the results on the gel, we concluded that RCP was likely produced with both concentrations as the gel exhibited fluorescent bands of DNA very close to the wells, while no band was expressed in the control (see Fig. 12). As a result, we can infer that cpRCA was successful after 4 hours of amplification and at only 50 molecules of miRNA, which is more efficient than the 8 hour amplification of traditional RCA, and increases the sensitivity of the assay.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/screenshot-2023-10-11-at-3-21-24-am.png" style="max-width:[300px]" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 12. Lane 1: ladder, Lanes 2-3: cpRCA with 1.66 fM miR-1, Lanes 4-5: cpRCA with 40.8 pM miR-1, Lanes 6-7: control; 2% agarose gel ran for 1 hour at 48V</div></div>
+<p>In the future, we plan to conduct further experimentation with lower miRNA concentrations to better visualize the fluorescent regions in the capillary tube, as well as characterize the full range of relevant miRNA concentrations for CAD.</p>
+<h1 id="inclusivity-estrogen-rca-results">Inclusivity Estrogen RCA Results</h1>
+<p>We performed RCA on miR-20b, miR-328, miR-146a using our respective padlock probe designs (<a href="http://parts.igem.org/Part:BBa_K4683008">BBa_K4683008</a>; <a href="http://parts.igem.org/Part:BBa_K4683024">BBa_K4683024</a>; <a href="http://parts.igem.org/Part:BBa_K4683028">BBa_K4683028</a>) and ran the subsequent rolling circle product (RCP) on a 1% agarose gel (see Fig. 2). The gel exhibited a fluorescent band of DNA very close to the well, indicating that a long strand of DNA greater than 1 kB - our RCP - was produced. Therefore, we can validate that our reaction was successful. In the future, we plan to expand our collection of biosensors to detect more miRNAs related to CAD and other demographics.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/human-practices/igem-estrogen-label.png" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 13. RCP from RCA reaction with miR-20b, miR-328,miR-146; 2% agarose gel ran for 1 hour at 48V</div></div>
+<h1 id="phi29-dna-polymerase-protein-purification-results">phi29 DNA Polymerase Protein Purification Results</h1>
+<p>After running our purified phi29 DNA polymerase from immobilized metal affinity chromatography (IMAC) on the Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE)<a href="https://2023.igem.wiki/lambert-ga/experiments/">see Experiments: Protein Purification</a>, Lambert iGEM saw bands around 50kD ~ 75kD. When we compared this band to the commercial phi29 DNA polymerase from New England Biolabs, it indicated that the protein was purified as the bands appeared in the same area (see Fig. 14).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/frame-10-1.png" style="max-width:800" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 14. SDS-PAGE with purified phi29 DNA polymerase, displaying bands at 50kD ~ 75kD</div></div>
+<p>Our team tested the efficiency of the purified phi29 DNA polymerase in rolling circle amplification (RCA) by comparing the rolling circle products (RCP) of the commercial and purified phi29 DNA polymerase. We added the same volume of both to RCA reactions and ran the rolling circle products on a gel. We saw fluorescent bands of DNA very close to the wells, indicating that both the RCA assay utilizing commercial and purified enzyme reactions were successful (see Fig. 15).</p>
+<p>If we buy phi29 DNA polymerase from New England Biolabs, it will cost $251.00 for 1,250 units. However, after we utilized the NEBExpress Ni Spin Columns ($10 per prep to purify the enzyme), we were able to produce 200ul in one prep, or approximately 2,000 units. Because, it can be assumed that the unit between the commercially bought enzyme and our purified enzyme is similar, if commercially bought, this 200ul amount would cost around $400. Therefore, we can determine that our purified protein is significantly more affordable than the commercially bought protein.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/hardware/screenshot-2023-10-12-at-10-37-15-am.png" style="max-width:800" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 15. Gel comparing the rolling circle product (RCP) between the team’s purified phi29 DNA polymerase and that commercially available phi29</div></div>
+<p>In the future, we plan to more accurately quantify the activity of the purified units of phi29 DNA polymerase. Additionally, we hope to test the accuracy of our enzyme by quantifying the RCP using linear DNA probes (see <a href="https://2023.igem.wiki/lambert-ga/rca/">RCA: outputs</a>) to validate its use of point-of-care testing.</p>
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+<a href="https://doi.org/10.1039/c4ra14467h">https://doi.org/10.1039/c4ra14467h</a></p></div>
+ </astro-slot></div></div></div></body></astro-island>
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+ <h1 id="laboratory">Laboratory</h1>
+<p>Safety is a fundamental aspect of our experimentation, spanning from the wet lab to hardware applications. This page serves as a comprehensive guide outlining the safety measures implemented by Lambert iGEM to establish a secure laboratory environment.</p>
+<h1 id="team-safety">Team Safety</h1>
+<ul>
+<li>Wash hands coming in and out of the laboratory.</li>
+<li>Protect eyes, mucous membranes, open cuts, and wounds from contact with biohazard material.</li>
+<li>Do not eat or drink when in the lab area.</li>
+<li>Always use gloves and splash-proof goggles.</li>
+<li>Tie back loose or long hair.</li>
+<li>Disinfect all surfaces with 70% ethanol before working.</li>
+<li>Disinfect all disposable tips, glassware, and tubes by soaking in 10% bleach solution for 20 minutes and then disposing in normal waste.</li>
+<li>Dispose of growth plates by disposing them into a biohazard container which gets autoclaved.</li>
+<li>Check all equipment for good working order: no chips, torn chords, or cracks. Report any issues to an instructor immediately.</li>
+<li>When pipetting, do not touch the tip to the side of the container.</li>
+<li>Do not lay caps of tubes upside down. Use masking tape to hold the bottom of the cabinet.</li>
+<li>Clean work area with 70% ethanol after working.</li>
+<li>Clean up all glassware and labware before leaving the lab.</li>
+<li>Place all backpacks and stools to the side of the lab to keep walkways clear.</li>
+<li>Always know the correct procedure for the disposal of lab materials.</li>
+</ul>
+<h1 id="project-safety">Project Safety</h1>
+<h2 id="overview">Overview</h2>
+<p>Current detection methods for coronary artery disease (CAD) are costly and invasive. To address these issues, 2022 Lambert iGEM created CADlock: a more accessible, point-of-care screening tool that detects microRNA upregulated in patients with CAD. In 2023, we plan to further improve CADlock’s affordability, accuracy, and accessibility, ultimately increasing its inclusivity by targeting multiple microRNAs that can be measured conjointly with current diagnostic tools.</p>
+<h2 id="wetlab">Wetlab</h2>
+<p><strong>Nonpathogenic <em>E.coli</em> Chassis</strong></p>
+
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+<table><thead><tr><th>E.coli Strain</th><th>Purpose</th><th>Pathogenicity</th><th>Health/Environmental hazards</th></tr></thead><tbody><tr><td>BL21</td><td>Transformation</td><td>Nonpathogenic-for lab cloning</td><td>Extremely limited-Biosafety Level 1 Lab</td></tr><tr><td>DH5-alpha</td><td>Transformation</td><td>Nonpathogenic-for lab cloning</td><td>Extremely limited-Biosafety Level 1 Lab</td></tr></tbody></table>
+<h2 id="discarding-cells-in-the-field">Discarding Cells in the Field</h2>
+<p>A solution of 1% sodium hypochlorite and 70% ethanol will kill biosensor cells. Results show that a 1% sodium hypochlorite solution sprayed on the surface and let sit for 10 minutes will effectively remove all DNA, saliva, blood, semen, and skin cells from any smooth or pitted surface when wiped down with 70% ethanol afterward. However, sodium hypochlorite solution followed by ethanol can produce amounts of gaseous chlorine above recommended exposure levels. As a result, 1% sodium hypochlorite followed by distilled water was tested and proven to be effective as well (Kaye et al., 2015).</p>
+<h2 id="software">Software</h2>
+<p>We developed CADmir, a heart disease-related miRNA database that uses large language models (LLMs) to curate the current research on heart disease-related miRNAs. CADmir only uses open-access papers that allow for text mining from PubMed. To ensure proper usage of LLMs and AI, we used OpenAI’s GPT-3.5 to process all of our data and power CADmir. OpenAI uses safety measures like reviewing use cases and fine-tuning models to ensure GPT-3.5 is used properly (OpenAI, 2022). We also analyzed the outputed results to ensure that all information is true and comes from reliable sources.</p>
+<h2 id="electronic-devices">Electronic Devices</h2>
+<p>Micro-Q and LabPilot were built and prototyped following proper safety precautions while working with the electronic devices. Whenever working with electronics such as the Arduino or an ESP-32 microcontroller, the devices are placed on antistatic materials. When using power supplies or AC adapters, we take precautions such as checking the devices for damage before use and making sure all liquids, food items, and potentially flammable materials are kept away. While soldering electronics, team members wear safety glasses, use electrically and thermally insulated gloves, and work in a well-ventilated and isolated environment. We utilized proper eye protection and thermal management procedures whilst working with LEDs to ensure our safety. After working with any electronic device, we make sure it is powered off, and disconnected from the power source; including removing batteries in certain devices.</p>
+<h2 id="drills">Drills</h2>
+<p>To assemble the separate parts of LabPilot, Lambert iGEM used a drill. The drill has the potential to get tangled in personal belongings and to cause serious harm.
+Precautions</p>
+<ul>
+<li>Wore PPE, including gloves and protective eyewear</li>
+<li>Turned it off when not using</li>
+<li>Tucked in all loose belongings</li>
+</ul>
+<h2 id="glass-capillary-tubes">Glass Capillary Tubes</h2>
+<p>During the development of our capillary-tube based RCA assay, we used glass capillary tubes. These tubes pose a safety risk due to their potential to shatter and produce glass shards, which have the potential to pierce the skin and cause lacerations.</p>
+<p><strong>Precautions:</strong></p>
+<ul>
+<li>Handling tubes gently using tweezers to avoid direct contact</li>
+<li>Wearing PPE, including eye protection and gloves, to cover skin and prevent injury</li>
+</ul>
+<h2 id="3d-printer">3D Printer</h2>
+<p>We prototyped Micro-Q and our frugal incubator using 3D printers, which pose potential safety risks because certain parts, like the plastic-melting nozzle, can heat up to 220°C. Furthermore, leaving the 3D printer unattended would be dangerous because its high temperatures could cause a fire.</p>
+<p><strong>Precautions:</strong></p>
+<ul>
+<li>Crash detection and heat overload software built into the printers</li>
+<li>Team member or PI is present during printing to intervene in case of an accident</li>
+</ul>
+<h2 id="human-practices-and-education">Human Practices and Education</h2>
+<p>Participants in all surveys provided signed consent for the release of their responses from themselves or a legal guardian. Additionally, participants in all events hosted by Lambert iGEM provided consent for photo and video release. Proper safety instructions and procedures were given during the in-person and virtual camp activities.</p>
+<h1 id="references">References</h1>
+<div class="pl-6 -indent-6"><p>Ballantyne, K. N., Salemi, R., Guarino, F., Pearson, J. R., Garlepp, D.,
+Fowler, S., & van Oorschot, R. A. (2015). DNA contamination
+minimisation–finding an effective cleaning method. Australian Journal of
+Forensic Sciences, 47(4), 428-439. Retrieved from
+<a href="https://doi.org/10.1080/00450618.2015.1004195">https://doi.org/10.1080/00450618.2015.1004195</a></p></div>
+<div class="pl-6 -indent-6"><p>National Institute of Health. (2014). Biosafety and biosecurity in the United
+States. Federal Select Agent Program. Retrieved from
+<a href="https://www.nih.gov/sites/default/files/research-training/usg-safety-factsheet-2014.pdf">https://www.nih.gov/sites/default/files/research-training/usg-safety-factsheet-2014.pdf</a></p></div>
+<div class="pl-6 -indent-6"><p>OpenAI. (2022). Lessons learned on language model safety and misuse.
+Openai.com. <a href="https://openai.com/research/language-model-safety-and-misuse">https://openai.com/research/language-model-safety-and-misuse</a></p></div>
+<div class="pl-6 -indent-6"><p>Rochester Institute of Technology. (2019). 3-D Printer Safety. Retrieved from
+<a href="https://www.rit.edu/fa/grms/ehs/content/3-d-printer-safety">https://www.rit.edu/fa/grms/ehs/content/3-d-printer-safety</a>.</p></div>
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+ <h1 id="cadmir">CADmir</h1>
+<h2 id="overview">Overview</h2>
+<p>The discovery of the first microRNAs (miRNAs) dates back to 1993; yet, it was not until the early 21st century that research on miRNAs gained significant momentum (Bhaskaran et al., 2014). Even with this emerging state, detailed information about each miRNA is scattered throughout scientific literature. Previous attempts by miRNA databases such as miRBase to extract and organize such information using text mining methods are insufficient, as their filtering and word cloud techniques only capture a fraction of the data from the literature (see Fig. 1) (Kozomara et al., 2019).</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/software/wordcloud.png" style="max-width:xs" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 1. miRBase’s word clouds for miR-21: the only functional information regarding the miRNA.</div></div>
+<p>Today’s system of publishing literature is based on journals, which are the established way of academic communication and exchange of research findings. Publishing in journals started in the early 17th century to facilitate scientific information exchange between scholars. What started off as around 500 journals publishing 2-3 times per year has boomed into at least 30,000 journals today publishing more than 5 million papers each year (Tress Academic, 2019). Publishing through journals allows scholars quick access to high-quality peer-reviewed academic literature, but as the volume of papers published grew exponentially, it has become increasingly difficult to keep up with scientific literature and conduct literature reviews, even within a specialized field. This difficulty is especially relevant for the field of miRNAs as not only do researchers have to look through and compare numerous papers, but they also have to keep track of the different methods that other researchers use to measure miRNAs.</p>
+<p>While researching miRNAs for CADlock, Lambert iGEM experienced firsthand the disorganization of data surrounding miRNAs. To confirm whether other individuals faced this issue, we consulted Dr. Christian Delles, a miRNA researcher from the University of Glasgow, who verified this lack of organization. Furthermore, Dr. Charles Searles, a cardiologist from Emory School of Medicine, reaffirmed the need for a database to streamline information on miRNAs related to coronary artery disease (CAD).</p>
+<p>To address the need for a database that extracts functional miRNA information and organizes the vast volume of scientific literature regarding the subject, we developed CADmir: a miRNA search engine utilizing large language models to process and organize the extensive information about miRNAs. With CADmir, most of the relevant data about specific miRNAs are consolidated in one place, improving information accessibility and reducing the time barrier associated with obtaining high-quality scientific information. CADmir works alongside existing databases such as miRBase to provide links to relevant scientific literature and incorporate sequence and genomic data. CADmir aims to expand research in miRNAs and create a new precedent for the organization of vital scientific information.</p>
+<h2 id="design">Design</h2>
+<style>astro-island,astro-slot,astro-static-slot{display:contents}</style><script>(()=>{var e=async t=>{await(await t())()};(self.Astro||(self.Astro={})).load=e;window.dispatchEvent(new Event("astro:load"));})();;(()=>{var d;{let p={0:t=>u(t),1:t=>l(t),2:t=>new RegExp(t),3:t=>new Date(t),4:t=>new Map(l(t)),5:t=>new Set(l(t)),6:t=>BigInt(t),7:t=>new URL(t),8:t=>new Uint8Array(t),9:t=>new Uint16Array(t),10:t=>new Uint32Array(t)},h=t=>{let[e,n]=t;return e in p?p[e](n):void 0},l=t=>t.map(h),u=t=>typeof t!="object"||t===null?t:Object.fromEntries(Object.entries(t).map(([e,n])=>[e,h(n)]));customElements.get("astro-island")||customElements.define("astro-island",(d=class extends HTMLElement{constructor(){super(...arguments);this.hydrate=async()=>{var i;if(!this.hydrator||!this.isConnected)return;let e=(i=this.parentElement)==null?void 0:i.closest("astro-island[ssr]");if(e){e.addEventListener("astro:hydrate",this.hydrate,{once:!0});return}let n=this.querySelectorAll("astro-slot"),o={},a=this.querySelectorAll("template[data-astro-template]");for(let r of a){let s=r.closest(this.tagName);s!=null&&s.isSameNode(this)&&(o[r.getAttribute("data-astro-template")||"default"]=r.innerHTML,r.remove())}for(let r of n){let s=r.closest(this.tagName);s!=null&&s.isSameNode(this)&&(o[r.getAttribute("name")||"default"]=r.innerHTML)}let c;try{c=this.hasAttribute("props")?u(JSON.parse(this.getAttribute("props"))):{}}catch(r){let s=this.getAttribute("component-url")||"<unknown>",y=this.getAttribute("component-export");throw y&&(s+=` (export ${y})`),console.error(`[hydrate] Error parsing props for component ${s}`,this.getAttribute("props"),r),r}await this.hydrator(this)(this.Component,c,o,{client:this.getAttribute("client")}),this.removeAttribute("ssr"),this.dispatchEvent(new CustomEvent("astro:hydrate"))}}connectedCallback(){!this.hasAttribute("await-children")||this.firstChild?this.childrenConnectedCallback():new MutationObserver((e,n)=>{n.disconnect(),setTimeout(()=>this.childrenConnectedCallback(),0)}).observe(this,{childList:!0})}async childrenConnectedCallback(){let e=this.getAttribute("before-hydration-url");e&&await import(e),this.start()}start(){let e=JSON.parse(this.getAttribute("opts")),n=this.getAttribute("client");if(Astro[n]===void 0){window.addEventListener(`astro:${n}`,()=>this.start(),{once:!0});return}Astro[n](async()=>{let o=this.getAttribute("renderer-url"),[a,{default:c}]=await Promise.all([import(this.getAttribute("component-url")),o?import(o):()=>()=>{}]),i=this.getAttribute("component-export")||"default";if(!i.includes("."))this.Component=a[i];else{this.Component=a;for(let r of i.split("."))this.Component=this.Component[r]}return this.hydrator=c,this.hydrate},e,this)}attributeChangedCallback(){this.hydrate()}},d.observedAttributes=["props"],d))}})();</script><astro-island uid="Z1teEaz" prefix="r2" component-url="/lambert-ga/_astro/ImageCarousel.440b7446.js" component-export="default" renderer-url="/lambert-ga/_astro/client.4f88357e.js" props="{"caption":[0,"Figure 2. Screenshots of CADmir working."],"images":[1,[[0,"https://static.igem.wiki/teams/4683/wiki/software/cadmir-screenshot.png"],[0,"https://static.igem.wiki/teams/4683/wiki/software/cadmir-search.png"]]]}" ssr="" client="load" opts="{"name":"Image","value":true}" await-children=""><div class="flex w-full flex-col items-center justify-center p-1"><div class="carousel-root" tabindex="0"><div class="carousel carousel-slider" style="width:100%"><ul class="control-dots"><li class="dot selected" value="0" role="button" tabindex="0" aria-label="slide item 1"></li><li class="dot" value="1" role="button" tabindex="0" aria-label="slide item 2"></li></ul><button type="button" aria-label="previous slide / item" class="control-arrow control-prev control-disabled"></button><div class="slider-wrapper axis-horizontal"><ul class="slider animated" style="-webkit-transform:translate3d(0,0,0);-ms-transform:translate3d(0,0,0);-o-transform:translate3d(0,0,0);transform:translate3d(0,0,0);-webkit-transition-duration:350ms;-moz-transition-duration:350ms;-o-transition-duration:350ms;transition-duration:350ms;-ms-transition-duration:350ms"><li class="slide selected previous"><div class="slide"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/software/cadmir-screenshot.png"/></div></li><li class="slide"><div class="slide"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/software/cadmir-search.png"/></div></li></ul></div><button type="button" aria-label="next slide / item" class="control-arrow control-next"></button><p class="carousel-status">1 of 2</p></div><div class="carousel"><div class="thumbs-wrapper axis-vertical"><button type="button" class="control-arrow control-prev control-disabled" aria-label="previous slide / item"></button><ul class="thumbs animated" style="-webkit-transform:translate3d(0,0,0);-moz-transform:translate3d(0,0,0);-ms-transform:translate3d(0,0,0);-o-transform:translate3d(0,0,0);transform:translate3d(0,0,0);-ms-transform:translate3d(0,0,0);-webkit-transition-duration:350ms;-moz-transition-duration:350ms;-ms-transition-duration:350ms;-o-transition-duration:350ms;transition-duration:350ms;-ms-transition-duration:350ms"><li class="thumb selected" aria-label="slide item 1" style="width:80px" role="button" tabindex="0"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/software/cadmir-screenshot.png"/></li><li class="thumb" aria-label="slide item 2" style="width:80px" role="button" tabindex="0"><img alt="sample_file" src="https://static.igem.wiki/teams/4683/wiki/software/cadmir-search.png"/></li></ul><button type="button" class="control-arrow control-next control-disabled" aria-label="next slide / item"></button></div></div></div><div class="text-center italic">Figure 2. Screenshots of CADmir working.</div></div></astro-island>
+<p>In developing CADmir, Lambert iGEM had two goals: a user-friendly interface and an effective organization of the thousands of papers regarding microRNAs (miRNAs) and coronary artery disease (CAD). To achieve this, we leveraged new technologies, such as OpenAI’s GPT-3.5 Turbo large language model, to process and analyze thousands of articles (OpenAI, 2023). Furthermore, we employed modern user-experience design principles, so that CADmir is human-centric and intuitive. By combining powerful AI-powered tools with a carefully crafted design, CADmir empowers researchers to swiftly search for information regarding heart disease-related miRNAs and retrieve comprehensive details accompanied by proper citations.</p>
+<h3 id="gathering-data">Gathering Data</h3>
+<p>CADmir creates an efficient way of interfacing with the vast amount of literature by extracting information from the existing scientific literature and embedding them in a format that’s easily accessible. Scientific literature, like all forms of text, has semantic meaning that is hard for computers to understand. While there may be numerous papers regarding the same topic, all of their information is spread across numerous papers, making it tedious to compare and compile. Sentence embeddings are numerical representations of sentences and can encode semantic information mathematically, allowing computers to understand the meaning of the text (Google, 2022). By using embeddings, CADmir can automate the tedious task of reading and comparing papers manually.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/software/cadmirautomatedpipelineha.png" style="max-width:100%" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 3. A pipeline of CADmir’s data-gathering process</div></div>
+<p>CADmir utilizes an automated pipeline to extract and organize data (see Fig. 3). First, CADmir uses a script to gather a curated collection of miRNA research papers focused on CAD. The script uses a web scraper to obtain PDFs from PubMed with the keywords “miRNA†and “coronary arteryâ€. For each downloaded PDF, CADmir extracts the body of the paper and splits each paper into individual sentences. Next, each sentence is passed through OpenAI’s embedding model and transformed into an embedding, a mathematical representation that computers can understand, before being added to a specialized database called a vector database that can store and search embeddings (OpenAI, 2023; Schwaber-Cohen, 2023). Embeddings are useful for clustering and organizing large amounts of unstructured data since they capture semantic meaning and relationships between sentences. Embeddings that are closer to each other mathematically are more semantically similar; thus, they can be searched and synthesized to automate various natural language tasks (Google, 2022). OpenAI’s embedding model uses large language models’ understanding of natural language to intelligently embed sentences and mathematically represent their meaning (OpenAI, 2023). In total, CADmir processed 3,845 papers or 98,008 sentence embeddings.</p>
+<h3 id="building-the-knowledge-base">Building the knowledge base</h3>
+<p>After processing all of the CAD-related papers referencing miRNAs and storing them on a vector database, we built a search engine to allow researchers to quickly interface with CADmir’s database of information.</p>
+<div class="flex w-full flex-col items-center justify-center p-1"><img src="https://static.igem.wiki/teams/4683/wiki/software/cadmirsearchingpipelineha.png" style="max-width:100%" alt="" class="mx-2 rounded-xl max-w-[400px] "/><div class="text-center italic">Figure 4. Searching pipeline</div></div>
+<p>When users ask a question in the search bar, their search query will first be embedded into a vector with OpenAI’s embedding model. Once the query is in vector form, CADmir will use the k-nearest-neighbor (KNN) algorithm to find the top 15 pieces of information most similar to the search query (IBM, 2023). This query and the 15 search results will be used as context for OpenAI’s GPT-3.5 Turbo model to generate the final response in natural language form and present it to the user (see Fig. 4). We designed CADmir with information integrity in mind. Each piece of information in CADmir has a reference to its source that is peer-reviewed and credible. When generating a search response, CADmir will pinpoint the source that each piece of information came from and list the sources it used to generate a search response.</p>
+<p>CADmir is a living database of miRNAs, and will be regularly updated with new papers. New papers added to the database will be automatically included in future search queries.</p>
+<h3 id="using-cadmir">Using CADmir</h3>
+<p>To get started, users can search for anything relevant to heart disease related miRNAs from the home page. The search will generate a response and sources to back up the information providing researchers with the flexibility to interface with CADmir’s repository of information flexibly without constraints. CADmir is available for anyone to use publicly <a href="http://cadmir.org/">here</a>.</p>
+<p>CADmir’s code is available opensource under a MIT license on <a href="https://github.com/Lambert-iGEM-2023/cadmir">github</a>.</p>
+<h2 id="impact">Impact</h2>
+<p>MicroRNA (miRNA) research is relatively new, making it difficult for researchers to find data regarding their functions. With CADmir, researchers can quickly find information regarding their desired miRNA. Lambert iGEM presented this database to miRNA researcher Dr. Charles Searles, a cardiologist at Emory Healthcare Hospital, who was excited about the increased efficiency CADmir brings. In the future, we plan on expanding CADmir to support more fields, not just miRNAs related to coronary artery disease.</p>
+<h3 id="references">References</h3>
+<div class="pl-6 -indent-6"><p>Bhaskaran, M., & Mohan, M. (2014). MicroRNAs: history, biogenesis, and their
+evolving role in animal development and disease: History, biogenesis, and
+their evolving role in animal development and disease. Veterinary Pathology,
+51(4), 759–774. <a href="https://doi.org/10.1177/0300985813502820">https://doi.org/10.1177/0300985813502820</a></p></div>
+<div class="pl-6 -indent-6"><p>Google. (2022). Embeddings. Google for Developers.
+<a href="https://developers.google.com/machine-learning/crash-course/embeddings/video-lecture">https://developers.google.com/machine-learning/crash-course/embeddings/video-lecture</a></p></div>
+<div class="pl-6 -indent-6"><p>IBM. (2023). What is the k-nearest neighbors algorithm? | IBM. Ibm.com.
+<a href="https://www.ibm.com/topics/knn#:~:text=The%20k%2Dnearest%20neighbors%20algorithm%2C%20also%20known%20as%20KNN%20or,of%20an%20individual%20data%20point">https://www.ibm.com/topics/knn#:~:text=The%20k%2Dnearest%20neighbors%20algorithm%2C%20also%20known%20as%20KNN%20or,of%20an%20individual%20data%20point</a>.</p></div>
+<div class="pl-6 -indent-6"><p>Kozomara, A., Birgaoanu, M., & Griffiths-Jones, S. (2019). miRBase: from
+microRNA sequences to function. Nucleic Acids Research, 47(D1), D155–D162.
+<a href="https://doi.org/10.1093/nar/gky1141">https://doi.org/10.1093/nar/gky1141</a></p></div>
+<div class="pl-6 -indent-6"><p>OpenAI. (2023). OpenAI GPT-3 API [gpt-3.5-turbo]. Available at:
+<a href="https://platform.openai.com/docs/models">https://platform.openai.com/docs/models</a></p></div>
+<div class="pl-6 -indent-6"><p>Schwaber-Cohen, R. (2023). What is a Vector Database? | Pinecone. Pinecone.io.
+<a href="https://www.pinecone.io/learn/vector-database/">https://www.pinecone.io/learn/vector-database/</a></p></div>
+<div class="pl-6 -indent-6"><p>Tress Academic. (2019, June 4). #13: Writing journal papers: Pros and cons |
+Tress Academic. Tress Academic.
+<a href="https://tressacademic.com/writing-journal-papers-pros-and-cons/">https://tressacademic.com/writing-journal-papers-pros-and-cons/</a></p></div>
+<p>‌</p>
+ </astro-slot></div></div></div></body></astro-island>
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w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/avani.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Avani Karthik</h3><p class="text-base text-body-color font-sans">Member</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/avi.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Avi Karamchandani</h3><p class="text-base text-body-color font-sans">Member</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/beige.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Beige Bae</h3><p class="text-base text-body-color font-sans">Member</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/christina.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Christina Cho</h3><p class="text-base text-body-color font-sans">Co-Captain</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/kashish.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Kashish Palla</h3><p class="text-base text-body-color font-sans">Member</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/kavya.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Kavya Parimi</h3><p class="text-base text-body-color font-sans">Member</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/natalie.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Natalie Tan</h3><p class="text-base text-body-color font-sans">Member</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/nick.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Nicholas Lockhart</h3><p class="text-base text-body-color font-sans">Member</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/ryan.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Ryan Du</h3><p class="text-base text-body-color font-sans">Co-Captain</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/sahasra.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Sahasra Tatavarti</h3><p class="text-base text-body-color font-sans">Member</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/sarvajith.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Sarvajith Kujuluva</h3><p class="text-base text-body-color font-sans">Member</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/srijyoth.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Srijyoth Ashok</h3><p class="text-base text-body-color font-sans">Member</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/srishti.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Srishti Iyer</h3><p class="text-base text-body-color font-sans">Member</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/vandana.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Vandana Vijay Kumar</h3><p class="text-base text-body-color font-sans">Member</p></div></div></button></div><h2 class="font-serif text-3xl text-center py-8">Advisors</h2><div class="flex flex-wrap items-start justify-center -mx-4"><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/sharer.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Catherine Sharer</h3><p class="text-base text-body-color font-sans">Primary Investigator</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/cantrell.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Dr. Brittney Denier Cantrell</h3><p class="text-base text-body-color font-sans">Secondary Investigator</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/standy.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Janet Standeven</h3><p class="text-base text-body-color font-sans">Secondary Investigator</p></div></div></button><button class="w-full px-4 md:w-1/2 xl:w-1/4"><div class="mx-auto mb-10 w-full max-w-[370px]"><div class="relative overflow-hidden rounded-lg"><img src="https://static.igem.wiki/teams/4683/wiki/teampage/seely.jpg" alt="https://static.igem.wiki/teams/4683/wiki/teampage/.jpg" style="width:257px;height:312px;object-fit:cover"/></div><div class="pt-3"><h3 class="text-xl font-bold text-dark font-serif ">Pam Seely</h3><p class="text-base text-body-color font-sans">Advisor</p></div></div></button></div></div></section></astro-island>
+ </main>
+
+ </div>
+
+ </div>
+
+ </div>
+
+
+
+
+
+</html>
+ <footer class="relative bg-[#292524] pt-8 pb-6 w-screen"><div class="w-full max-w-5xl mx-16 mt-8 container mx-auto px-4 py-10"><div class="flex flex-wrap text-left lg:text-left"><div class="w-full lg:w-6/12 px-4"><h1 class="text-4xl text-white font-serif font-light">Lambert-GA</h1><br/><h3 class="text-xs w-3/4 mt-0 mb-2 font-sans text-white">© <span id="get-current-year">2023</span> - Content on this site is licensed under a<a href="https://creativecommons.org/licenses/by/4.0/" class="underline hover:text-gray-300" target="_blank">Creative Commons Attribution 4.0 International license.</a><br/><br/>The repository used to create this website is available at<a href="https://gitlab.igem.org/2023/lambert-ga" class="underline hover:text-gray-300" target="_blank">https://gitlab.igem.org/2023/lambert-ga</a></h3></div><div class="w-full lg:w-5/12 px-4"><div class="flex flex-wrap items-top mb-6"><div class="w-full lg:w-4/12 px-4 ml-auto"><ul class="w-full list-unstyled"><li class="flex"><svg width="25" height="25" viewBox="0 0 25 25" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M18.7498 2.0835H15.6248C14.2435 2.0835 12.9187 2.63223 11.942 3.60898C10.9652 4.58573 10.4165 5.91049 10.4165 7.29183V10.4168H7.2915V14.5835H10.4165V22.9168H14.5832V14.5835H17.7082L18.7498 10.4168H14.5832V7.29183C14.5832 7.01556 14.6929 6.75061 14.8883 6.55526C15.0836 6.35991 15.3486 6.25016 15.6248 6.25016H18.7498V2.0835Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">Lambert iGEM</a></li><li class="flex"><svg width="25" height="28" viewBox="0 0 25 28" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M22.9168 4.52889C22.9168 4.52889 22.1877 6.90661 20.8335 8.37853C22.5002 19.701 11.0418 27.9664 2.0835 21.5126C4.37516 21.6258 6.66683 20.8332 8.3335 19.2481C3.12516 17.5497 0.520996 10.8695 3.12516 5.66114C5.41683 8.60498 8.9585 10.3033 12.5002 10.1901C11.5627 5.43469 16.6668 2.7173 19.7918 5.88759C20.9377 5.88759 22.9168 4.52889 22.9168 4.52889Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">@lamberths_iGEM</a></li><li class="flex "><svg width="25" height="25" viewBox="0 0 25 25" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M17.7085 2.21484H7.29183C4.41535 2.21484 2.0835 4.49911 2.0835 7.31688V17.521C2.0835 20.3387 4.41535 22.623 7.29183 22.623H17.7085C20.585 22.623 22.9168 20.3387 22.9168 17.521V7.31688C22.9168 4.49911 20.585 2.21484 17.7085 2.21484Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path><path d="M16.6667 11.7759C16.7952 12.6251 16.6472 13.4924 16.2435 14.2545C15.8399 15.0165 15.2012 15.6345 14.4184 16.0205C13.6355 16.4064 12.7484 16.5408 11.8831 16.4044C11.0179 16.268 10.2185 15.8678 9.59881 15.2608C8.9791 14.6537 8.57057 13.8707 8.43134 13.0231C8.29211 12.1754 8.42926 11.3064 8.82328 10.5395C9.2173 9.77269 9.84813 9.14706 10.626 8.75165C11.404 8.35625 12.2893 8.21119 13.1563 8.33712C14.0406 8.46558 14.8592 8.86923 15.4914 9.48846C16.1235 10.1077 16.5356 10.9096 16.6667 11.7759Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">@lambertigem</a></li><li class="flex"><svg width="25" height="24" viewBox="0 0 25 24" fill="none" xmlns="http://www.w3.org/2000/svg"><path d="M12.5002 18.2922C10.1043 18.2922 5.8335 18.1028 4.06266 17.7241C3.3335 17.5347 2.81266 17.0612 2.60433 16.3983C2.29183 15.3566 2.0835 13.1786 2.0835 11.6635C2.0835 10.1483 2.29183 7.97027 2.60433 6.92861C2.81266 6.26573 3.3335 5.79224 4.06266 5.60285C5.8335 5.22406 10.1043 5.03467 12.5002 5.03467C14.896 5.03467 19.1668 5.22406 20.9377 5.60285C21.6668 5.79224 22.1877 6.26573 22.396 6.92861C22.7085 7.97027 22.9168 10.1483 22.9168 11.6635C22.9168 13.1786 22.7085 15.3566 22.396 16.3983C22.1877 17.0612 21.6668 17.5347 20.9377 17.7241C19.1668 18.1028 14.896 18.2922 12.5002 18.2922Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path><path d="M10.4165 14.5046L15.6248 11.6637L10.4165 8.82275V14.5046Z" stroke="white" stroke-width="2" stroke-linecap="round" stroke-linejoin="round"></path></svg><a class="text-white hover:text-gray-300 font-semibold block pb-2 text-sm flex-grow" href="">Lambert iGEM</a></li></ul></div></div></div></div></div></footer>
+</div>
\ No newline at end of file
diff --git a/src/components/index.tsx b/src/components/index.tsx
index 413bd3c..df553d8 100644
--- a/src/components/index.tsx
+++ b/src/components/index.tsx
@@ -436,7 +436,7 @@ function HomePage() {
</div>
<div className="accuracy-cards flex absolute z-20">
<CommitteeCard
- title="RCA Optimize"
+ title="RCA Optimization"
description="We optimized rolling circle amplification from last year by decreasing our reaction time and testing different reporter outputs. "
colorClass={["border-green-6", "bg-green-9"]}
href="https://2023.igem.wiki/lambert-ga/rca/"
diff --git a/src/pages/contribution.mdx b/src/pages/contribution.mdx
index f974fca..153b997 100644
--- a/src/pages/contribution.mdx
+++ b/src/pages/contribution.mdx
@@ -44,7 +44,7 @@ LabPilot is an automated liquid handler compatible with micropipettes. It utiliz
### Frame
-Our team designed Labpilot’s frame to be small enough to fit inside a flow hood and large enough to fit standard laboratory containers on its bed (e.g. microcentrifuge tube racks, micropipette tip boxes, and well plates). We designed the frame by creating a linear rail extrusion, as shown in Figure W, typically found on commercial 3D printers. We used linear extrusions to make the cage-like design of LabPilot, inspired by Ender 3D printers like the Ender 5 (see Table 1) (Ender 5 S1 3D Printer). The finalized XYZ dimensions of LabPilot are 366mm x 296mm x 356mm (see Fig. 2). To provide stability to LabPilot’s frame, we added angle brackets that connect two of the extrusions that run perpendicular to each other at all 16 corners of the frame.
+Our team designed Labpilot’s frame to be small enough to fit inside a flow hood and large enough to fit standard laboratory containers on its bed (e.g. microcentrifuge tube racks, micropipette tip boxes, and well plates). We designed the frame by creating a linear rail extrusion, as shown in Figure 1, typically found on commercial 3D printers. We used linear extrusions to make the cage-like design of LabPilot, inspired by Ender 3D printers like the Ender 5 (see Table 1) (Ender 5 S1 3D Printer). The finalized XYZ dimensions of LabPilot are 366mm x 296mm x 356mm (see Fig. 2). To provide stability to LabPilot’s frame, we added angle brackets that connect two of the extrusions that run perpendicular to each other at all 16 corners of the frame.
We spoke to Priya Soneji, an undergraduate researcher at the Georgia Institute of Technology, who is a part of the Bhamla Lab. For the future, she recommended the switch to metal frames if the 3D-printed parts were imprecise. She confirmed the validity of our overall plan for controlling each motor axis on the XYZ plane and methods for motor movement.
<Image
@@ -108,7 +108,7 @@ The app provides instructions to the microcontroller via the Arduino serial USB
### How to use
-To use LabPilot, users must connect a laptop to LabPilot via a USB cable and open its web dashboard. Once the dashboard is open, there is a simple setup process where users can drag and drop components from the sidebar to recreate the physical layout of their LabPilot device, as seen in Figure Y. After setup, the app digitally recreates the layout of this device from data gathered in the setup process, enabling adequate simulation and control. Then, the users can queue and perform pipetting actions.
+To use LabPilot, users must connect a laptop to LabPilot via a USB cable and open its web dashboard. Once the dashboard is open, there is a simple setup process where users can drag and drop components from the sidebar to recreate the physical layout of their LabPilot device, as seen in Figure 8. After setup, the app digitally recreates the layout of this device from data gathered in the setup process, enabling adequate simulation and control. Then, the users can queue and perform pipetting actions.
<Image
src="https://static.igem.wiki/teams/4683/wiki/image-10-10-23-at-8-43-pm.jpg"
diff --git a/src/pages/hardware.mdx b/src/pages/hardware.mdx
index 1d007cb..769727a 100644
--- a/src/pages/hardware.mdx
+++ b/src/pages/hardware.mdx
@@ -44,7 +44,7 @@ LabPilot is an automated liquid handler compatible with micropipettes. It utiliz
### Frame
-Our team designed Labpilot’s frame to be small enough to fit inside a flow hood and large enough to fit standard laboratory containers on its bed (e.g. microcentrifuge tube racks, micropipette tip boxes, and well plates). We designed the frame by creating a linear rail extrusion, as shown in Figure W, typically found on commercial 3D printers. We used linear extrusions to make the cage-like design of LabPilot, inspired by Ender 3D printers like the Ender 5 (see Table 1) (Ender 5 S1 3D Printer). The finalized XYZ dimensions of LabPilot are 366mm x 296mm x 356mm (see Fig. 2). To provide stability to LabPilot’s frame, we added angle brackets that connect two of the extrusions that run perpendicular to each other at all 16 corners of the frame.
+Our team designed Labpilot’s frame to be small enough to fit inside a flow hood and large enough to fit standard laboratory containers on its bed (e.g. microcentrifuge tube racks, micropipette tip boxes, and well plates). We designed the frame by creating a linear rail extrusion, as shown in Figure 1, typically found on commercial 3D printers. We used linear extrusions to make the cage-like design of LabPilot, inspired by Ender 3D printers like the Ender 5 (see Table 1) (Ender 5 S1 3D Printer). The finalized XYZ dimensions of LabPilot are 366mm x 296mm x 356mm (see Fig. 2). To provide stability to LabPilot’s frame, we added angle brackets that connect two of the extrusions that run perpendicular to each other at all 16 corners of the frame.
We spoke to Priya Soneji, an undergraduate researcher at the Georgia Institute of Technology, who is a part of the Bhamla Lab. For the future, she recommended the switch to metal frames if the 3D-printed parts were imprecise. She confirmed the validity of our overall plan for controlling each motor axis on the XYZ plane and methods for motor movement.
<Image
@@ -108,7 +108,7 @@ The app provides instructions to the microcontroller via the Arduino serial USB
### How to use
-To use LabPilot, users must connect a laptop to LabPilot via a USB cable and open its web dashboard. Once the dashboard is open, there is a simple setup process where users can drag and drop components from the sidebar to recreate the physical layout of their LabPilot device, as seen in Figure Y. After setup, the app digitally recreates the layout of this device from data gathered in the setup process, enabling adequate simulation and control. Then, the users can queue and perform pipetting actions.
+To use LabPilot, users must connect a laptop to LabPilot via a USB cable and open its web dashboard. Once the dashboard is open, there is a simple setup process where users can drag and drop components from the sidebar to recreate the physical layout of their LabPilot device, as seen in Figure 8. After setup, the app digitally recreates the layout of this device from data gathered in the setup process, enabling adequate simulation and control. Then, the users can queue and perform pipetting actions.
<Image
src="https://static.igem.wiki/teams/4683/wiki/image-10-10-23-at-8-43-pm.jpg"
diff --git a/src/pages/human-practices.mdx b/src/pages/human-practices.mdx
index ac52023..2dd7797 100644
--- a/src/pages/human-practices.mdx
+++ b/src/pages/human-practices.mdx
@@ -9,89 +9,114 @@ import Wip from "../components/md_components/Wip";
import Image from "../components/md_components/Image";
import Reference from "../components/md_components/Reference";
-
-
# Human Practices
-
## Overview
-Last year, Lambert iGEM identified patients as the end goal of CADlock’s implementation. In order to accomplish this, we interviewed multiple leading microRNA researchers and cardiologists, who contributed to identifying the direction of our project as a point-of-care diagnostic screening tool to track the progression of coronary artery disease (CAD). Inspired to expand last year’s project with a focus on inclusivity, we improved and implemented wet lab mechanisms, including exponential rolling circle amplification (eRCA) from RCA [see RCA](https://2023.igem.wiki/lambert-ga/rca/). We also used protein purification to create our own phi29 DNA polymerase to minimize reaction costs. In addition, we expanded upon MicroQ, implementing an adaptation called MicroQ Pro. Furthermore, after talking with a graduate student, we developed LabPilot, an automated pipetter. We created a podcast and spoke to multiple doctors and professionals about the direction of our project. Drawing on the expertise of exceptional stakeholders, our journey transformed from conceptualization to implementation (see Fig. 1).
-
-<Image src='https://static.igem.wiki/teams/4683/wiki/human-practices/hp-graphic-1.png'
-size="800"
-caption='Figure 1. Overview graphic of the 2023 inclusivity approach.' />
+Last year, Lambert iGEM identified patients as the end goal of CADlock’s implementation. In order to accomplish this, we interviewed multiple leading microRNA researchers and cardiologists, who contributed to identifying the direction of our project as a point-of-care diagnostic screening tool to track the progression of coronary artery disease (CAD). Inspired to expand last year’s project with a focus on inclusivity, we improved and implemented wet lab mechanisms, including exponential rolling circle amplification (eRCA) from RCA [see RCA](https://2023.igem.wiki/lambert-ga/rca/). We also used protein purification to create our own phi29 DNA polymerase to minimize reaction costs. In addition, we expanded upon MicroQ, implementing an adaptation called MicroQ Pro. Furthermore, after talking with a graduate student, we developed LabPilot, an automated pipetter. We created a podcast and spoke to multiple doctors and professionals about the direction of our project. Drawing on the expertise of exceptional stakeholders, our journey transformed from conceptualization to implementation (see Fig. 1).
+<Image
+ src="https://static.igem.wiki/teams/4683/wiki/human-practices/hp-graphic-1.png"
+ size="800"
+ caption="Figure 1. Overview graphic of the 2023 inclusivity approach."
+/>
## Doctors
### Dr. Gentry
-We spoke to Dr. Mindy B. Gentry, a cardiologist in Marietta, Georgia, about sex inclusivity and CAD. We originally planned to use a gene called SVEP1, a gene that influences atherosclerosis. Specifically, SVEP1 expresses a protein that stimulates the development of plaque in arteries. This gene has a negative correlation with a miRNA named miR-1269b. Instead, she guided us to look at CAD correlation with an emphasis on estrogen-induced pathways. With her help, we were able to find a correlation between CAD and an estrogen-specific miRNA. Dr. Gentry, who runs a female cardiac health center, expressed the need for proactive measures to be taken to preserve heart health for females. The incorporation of Lambert iGEM’s estrogen initiative, guided by her aid, strengthens the proactive approach by making a diagnostic for a specialized risk group.
+We spoke to Dr. Mindy B. Gentry, a cardiologist in Marietta, Georgia, about sex inclusivity and CAD. We originally planned to use a gene called SVEP1, a gene that influences atherosclerosis. Specifically, SVEP1 expresses a protein that stimulates the development of plaque in arteries. This gene has a negative correlation with a miRNA named miR-1269b. Instead, she guided us to look at CAD correlation with an emphasis on estrogen-induced pathways. With her help, we were able to find a correlation between CAD and an estrogen-specific miRNA. Dr. Gentry, who runs a female cardiac health center, expressed the need for proactive measures to be taken to preserve heart health for females. The incorporation of Lambert iGEM’s estrogen initiative, guided by her aid, strengthens the proactive approach by making a diagnostic for a specialized risk group.
-<Image src='https://static.igem.wiki/teams/4683/wiki/human-practices/gentry.png'
-caption='Figure 2. Dr. Mindy B. Gentry' />
+<Image
+ src="https://static.igem.wiki/teams/4683/wiki/human-practices/gentry.png"
+ caption="Figure 2. Dr. Mindy B. Gentry"
+/>
### Dr. Fairweather
Dr. DeLisa Fairweather is a director of research at the Department of Cardiovascular Diseases at Mayo Clinic's Florida campus. She conducts research on individualized therapies and improved diagnosis in the Cardiovascular field. Due to her specialized and intensive research on estrogen in cardiovascular disease, we spoke to her about our initiatives in trying to incorporate sex inclusivity in our project. She informed us that estrogen increases autoimmune disease in women, and inflammation associated with it leads to CAD in women. Initially, we had the understanding that miR-20b should protect women from CAD, but she clarified when estrogen drops with menopause, the Estrogen Receptors Alpha pathway would be so low that miR-20b would have no effect. On the other hand, miR-20b is a viable indicator for CAD in men. She guided us to expand our focus to this miR to encapsulate men, as well as have a specialized risk group for women who have symptoms of inflammation. Thus, we were able to not only target our proactive initiative towards women but men as well.
-<Image src='https://static.igem.wiki/teams/4683/wiki/human-practices/fairweather.png' caption='Figure 3. Dr. DeLisa Fairweather' />
+<Image
+ src="https://static.igem.wiki/teams/4683/wiki/human-practices/fairweather.png"
+ caption="Figure 3. Dr. DeLisa Fairweather"
+/>
## Researches
### Dr. Bhamla
-Dr. Saad Bhamla, head of the Bhamla Lab at the Georgia Institute of Technology, has been an invaluable collaborator and supporter of Lambert iGEM. Through projects like the Frugal Science Academy (done in partnership with Lambert iGEM advisor Janet Standeven), Dr. Bhamla has provided funding, feedback, and opportunities to test designs in real-world settings. Last year, his input enabled Lambert iGEM to develop Micro-Q, a low-cost fluorometer, and test it in Thailand. This year, Dr. Bhamla's insights allowed further iteration and adaptation of Micro-Q Pro to better its accuracy and facilitate the real-world impact of many Lambert iGEM projects.
-
-<Image src='https://static.igem.wiki/teams/4683/wiki/human-practices/bhamla.png' caption='Figure 4. Dr. Saad Bhamla' />
+Dr. Saad Bhamla, head of the Bhamla Lab at the Georgia Institute of Technology, has been an invaluable collaborator and supporter of Lambert iGEM. Through projects like the Frugal Science Academy (done in partnership with Lambert iGEM advisor Janet Standeven), Dr. Bhamla has provided funding, feedback, and opportunities to test designs in real-world settings. Last year, his input enabled Lambert iGEM to develop Micro-Q, a low-cost fluorometer, and test it in Thailand. This year, Dr. Bhamla's insights allowed further iteration and adaptation of Micro-Q Pro to better its accuracy and facilitate the real-world impact of many Lambert iGEM projects.
+<Image
+ src="https://static.igem.wiki/teams/4683/wiki/human-practices/bhamla.png"
+ caption="Figure 4. Dr. Saad Bhamla"
+/>
### Rajas Poorna, (a grad student at GT)
-We spoke to Rajas Poorna, a graduate student working for the Bhamla Lab at the Georgia Institute of Technology, regarding alternatives to our existing RCA methodology to increase its accessibility in the field. Rajas informed us about existing capillary-tube-based diagnostics methods that used PCR and suggested modifying RCA to use capillary tubes. During our testing, we encountered problems with our experiments, such as capping the tubes and drawing up liquid. Some of his suggestions included using a biocompatible wax sealant to effectively seal the tubes and maintain the sample's integrity by tilting the PCR tubes sideways to counteract the force of gravity on the liquid as it rose in the tube. Our conversations with Rajas gave us insight into developing a novel method of quantifying that does not require a fluorometer, enabling greater inclusivity and accessibility of our diagnostic tool.
+We spoke to Rajas Poorna, a graduate student working for the Bhamla Lab at the Georgia Institute of Technology, regarding alternatives to our existing RCA methodology to increase its accessibility in the field. Rajas informed us about existing capillary-tube-based diagnostics methods that used PCR and suggested modifying RCA to use capillary tubes. During our testing, we encountered problems with our experiments, such as capping the tubes and drawing up liquid. Some of his suggestions included using a biocompatible wax sealant to effectively seal the tubes and maintain the sample's integrity by tilting the PCR tubes sideways to counteract the force of gravity on the liquid as it rose in the tube. Our conversations with Rajas gave us insight into developing a novel method of quantifying that does not require a fluorometer, enabling greater inclusivity and accessibility of our diagnostic tool.
-<Image src='https://static.igem.wiki/teams/4683/wiki/human-practices/poorna.png' caption='Figure 5. Rajas Poorna' />
+<Image
+ src="https://static.igem.wiki/teams/4683/wiki/human-practices/poorna.png"
+ caption="Figure 5. Rajas Poorna"
+/>
### Dr. Sliverman
-This year, Lambert iGEM wanted to produce phi29 DNA Polymerase, a protein involved in the amplification process of RCA, by utilizing the Immobilized Metal Affinity Chromatography (IMAC) mechanism[see Protein Purification](https://2023.igem.wiki/lambert-ga/protein-purification/). However, after talking to professors at Georgia Tech, they informed us to consider obtaining it through the cell-free system, as the cells will be able to intake the plasmid better than in the in vivo system while creating the optimal amount of phi29 DNA Polymerase needed in our lab. We then spoke to Dr. Adam Sliverman, a Scientist at Sherlock Biosciences, who guided us in choosing either cellfree or IMAC. He informed us about the inability to scale large amounts of protein in vitro. Therefore, he suggested our team grow a liter of E. coli with phi29 DNA Polymerase in a pET expression vector, inducing it with IPTG and outgrowing it overnight. He also assured us that the purification of the protein from that point is relatively straightforward. With this information, we continued our research on the IMAC mechanism to selectively extract the his-tagged phi29 DNA polymerase from the lysate to obtain the purified phi29 DNA polymerase for the RCA mechanism.
-Aside from protein purification, we also wanted to pursue exponential rolling circle amplification (eRCA). Dr. Silverman has previously experimented with RCA and believed eRCA to be a sufficient mechanism for our sensor’s sensitivity. As eRCA exponentially increases output, he advised us to include additional controls to ensure we are not amplifying errors. He suggested using an ON-state reporter mechanism, as it would reduce error/noise by promoting signal gain. While we had been experimenting with the ON-state lettuce DNA aptamers, Dr. Silverman also proposed using G-quadruplexes or SYBR Green instead, as DNA aptamers are not very fluorescent. Although we considered these mechanisms, we found that the efficiency and cost of linear probes -- an OFF-state reporter -- was more suitable for our project.
+This year, Lambert iGEM wanted to produce phi29 DNA Polymerase, a protein involved in the amplification process of RCA, by utilizing the Immobilized Metal Affinity Chromatography (IMAC) mechanism [see Protein Purification](https://2023.igem.wiki/lambert-ga/protein-purification/). However, after talking to professors at Georgia Tech, they informed us to consider obtaining it through the cell-free system, as the cells will be able to intake the plasmid better than in the in vivo system while creating the optimal amount of phi29 DNA Polymerase needed in our lab. We then spoke to Dr. Adam Sliverman, a Scientist at Sherlock Biosciences, who guided us in choosing either cellfree or IMAC. He informed us about the inability to scale large amounts of protein in vitro. Therefore, he suggested our team grow a liter of E. coli with phi29 DNA Polymerase in a pET expression vector, inducing it with IPTG and outgrowing it overnight. He also assured us that the purification of the protein from that point is relatively straightforward. With this information, we continued our research on the IMAC mechanism to selectively extract the his-tagged phi29 DNA polymerase from the lysate to obtain the purified phi29 DNA polymerase for the RCA mechanism.
+Aside from protein purification, we also wanted to pursue exponential rolling circle amplification (eRCA). Dr. Silverman has previously experimented with RCA and believed eRCA to be a sufficient mechanism for our sensor’s sensitivity. As eRCA exponentially increases output, he advised us to include additional controls to ensure we are not amplifying errors. He suggested using an ON-state reporter mechanism, as it would reduce error/noise by promoting signal gain. While we had been experimenting with the ON-state lettuce DNA aptamers, Dr. Silverman also proposed using G-quadruplexes or SYBR Green instead, as DNA aptamers are not very fluorescent. Although we considered these mechanisms, we found that the efficiency and cost of linear probes -- an OFF-state reporter -- was more suitable for our project.
We also considered multiplexed detection of miRNAs using RCA, however, Dr. Silverman advised us against this due to the low efficiency of SplintR Ligase. He suggested attempting multiplexing with pre-ligated padlock probes first to eliminate the potential error created by the enzyme, but we plan to test this idea in the future.
Dr. Silverman’s feedback in improving the affordability and accuracy of our biosensor enabled us to create a higher-quality assay that makes preliminary care more accessible for all patients.
-
-<Image src='https://static.igem.wiki/teams/4683/wiki/human-practices/silverman.png' caption='Figure 6. Dr. Adam Silverman' />
+<Image
+ src="https://static.igem.wiki/teams/4683/wiki/human-practices/silverman.png"
+ caption="Figure 6. Dr. Adam Silverman"
+/>
### Dr. Charles Searles
-Dr. Charles Searles is an associate professor of medicine at Emory Healthcare Hospital and a cardiologist specializing in biomarkers correlated with CAD. Last year, he provided us with hsa-miR-451a -- unrelated to the pathophysiology of CAD -- as a control for our proof-of-concept experimentation. Dr. Searles has tested our biosensors in his lab and offered to test them with actual patient serum samples in the future. He also gave us helpful insights on what would be useful in a miRNA database for CAD and validated the need for a database like CADmir. After we built CADmir, we shared it with Dr. Searles, and he gave us valuable feedback on improvements we could make to the platform. We iterated on his input and modified CADmir with filtration and testing on our publications. With his help, we were able to develop a miRNA database for CAD that helps miRNA researchers find information faster.
+Dr. Charles Searles is an associate professor of medicine at Emory Healthcare Hospital and a cardiologist specializing in biomarkers correlated with CAD. Last year, he provided us with hsa-miR-451a -- unrelated to the pathophysiology of CAD -- as a control for our proof-of-concept experimentation. Dr. Searles has tested our biosensors in his lab and offered to test them with actual patient serum samples in the future. He also gave us helpful insights on what would be useful in a miRNA database for CAD and validated the need for a database like CADmir. After we built CADmir, we shared it with Dr. Searles, and he gave us valuable feedback on improvements we could make to the platform. We iterated on his input and modified CADmir with filtration and testing on our publications. With his help, we were able to develop a miRNA database for CAD that helps miRNA researchers find information faster.
-<Image src='https://static.igem.wiki/teams/4683/wiki/human-practices/searles.png' caption='Figure 7. Dr. Charles Searles' />
+<Image
+ src="https://static.igem.wiki/teams/4683/wiki/human-practices/searles.png"
+ caption="Figure 7. Dr. Charles Searles"
+/>
### Kimberly Ann Rooney
-Kimberly Ann Rooney, a lab technician at the Searles Cardiovascular Lab led by Dr. Charles Searles, ran RCA reactions at Emory utilizing our protocol to test whether our biosensor could be practical and applicable as a diagnostic tool. Ms. Rooney gave us feedback on our methodology, advising that we reduce the number of variables used as controls and use serial dilutions to obtain more precise miRNA concentrations. She guided us towards optimizing the overall time of our assay, allowing us to further emphasize proactive care by decreasing the time it takes for hospitals to communicate results to patients.
+Kimberly Ann Rooney, a lab technician at the Searles Cardiovascular Lab led by Dr. Charles Searles, ran RCA reactions at Emory utilizing our protocol to test whether our biosensor could be practical and applicable as a diagnostic tool. Ms. Rooney gave us feedback on our methodology, advising that we reduce the number of variables used as controls and use serial dilutions to obtain more precise miRNA concentrations. She guided us towards optimizing the overall time of our assay, allowing us to further emphasize proactive care by decreasing the time it takes for hospitals to communicate results to patients.
-<Image src='https://static.igem.wiki/teams/4683/wiki/human-practices/rooney.png' caption='Figure 8. Kimberly Ann Rooney' />
+<Image
+ src="https://static.igem.wiki/teams/4683/wiki/human-practices/rooney.png"
+ caption="Figure 8. Kimberly Ann Rooney"
+/>
### Dr. Mark Styczynski
Dr. Mark Styczynski, a researcher from the Georgia Institute of Technology, told us that eRCA is a plausible mechanism. If the error with the RCA assay is from the technological aspect, eRCA should perform as expected. However, it could potentially amplify the discrepancy in our data if the error is due to the biological process of RCA. Dr. Styczinski also pointed out that using an OFF-state reporter, such as linear DNA probes, has a limited dynamic range as it is losing signal. He suggested using an ON-state sensor instead, as it would address this fundamental limitation. While we had been working with split lettuce DNA aptamers, Dr. Styczynski urged us away from this mechanism as they are more unreliable than other reporters. We then proposed producing whole aptamers through RCA. He was against this idea as the secondary structures of multiple aptamers' would likely interact poorly on a single strand of DNA. Therefore, we utilized eRCA to produce whole lettuce aptamers, as this mechanism produces separate, isolated strands of DNA rather than a single, long strand.
-<Image src='https://static.igem.wiki/teams/4683/wiki/human-practices/styczynski.png' caption='Figure 9. Dr. Mark Styczynski' />
+<Image
+ src="https://static.igem.wiki/teams/4683/wiki/human-practices/styczynski.png"
+ caption="Figure 9. Dr. Mark Styczynski"
+/>
### Megan McSweeney
-Megan McSweeney is a graduate student at the Georgia Institute of Technology and part of the Styczynski lab. She stated that the efficiency of eRCA would depend on the source of the error, paralleling Dr. Styczynski’s input. eRCA could be more accurate than RCA if it does not amplify the error. She was also concerned that our discrepancy could be due to contamination, leading us to take extra precautions to ensure an aseptic and RNase-free environment. Her feedback helped us increase the accuracy of our biosensor, therefore increasing its proactiveness and applicability in point-of-care testing.
+Megan McSweeney is a graduate student at the Georgia Institute of Technology and part of the Styczynski lab. She stated that the efficiency of eRCA would depend on the source of the error, paralleling Dr. Styczynski’s input. eRCA could be more accurate than RCA if it does not amplify the error. She was also concerned that our discrepancy could be due to contamination, leading us to take extra precautions to ensure an aseptic and RNase-free environment. Her feedback helped us increase the accuracy of our biosensor, therefore increasing its proactiveness and applicability in point-of-care testing.
-<Image src='https://static.igem.wiki/teams/4683/wiki/human-practices/mcsweeney.png' caption='Figure 10. Megan McSweeney' />
+<Image
+ src="https://static.igem.wiki/teams/4683/wiki/human-practices/mcsweeney.png"
+ caption="Figure 10. Megan McSweeney"
+/>
### Priya Soneji
Priya Soneji is an undergraduate researcher at the Georgia Institute of Technology and part of the Bhamla Lab. She designed and built a device called Trackoscope, a frugal tracking microscope for tracking the movement of microorganisms. It has a similar frame and structural design we intended to use for LabPilot, which is the inexpensive, automated micropipetter we are creating to aid labwork. We proposed our design on Fusion 360, a computer-aided design (CAD) software tool, to Priya, who verified that this software was the best option for CAD design. She also recommended that we try aluminum supports in case the 3D printed parts were time-consuming and imprecise issues. Furthermore, Priya uses the Nema 17 motors in her Trackoscope design, which includes a micro-stepping feature that enables precise motions. We then asked her if using Nema 17 motors seemed appropriate for our build, and she said they are very reliable and accurate motors. She confirmed the validity of our overall plan for controlling each motor axis on the XYZ plane and methods for motor movement. Priya greatly improved our understanding of the accuracy of our pipette mechanism and also allowed us to look into the affordability of some elements.
-<Image src='https://static.igem.wiki/teams/4683/wiki/human-practices/priya.png' caption='Figure 11. Priya Soneji' />
+<Image
+ src="https://static.igem.wiki/teams/4683/wiki/human-practices/priya.png"
+ caption="Figure 11. Priya Soneji"
+/>
## Institutions/Broader Community
@@ -99,14 +124,16 @@ Priya Soneji is an undergraduate researcher at the Georgia Institute of Technolo
We also spoke to Dr. Shana Scott, one of the leading members of the American Heart Association (AHA). She helped us establish and co-hosted the September Heart Health Month. She also guided us towards initiating an updated CERP (Cardiac Emergency Response Plan) at our school in order to cater to a more inclusive community. Dr. Shana Scott was also integral in promoting and increasing our outreach countywide. Lambert iGEM originally planned school-wide September Heart Health webinars and only planned CPR/First Aid education. With Dr. Scott's guidance, we were able to increase our outreach and expand our CPR program to a policy.
-<Image src='https://static.igem.wiki/teams/4683/wiki/human-practices/scott.png' caption='Figure 12. Dr. Shana Scott' />
+<Image
+ src="https://static.igem.wiki/teams/4683/wiki/human-practices/scott.png"
+ caption="Figure 12. Dr. Shana Scott"
+/>
### Mrs. Michelle Routhenstein
-Mrs. Routhenstein, a nutritionist, inspired our initiative to shift towards a more proactive approach. As a guest on our podcast, she detailed the importance of maintaining a balanced diet and implementing exercise in a daily routine as crucial practices in preventing heart disease altogether. She discussed general trends she’d seen in her patients with CAD, such as a poor diet filled with saturated fats, and a lack of an active lifestyle. She explained how as a nutritionist, her minor changes to these patients’ diets improved their condition tremendously. She even inspired us to conduct our September Heart Health webinar to encourage the message of the importance of diet and nutrition in preventing CAD to a broad and diverse audience.
-
-<Image src='https://static.igem.wiki/teams/4683/wiki/human-practices/routhenstein.png' caption='Figure 13. Mrs. Michelle Routhenstein' />
-
-
-
+Mrs. Routhenstein, a nutritionist, inspired our initiative to shift towards a more proactive approach. As a guest on our podcast, she detailed the importance of maintaining a balanced diet and implementing exercise in a daily routine as crucial practices in preventing heart disease altogether. She discussed general trends she’d seen in her patients with CAD, such as a poor diet filled with saturated fats, and a lack of an active lifestyle. She explained how as a nutritionist, her minor changes to these patients’ diets improved their condition tremendously. She even inspired us to conduct our September Heart Health webinar to encourage the message of the importance of diet and nutrition in preventing CAD to a broad and diverse audience.
+<Image
+ src="https://static.igem.wiki/teams/4683/wiki/human-practices/routhenstein.png"
+ caption="Figure 13. Mrs. Michelle Routhenstein"
+/>
diff --git a/src/pages/inclusivity.mdx b/src/pages/inclusivity.mdx
index efbeeb3..f3e54b8 100644
--- a/src/pages/inclusivity.mdx
+++ b/src/pages/inclusivity.mdx
@@ -8,19 +8,17 @@ import Wip from "../components/md_components/Wip";
import Image from "../components/md_components/Image";
import Reference from "../components/md_components/Reference";
-
-
# Overview
-Healthcare is broken down into one of two categories: reactive and proactive. The reactive approach utilizes medical intervention to treat already present symptoms and conditions (Waldman, 2019). Unfortunately, the United States healthcare system focuses strictly on reactive treatment, accounting for more than 75% of healthcare spending in the U.S. (Mercury Healthcare, 2018). These reactive practices occur in coronary artery disease (CAD) diagnosis; patients undergo invasive tests after showing observable symptoms (Skelly, Hashimoto, & Buckley et al., 2016). Although there is a shift from reactive to proactive healthcare regarding age-related diseases, patient retention remains an issue (Mercury Healthcare, 2018). While people visit facilities for early testing, the delayed process of receiving lab results- whether it be from outsourcing samples to different labs, scheduling, etc.- has the effect of fewer people returning to healthcare facilities for preventative care (Mercury Healthcare, 2018) . Furthermore, four out of seven studies reported missed diagnoses of malignancy, hypothyroidism, hyperthyroidism, osteoporosis, and microbiological results (Callen et al., 2011). CADlock aims to increase the accessibility to preventative healthcare by emphasizing affordable screening, early diagnostics, and accessibility for all. This approach will ensure care for individuals of all demographics and socioeconomic statuses. Therefore, our team’s goal was a multifaceted approach using synthetic biology to develop diagnostic solutions, to educate our community about proactive healthcare, and to include underrepresented populations within our initiatives (see Fig. 1).
+Healthcare is broken down into one of two categories: reactive and proactive. The reactive approach utilizes medical intervention to treat already present symptoms and conditions (Waldman, 2019). Unfortunately, the United States healthcare system focuses strictly on reactive treatment, accounting for more than 75% of healthcare spending in the U.S. (Mercury Healthcare, 2018). These reactive practices occur in coronary artery disease (CAD) diagnosis; patients undergo invasive tests after showing observable symptoms (Skelly, Hashimoto, & Buckley et al., 2016). Although there is a shift from reactive to proactive healthcare regarding age-related diseases, patient retention remains an issue (Mercury Healthcare, 2018). While people visit facilities for early testing, the delayed process of receiving lab results- whether it be from outsourcing samples to different labs, scheduling, etc.- has the effect of fewer people returning to healthcare facilities for preventative care (Mercury Healthcare, 2018) . Furthermore, four out of seven studies reported missed diagnoses of malignancy, hypothyroidism, hyperthyroidism, osteoporosis, and microbiological results (Callen et al., 2011). CADlock aims to increase the accessibility to preventative healthcare by emphasizing affordable screening, early diagnostics, and accessibility for all. This approach will ensure care for individuals of all demographics and socioeconomic statuses. Therefore, our team’s goal was a multifaceted approach using synthetic biology to develop diagnostic solutions, to educate our community about proactive healthcare, and to include underrepresented populations within our initiatives (see Fig. 1).
<Image
src="https://static.igem.wiki/teams/4683/wiki/inclusivity/hp-hierarchy.png"
-size="600"
+ size="600"
caption="Figure 1. The expansion of our inclusivity focus this year, combining accessibility, affordability, and accuracy under proactivity."
/>
-Our inclusive preventative approach begins by diagnosing CAD via microRNAs. We expanded Rolling Circle Amplification (RCA) to exponential RCA (eRCA) which increases signal strength and decreases time to detection from picomolar concentrations of miRNA in serum samples. This approach directly targets the issue of patient retention by increasing the accessibility of knowledge patients have about their condition. This early diagnosis and consultation brings patients back in since they know they may be susceptible to a disease, and thus may prevent it before their condition worsens. In addition to using eRCA, we also demonstrate using capillary tubes to perform RCA reactions with the goal of incorporating a future all-in-one reaction/visualization device for point-of-care testing. To address the high cost of phi29 polymerase we experimented with cloning and purifying our own proteins. Under the advice of Dr. Charles Searles, in conjuction to changing the RNA detection methods, we also expanded our library of miRNAs to include multiple novel sequences shown to be indicative of CAD in African American populations in Atlanta, GA (Dluzen et al., 2016). We envision an eventual library of miRNA padlocks available to clinicians to provide individualized medical diagnostics, thus enabling clinicians to directly provide individualized early diagnostics to their patient populations. Essentially, the increase in the rate at which patients receive their test results is positively correlated with proactive care because patients are more willing to take precautionary measures when they are given optimal accessibility to care (Mercury Healthcare, 2018).
+Our inclusive preventative approach begins by diagnosing CAD via microRNAs. We expanded Rolling Circle Amplification (RCA) to exponential RCA (eRCA) which increases signal strength and decreases time to detection from picomolar concentrations of miRNA in serum samples. This approach directly targets the issue of patient retention by increasing the accessibility of knowledge patients have about their condition. This early diagnosis and consultation brings patients back in since they know they may be susceptible to a disease, and thus may prevent it before their condition worsens. In addition to using eRCA, we also demonstrate using capillary tubes to perform RCA reactions with the goal of incorporating a future all-in-one reaction/visualization device for point-of-care testing. To address the high cost of phi29 polymerase we experimented with cloning and purifying our own proteins. Under the advice of Dr. Charles Searles, in conjuction to changing the RNA detection methods, we also expanded our library of miRNAs to include multiple novel sequences shown to be indicative of CAD in African American populations in Atlanta, GA (Dluzen et al., 2016). We envision an eventual library of miRNA padlocks available to clinicians to provide individualized medical diagnostics, thus enabling clinicians to directly provide individualized early diagnostics to their patient populations. Essentially, the increase in the rate at which patients receive their test results is positively correlated with proactive care because patients are more willing to take precautionary measures when they are given optimal accessibility to care (Mercury Healthcare, 2018).
During our research into miRNAs, it was evident that a lack of a database for miRNAs specifically indicated in coronary diseases was needed. To address the gap, we developed CADmir, a large language model (LLM) powered web app that synthesizes scientific information backed up by open-source, peer reviewed miRNA research (see Fig. 2). This will allow anyone with computer access to quickly conduct a literature review on heart disease related miRNA research, increasing accessibility to scientific knowledge.
@@ -29,7 +27,7 @@ During our research into miRNAs, it was evident that a lack of a database for mi
caption="Figure 2. Image of the CADmir user interface"
/>
-Pipetting multiple reactions for trials in triplicate can be tedious and lead to errors that interfere with data analysis or worse, having to repeat costly experiments. We developed LabPilot, an automated pipette system to increase pipet reliability and increase the researcher's time efficiency. According to multiple interviews with current Ph.D. and Postdoctoral researchers, pipetting is time consuming, error prone, and seen as a hindrance to their “real†work LabPilot is a low-cost alternative to commercial liquid handling systems that cost $10,000+, limiting their use to well funded labs (see Fig. 3).
+Pipetting multiple reactions for trials in triplicate can be tedious and lead to errors that interfere with data analysis or worse, having to repeat costly experiments. We developed LabPilot, an automated pipette system to increase pipet reliability and increase the researcher's time efficiency. According to multiple interviews with current Ph.D. and Postdoctoral researchers, pipetting is time consuming, error prone, and seen as a hindrance to their “real†work LabPilot is a low-cost alternative to commercial liquid handling systems that cost $10,000+, limiting their use to well funded labs (see Fig. 3).
<Image
src="https://static.igem.wiki/teams/4683/wiki/inclusivity/labpilot.png"
@@ -40,12 +38,11 @@ In our current use, RCA reactions output a fluorescent signal leading to the nee
Preventative care is two-fold, relying on early detection and educating patients. By implementing different RNA detection methods, software, and novel hardware, we aim to increase early detection. Educating local and dispersed populations was another major initiative this year. We held a local Heart Healthy Awareness month and contributed to a state-wide teacher professional development. To reach a broader audience, we created a podcast called SynbioOutLoud, and collaborated with Flowers for the Future, an international organization dedicated to supplying resources in order to promote STEM education for girls in Kabul, Afghanistan.
-
# Wetlab
## RCA
-Furthering last year's approach with accessibility, affordability, and accuracy, we continued utilizing and improving RCA to assess CAD risk groups while also addressing underrepresented demographics of people. Improvements to last year’s RCA mechanism include testing the biosensor’s specificity, comparing reporter mechanisms, and optimizing reaction times. In addition to RCA modifications, Lambert iGEM pursued a new wet-lab initiative called exponential rolling circle amplification (eRCA). eRCA provides a more sensitive reading of microRNAs with a faster reading time. The emphasis on additional accuracy through eRCA and affordability to end users enables CADlock to be accessible to a wide range of demographics, not limited to race, sex, and economic status (See [RCA: eRCA](https://2023.igem.wiki/lambert-ga/rca/)).
+Furthering last year's approach with accessibility, affordability, and accuracy, we continued utilizing and improving RCA to assess CAD risk groups while also addressing underrepresented demographics of people. Improvements to last year’s RCA mechanism include testing the biosensor’s specificity, comparing reporter mechanisms, and optimizing reaction times. In addition to RCA modifications, Lambert iGEM pursued a new wet-lab initiative called exponential rolling circle amplification (eRCA). eRCA provides a more sensitive reading of microRNAs with a faster reading time. The emphasis on additional accuracy through eRCA and affordability to end users enables CADlock to be accessible to a wide range of demographics, not limited to race, sex, and economic status See [RCA: eRCA](https://2023.igem.wiki/lambert-ga/rca/).
## Capillary
@@ -53,28 +50,29 @@ Lambert iGEM explored optimizing the reaction time and resources by running the
<Image
src="https://static.igem.wiki/teams/4683/wiki/inclusivity/screenshot-2023-10-11-102911.png"
-size="500"
+ size="500"
caption="Figure 4. A representation of the difference between the RCA reaction in the PCR tube vs. the capillary tube."
/>
## Protein Purification
-In addition to frugal technologies, Lambert iGEM aimed to decrease the cost of Rolling Circle Amplification (RCA) reagents by producing the enzymes required through protein purification. Phi29 DNA polymerase, priced at $251.00 for 1,250 units from New England Biolabs, not only covers the majority of the cost of RCA reagents but also possesses invaluable qualities such as strand displacement and processive synthesis properties that are necessary for RCA (New England Biolabs, n.d). By producing phi29 DNA polymerase through protein purification, combined with the accuracy of exponential RCA, Lambert iGEM aims to increase the affordability and in turn accessibility, both of which are contributing factors to inclusive care. This allows for more tests because the proteins are much more affordable, increasing the preventative aspect of healthcare. [See Wetlab: Protein Purification](https://2023.igem.wiki/lambert-ga/protein-purification/).
+In addition to frugal technologies, Lambert iGEM aimed to decrease the cost of Rolling Circle Amplification (RCA) reagents by producing the enzymes required through protein purification. Phi29 DNA polymerase, priced at $251.00 for 1,250 units from New England Biolabs, not only covers the majority of the cost of RCA reagents but also possesses invaluable qualities such as strand displacement and processive synthesis properties that are necessary for RCA (New England Biolabs, n.d). By producing phi29 DNA polymerase through protein purification, combined with the accuracy of exponential RCA, Lambert iGEM aims to increase the affordability and in turn accessibility, both of which are contributing factors to inclusive care. This allows for more tests because the proteins are much more affordable, increasing the preventative aspect of healthcare. [See Wetlab: Protein Purification](https://2023.igem.wiki/lambert-ga/protein-purification/).
## Estrogen
-While researching CAD risk factors and statistics, we came across literature that informed us about disparities among the sexes concerning early CAD diagnosis. We first conferred with Dr. Mindy Gentry, a women’s cardiologist, to get her opinion on the sex differences in cardiovascular health. She confirmed our insights about estrogen being a factor in increased susceptibility to CAD in women. To further our scientific research, we spoke to Dr. DeLisa Fairweather, a director of translational research in the Department of Cardiovascular Medicine at Mayo Clinic's Florida campus. She educated us on estrogen and its role as a CAD suppressor, and explained how women after menopause are more likely to develop heart disease because estrogen levels drastically decrease after menopause, causing women to be more likely to develop inflammation. Dr. Fairweather detailed that women who have inflammation in the heart are more likely to develop autoimmune disease and CAD. Additionally, she also guided us in the further development of our inclusivity by discussing how the hormone affects men as well. She told us that since men have a low estrogen level that also remains stable, they would be an ideal demographic to test for consistency in results, unlike women who have fluctuating levels of the hormone. Under the guidance of Dr. Fairweather, we tested miRNA-20b using Rolling Circle Amplification. In the presence of estrogen, CAD suppressors are activated and miRNA-20b is produced. miR-20b causes a negative feedback loop and suppresses estrogen, which will in turn inhibit the CAD suppressors. Dr. Fairweather explained how in men, a high level of miR-20b means that they are likely to develop CAD, however, in premenopausal women with inflammation symptoms, miR-20b protects them from developing CAD. She explained how if there is a high level of 20b, it will inhibit the estrogen from causing a catalyst between the developing autoimmune disease and CAD. We tested miR-20b with the RCA mechanism and ran a 1% gel to validate the presence of our product (see Fig. 5). Ultimately, increasing the factors that can be tested to signify CAD correlation encompasses a wider yet specified group, allowing for quality proactive care to be implemented. [See Wetlab: Inclusivity](https://2023.igem.wiki/lambert-ga/rca/).
+While researching CAD risk factors and statistics, we came across literature that informed us about disparities among the sexes concerning early CAD diagnosis. We first conferred with Dr. Mindy Gentry, a women’s cardiologist, to get her opinion on the sex differences in cardiovascular health. She confirmed our insights about estrogen being a factor in increased susceptibility to CAD in women. To further our scientific research, we spoke to Dr. DeLisa Fairweather, a director of translational research in the Department of Cardiovascular Medicine at Mayo Clinic's Florida campus. She educated us on estrogen and its role as a CAD suppressor, and explained how women after menopause are more likely to develop heart disease because estrogen levels drastically decrease after menopause, causing women to be more likely to develop inflammation. Dr. Fairweather detailed that women who have inflammation in the heart are more likely to develop autoimmune disease and CAD. Additionally, she also guided us in the further development of our inclusivity by discussing how the hormone affects men as well. She told us that since men have a low estrogen level that also remains stable, they would be an ideal demographic to test for consistency in results, unlike women who have fluctuating levels of the hormone. Under the guidance of Dr. Fairweather, we tested miRNA-20b using Rolling Circle Amplification. In the presence of estrogen, CAD suppressors are activated and miRNA-20b is produced. miR-20b causes a negative feedback loop and suppresses estrogen, which will in turn inhibit the CAD suppressors. Dr. Fairweather explained how in men, a high level of miR-20b means that they are likely to develop CAD, however, in premenopausal women with inflammation symptoms, miR-20b protects them from developing CAD. She explained how if there is a high level of 20b, it will inhibit the estrogen from causing a catalyst between the developing autoimmune disease and CAD. We tested miR-20b with the RCA mechanism and ran a 1% gel to validate the presence of our product (see Fig. 5). Ultimately, increasing the factors that can be tested to signify CAD correlation encompasses a wider yet specified group, allowing for quality proactive care to be implemented. [See Wetlab: Inclusivity](https://2023.igem.wiki/lambert-ga/rca/).
<Image
src="https://static.igem.wiki/teams/4683/wiki/inclusivity/gel1.png"
caption="Figure 5. The working gel, two wells on the right of the ladder contains the estrogen RCP."
+ size="600px"
/>
# Software
## CADmir
-Lambert iGEM developed CADmir as a centralized microRNA (miRNA) database to enhance researchers’ access to functional information about miRNAs more efficiently. (see Fig. 6) Currently, valuable insights on miRNAs are dispersed throughout academic literature, posing a challenge for researchers seeking detailed information about specific miRNAs. CADmir aims to address these issues by offering a cohesive and easily accessible platform that organizes all of the current scientific literature about miRNAs, facilitating quick and efficient searches. Lambert iGEM hopes that with CADmir, more researchers will explore miRNA research and incorporate miRNAs into more projects without requiring substantial amounts of time dedicated to reading through papers. CADmir aims to advance the field of miRNA research by improving information accessibility and reducing the time barrier associated with obtaining high-quality scientific information on these topics. [See Drylab: Software](https://2023.igem.wiki/lambert-ga/software/).
+Lambert iGEM developed CADmir as a centralized microRNA (miRNA) database to enhance researchers’ access to functional information about miRNAs more efficiently. (see Fig. 6) Currently, valuable insights on miRNAs are dispersed throughout academic literature, posing a challenge for researchers seeking detailed information about specific miRNAs. CADmir aims to address these issues by offering a cohesive and easily accessible platform that organizes all of the current scientific literature about miRNAs, facilitating quick and efficient searches. Lambert iGEM hopes that with CADmir, more researchers will explore miRNA research and incorporate miRNAs into more projects without requiring substantial amounts of time dedicated to reading through papers. CADmir aims to advance the field of miRNA research by improving information accessibility and reducing the time barrier associated with obtaining high-quality scientific information on these topics. [See Drylab: Software](https://2023.igem.wiki/lambert-ga/software/).
<Image
src="https://static.igem.wiki/teams/4683/wiki/inclusivity/cadmir-time.png"
@@ -83,11 +81,11 @@ Lambert iGEM developed CADmir as a centralized microRNA (miRNA) database to enha
## LabPilot
-LabPilot was targeted towards underfunded laboratories and independent researchers to increase accessibility to this crucial equipment. The leading retailing liquid handler costs around \$10,000, prohibiting less-privileged laboratories from purchasing liquid handlers and instead manually pipetting (OT-2 Robot, 2023). LabPilot is primarily made with 3D printed parts, making it affordable and easily replicable by any community with a 3D printer at a build cost of \$250 and a pipette cost of \$409 (Eppendorf Research Plus Adjustable Volume, Single Channel Pipette, 2 - 20 uL, Yellow)(See Parts List), and about 6.5% the cost of a commercial device (OT-2 Robot). LabPilot upholds the standard of accuracy found in other liquid handlers by having a significant difference when comparing the volume of liquid pipetted by LabPilot and a human (See Proof of Concept). LabPilot’s intuitive software utilizes critical user experience (UX) principles: a user can quickly transfer liquid from one location to another with just three taps. Additionally, LabPilot’s software conforms to the Web Content Accessibility Guidelines 2.1 standards, ensuring a quality user experience for people with disabilities. (See Hardware: LabPilot) [See Drylab: Hardware](https://2023.igem.wiki/lambert-ga/hardware/).
+LabPilot was targeted towards underfunded laboratories and independent researchers to increase accessibility to this crucial equipment. The leading retailing liquid handler costs around \$10,000, prohibiting less-privileged laboratories from purchasing liquid handlers and instead manually pipetting (OT-2 Robot, 2023). LabPilot is primarily made with 3D printed parts, making it affordable and easily replicable by any community with a 3D printer at a build cost of \$250 and a pipette cost of \$409 (Eppendorf Research Plus Adjustable Volume, Single Channel Pipette, 2 - 20 uL, Yellow)(See Parts List), and about 6.5% the cost of a commercial device (OT-2 Robot). LabPilot upholds the standard of accuracy found in other liquid handlers by having a significant difference when comparing the volume of liquid pipetted by LabPilot and a human (See Proof of Concept). LabPilot’s intuitive software utilizes critical user experience (UX) principles: a user can quickly transfer liquid from one location to another with just three taps. Additionally, LabPilot’s software conforms to the Web Content Accessibility Guidelines 2.1 standards, ensuring a quality user experience for people with disabilities. [See Drylab: Hardware](https://2023.igem.wiki/lambert-ga/hardware/).
## Micro-Q
-Many medical diagnostics utilize fluorescence reporter mechanisms with mathematical models to quantify substances. However, commercial fluorometers are often bulky and cost over \$10,000, making these diagnostics inaccessible. Last year, Micro-Q exhibited single-tube green fluorescence quantification for \$16 but was limited to one sample. This year, Micro-Q Plus allows multi-sample, multi-spectral quantification with camera based fluorescence detection enabling automatic generation of fluorescence/protein expression curves. All these capabilities fit in a pocket-sized portable box, increasing accessibility to underfunded labs and the public. (See Measurement: MicroQ)[See Drylab: Measurement](https://2023.igem.wiki/lambert-ga/measurement/).
+Many medical diagnostics utilize fluorescence reporter mechanisms with mathematical models to quantify substances. However, commercial fluorometers are often bulky and cost over \$10,000, making these diagnostics inaccessible. Last year, Micro-Q exhibited single-tube green fluorescence quantification for \$16 but was limited to one sample. This year, Micro-Q Plus allows multi-sample, multi-spectral quantification with camera based fluorescence detection enabling automatic generation of fluorescence/protein expression curves. All these capabilities fit in a pocket-sized portable box, increasing accessibility to underfunded labs and the public. [See Drylab: Measurement](https://2023.igem.wiki/lambert-ga/measurement/).
# Education
@@ -95,11 +93,13 @@ Many medical diagnostics utilize fluorescence reporter mechanisms with mathemati
Lambert iGEM held a summer Biotechnology Bootcamp for students between the ages of 12-14 where students participated in hands-on experiments while learning fundamental scientific concepts and skills needed in preparation for high school. The students learned about DNA structure and functions, acids and bases, proteins and enzymes, and more through a biotechnology application lens. The students were given pre and post surveys every day to gauge their learning progress. For example, we presented statements like “I can describe the function and purpose of a gel electrophoresis.†and the campers would respond with how confident they were. After speaking with our advisor, Dr. Brittney Cantrell, she provided us with a standards alignment chart that is used by the Forsyth County Board of Education. We reorganized the chart to include a link to all presentations, activities, and pre-planned labs that Lambert iGEM has made so that teachers are able to follow the new biotechnology curriculum and find easily accessible materials. Alongside the teaching materials, we linked competitions and events that younger students who are passionate about science can participate in.
- <iframe
- src="https://static.igem.wiki/teams/4683/wiki/inclusivity/new-standards-alignment-1.pdf"
- height="1000px"
- width="120%"
- ></iframe>
+{" "}
+
+<iframe
+ src="https://static.igem.wiki/teams/4683/wiki/inclusivity/new-standards-alignment-1.pdf"
+ height="1000px"
+ width="120%"
+></iframe>
## Teacher training
@@ -112,7 +112,7 @@ To supplement the Standards Alignment teacher resources, Lambert iGEM attended t
## Podcast
-Human Practices involves speaking with various professionals, doctors, and researchers to gain insight into the direction and impact of our project. This year, Lambert iGEM made the knowledge available to anyone interested by creating a podcast on Spotify called SynbioOutLoud (see Fig. 8). In this podcast, we invited a diverse set of several guests, including cardiologists and nutritionists, to discuss barriers they have experienced in the STEM field and how they handled prospective prejudice. We took their primary accounts and feedback to inspire our project direction for this year (see Fig. 9). Dr. Mindy Gentry, a women's cardiac care specialist, expressed the lack of heart health awareness for women, enabling us to expand sex inclusivity to promote our ongoing research on estrogen’s correlation to CAD (see Estrogen, Inclusivity). Mrs. Saru Bhardwaj, Lambert High School’s AP chemistry teacher, expressed the lack of education regarding gender disparities, and the changes she encountered when she was a student. In addressing this, we conducted a meta-analysis on gender distributions spanning six years of iGEM teams and compiled the findings into a comprehensive spreadsheet. In the study, we found that although the demographic distributions on teams are equal, the specific occupations on the team are very segregated. For example, the females on the teams are primarily centered around human practices, while the majority of males work with hardware. We noticed this trend in our teams too, and so combatting this trend was an integral inspiration for our inclusivity this season. Dr. Cantrell, a member of the Forsyth County Schools Secondary Science Specialist, iGEM advisor, and guest on our podcast informed us about a lack of community involvement she has observed while trying to promote inclusivity in science education. She expressed the need for female role models in the current society. In order to synthesize and express what Lambert iGEM has learned from these conversations, we developed a final episode of our podcast where we talk about our personal account of being students and women, and what we have noted from this experience. We discussed the “why†of our initiatives this year and the ideas behind their implementations. Overall, the SynbioOutLoud podcast enabled Lambert iGEM to create products catering to the community's education and awareness (see Fig. 10).
+Human Practices involves speaking with various professionals, doctors, and researchers to gain insight into the direction and impact of our project. This year, Lambert iGEM made the knowledge available to anyone interested by creating a podcast on Spotify called SynbioOutLoud (see Fig. 8). In this podcast, we invited a diverse set of several guests, including cardiologists and nutritionists, to discuss barriers they have experienced in the STEM field and how they handled prospective prejudice. We took their primary accounts and feedback to inspire our project direction for this year (see Fig. 9). Dr. Mindy Gentry, a women's cardiac care specialist, expressed the lack of heart health awareness for women, enabling us to expand sex inclusivity to promote our ongoing research on estrogen’s correlation to CAD (see Estrogen, Inclusivity). Mrs. Saru Bhardwaj, Lambert High School’s AP chemistry teacher, expressed the lack of education regarding gender disparities, and the changes she encountered when she was a student. In addressing this, we conducted a meta-analysis on gender distributions spanning six years of iGEM teams and compiled the findings into a comprehensive spreadsheet. In the study, we found that although the demographic distributions on teams are equal, the specific occupations on the team are very segregated. For example, the females on the teams are primarily centered around human practices, while the majority of males work with hardware. We noticed this trend in our teams too, and so combatting this trend was an integral inspiration for our inclusivity this season. Dr. Cantrell, a member of the Forsyth County Schools Secondary Science Specialist, iGEM advisor, and guest on our podcast informed us about a lack of community involvement she has observed while trying to promote inclusivity in science education. She expressed the need for female role models in the current society. In order to synthesize and express what Lambert iGEM has learned from these conversations, we developed a final episode of our podcast where we talk about our personal account of being students and women, and what we have noted from this experience. We discussed the “why†of our initiatives this year and the ideas behind their implementations. Overall, the SynbioOutLoud podcast enabled Lambert iGEM to create products catering to the community's education and awareness (see Fig. 10).
<Image
src="https://static.igem.wiki/teams/4683/wiki/inclusivity/podcast.png"
@@ -130,10 +130,10 @@ Human Practices involves speaking with various professionals, doctors, and resea
/>
<iframe
- src="https://static.igem.wiki/teams/4683/wiki/inclusivity/real-gender-breakdown-2013.pdf"
- height="1000px"
- width="120%"
- ></iframe>
+ src="https://static.igem.wiki/teams/4683/wiki/inclusivity/real-gender-breakdown-2013.pdf"
+ height="1000px"
+ width="120%"
+></iframe>
## Heart Health Awareness month
@@ -141,38 +141,49 @@ Lambert iGEM planned to expand upon the SynbioOutLoud podcast by hosting an audi
<Image
src="https://static.igem.wiki/teams/4683/wiki/inclusivity/webinar-listenres.png"
-size="600"
+ size="600"
caption="Figure 11. Webinar participants at our school receive prizes for answering questions correctly. "
/>
## Flowers for the Future
-After a discussion with some of our members who are part of the Lambert Women in STEM club, we decided to contact an organization called Flowers for the Future. They are a student-led organization that helps girls in Afghanistan by providing resources for STEM education for girls. Some of the topics covered include chemistry, biology, electricity, cellular pathways, computer science, coding, and mathematics. We noticed that their curriculum does not include resources for biotechnology concepts such as electrochemistry, application, lab equipment, basics of central dogma, DNA extraction, the purpose of pipetting, PCR, and Gel electrophoresis. After a discussion with the Flowers for the Future members, we agreed to send them multiple videos and presentations of these concepts with additional labs that they could do with household items, as they were not allowed access to many items due to Taliban restrictions.
-In order to maintain safety and confidentiality for our students and partners, Flowers For the Future makes no mention of any names, locations, and/or specificities anywhere on websites, social media, or the internet (e.g. “learning academy,†not “[name] schoolâ€) to avoid the potentially dangerous path to the academy. The organization also fundraises to provide the girls with laptops and wifi so they can learn from home and not be seen or have their safety undermined. They also do not publicize the organizations they work with, and make sure that any and all released video lessons are put on the Flowers for the Future YouTube channel, with no other information given than the instructional topics being presented.
-
+After a discussion with some of our members who are part of the Lambert Women in STEM club, we decided to contact an organization called Flowers for the Future. They are a student-led organization that helps girls in Afghanistan by providing resources for STEM education for girls. Some of the topics covered include chemistry, biology, electricity, cellular pathways, computer science, coding, and mathematics. We noticed that their curriculum does not include resources for biotechnology concepts such as electrochemistry, application, lab equipment, basics of central dogma, DNA extraction, the purpose of pipetting, PCR, and Gel electrophoresis. After a discussion with the Flowers for the Future members, we agreed to send them multiple videos and presentations of these concepts with additional labs that they could do with household items, as they were not allowed access to many items due to Taliban restrictions.
+In order to maintain safety and confidentiality for our students and partners, Flowers For the Future makes no mention of any names, locations, and/or specificities anywhere on websites, social media, or the internet (e.g. “learning academy,†not “[name] schoolâ€) to avoid the potentially dangerous path to the academy. The organization also fundraises to provide the girls with laptops and wifi so they can learn from home and not be seen or have their safety undermined. They also do not publicize the organizations they work with, and make sure that any and all released video lessons are put on the Flowers for the Future YouTube channel, with no other information given than the instructional topics being presented.
# References
<Reference>
-Callen, J., Georgiou, A., Li, J., & Westbrook, J. I. (2011). The safety implications of missed test results for hospitalized patients: a systematic review. BMJ quality & safety, 20(2), 194–199. https://doi.org/10.1136/bmjqs.2010.044339
+ Callen, J., Georgiou, A., Li, J., & Westbrook, J. I. (2011). The safety
+ implications of missed test results for hospitalized patients: a systematic
+ review. BMJ quality & safety, 20(2), 194–199.
+ https://doi.org/10.1136/bmjqs.2010.044339
</Reference>
<Reference>
-Centers for Disease Control and Prevention. (2023, May 15). Women and heart disease. Centers for Disease Control and Prevention. https://www.cdc.gov/heartdisease/women.htm
+ Centers for Disease Control and Prevention. (2023, May 15). Women and heart
+ disease. Centers for Disease Control and Prevention.
+ https://www.cdc.gov/heartdisease/women.htm
</Reference>
<Reference>
-Dluzen, D. F., Noren Hooten, N., Zhang, Y., Kim, Y., Glover, F. E., Tajuddin, S. M., Jacob, K. D., Zonderman, A. B., & Evans, M. K. (2016). Racial differences in microRNA and gene expression in hypertensive women. Scientific reports, 6, 35815. https://doi.org/10.1038/srep35815
+ Dluzen, D. F., Noren Hooten, N., Zhang, Y., Kim, Y., Glover, F. E., Tajuddin,
+ S. M., Jacob, K. D., Zonderman, A. B., & Evans, M. K. (2016). Racial
+ differences in microRNA and gene expression in hypertensive women. Scientific
+ reports, 6, 35815. https://doi.org/10.1038/srep35815
</Reference>
<Reference>
-Introduction - noninvasive testing for coronary artery disease - NCBI ... (n.d.-a). https://www.ncbi.nlm.nih.gov/books/NBK361133/
+ Introduction - noninvasive testing for coronary artery disease - NCBI ...
+ (n.d.-a). https://www.ncbi.nlm.nih.gov/books/NBK361133/
</Reference>
<Reference>
-Moving away from Reactive Care Models | Mercury Healthcare. (n.d.). https://www.mercuryhealthcare.com/blog/from-reactive-to-proactive-health
+ Moving away from Reactive Care Models | Mercury Healthcare. (n.d.).
+ https://www.mercuryhealthcare.com/blog/from-reactive-to-proactive-health
</Reference>
<Reference>
-Waldman, S. A., & Terzic, A. (2019). Health Care Evolves From Reactive to Proactive. Clinical pharmacology and therapeutics, 105(1), 10–13. https://doi.org/10.1002/cpt.1295
-</Reference>
\ No newline at end of file
+ Waldman, S. A., & Terzic, A. (2019). Health Care Evolves From Reactive to
+ Proactive. Clinical pharmacology and therapeutics, 105(1), 10–13.
+ https://doi.org/10.1002/cpt.1295
+</Reference>
--
GitLab