@@ -14,9 +14,7 @@ import Reference from "../components/md_components/Reference";
### Linear DNA Probes
Fluorophore and quencher-tagged linear DNA probes were used to quantify the presence and concentration of target miRNA in samples (see [RCA: outputs](https://2023.igem.wiki/lambert-ga/rca/)). The decrease in fluorescence in the solution can be correlated with a specific concentration of miRNA through characterization. Changes in fluorescence are measured using a plate reader in experimentation.
Similar to last year, we characterized and quantified RCP through the linear probes reporting mechanism (see [Experiments: Linear DNA Probes with RCP](https://2023.igem.wiki/lambert-ga/experiments/)). There is a negative logarithmic correlation between the miRNA concentrations and the relative fluorescence units (RFU) (see Fig. 1).
Fluorophore ([BBa_K4245130](http://parts.igem.org/Part:BBa_K4245130)) and quencher-tagged linear DNA probes ([BBa_K4245132](http://parts.igem.org/Part:BBa_K4245132)) were used to quantify the presence and concentration of target microRNA (miRNA) in samples (see [RCA: outputs](https://2023.igem.wiki/lambert-ga/rca/)). 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 [Experiments: Linear DNA Probes with RCP](https://2023.igem.wiki/lambert-ga/experiments/)). 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).
@@ -25,9 +23,7 @@ Similar to last year, we characterized and quantified RCP through the linear pro
### Lettuce
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 RCA reaction, the dye is able to bind to the RCP and produce fluorescence, therefore an increase in miRNA concentration should correlate with an increase in fluorescence (see [RCA: outputs](https://2023.igem.wiki/lambert-ga/rca/)).
We characterized and quantified RCP through the Lettuce reporting mechanism (see [Experiments: Lettuce with RCP](https://2023.igem.wiki/lambert-ga/experiments/)). There is a positive logarithmic correlation between the miRNA concentrations and the relative fluorescence units (RFU) (see Fig. 2).
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 [RCA: outputs](https://2023.igem.wiki/lambert-ga/rca/)). We characterized and quantified RCP through the Lettuce reporting mechanism ([BBa_K4245134](http://parts.igem.org/Part:BBa_K4245134); [BBa_K4245135](http://parts.igem.org/Part:BBa_K4245135)) (see [Experiments: Lettuce with RCP](https://2023.igem.wiki/lambert-ga/experiments/)). 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).
@@ -42,19 +38,20 @@ Both reporter mechanisms resulted in significant SEM overlap between the lower m
## Specificity
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 ([BBa_K4245200](http://parts.igem.org/Part:BBa_K4245160)) in the presence of four different miRNA sequences (see [RCA: outputs](https://2023.igem.wiki/lambert-ga/rca/)). The first is the original miR-1 sequence ([BBa_K4245006](http://parts.igem.org/Part:BBa_K4245006)), 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 ([BBa_K4683003](http://parts.igem.org/Part:BBa_K4683003)) and one with three SNVs ([BBa_K4683004](http://parts.igem.org/Part:BBa_K4683004)). hsa-miR-133a-3p ([BBa_K4683004](http://parts.igem.org/Part:BBa_K4683004)) was also included to ensure the padlock would not ligate to any miRNA.
We ran RCA using the hsa-miR-1-3p padlock ([BBa_K4245200](http://parts.igem.org/Part:BBa_K4245200)) in the presence of four different miRNA sequences (see Fig. 1). The first is the original miR-1 sequence ([BBa_K4245006](http://parts.igem.org/Part:BBa_K4245006)), 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 ([BBa_K4683003](http://parts.igem.org/Part:BBa_K4683003)) and one with three SNVs ([BBa_K4683004](http://parts.igem.org/Part:BBa_K4683004)). hsa-miR-133a-3p ([BBa_K4245009](http://parts.igem.org/Part:BBa_K4245009)) was also included to ensure the padlock would not ligate to any miRNA.
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 [Experiments: blueGel™ with RCP](https://2023.igem.wiki/lambert-ga/experiments/)). We then tested the RCP with linear DNA probes and quantified the resultant fluorescence in a plate reader (see Fig. 4) (see [Experiments: Linear DNA Probes with RCP](https://2023.igem.wiki/lambert-ga/experiments/). 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.
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 [Experiments: blueGel™ with RCP](https://2023.igem.wiki/lambert-ga/experiments/)).
caption="Figure 3. Gel results: A: miR-1, B: 1 SNV, C: 3 SNVs, D: miR-133a- Image of an agarose gel run with RCP from RCA run with 40.8 pM of each miRNA in reaction."
caption="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"
/>
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 [Experiments: Linear DNA Probes with RCP](https://2023.igem.wiki/lambert-ga/experiments/). 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.
caption="Figure 4. Comparison of RCA with miR-1, 1SNV, 3SNVs, and 133a fluorescence output using linear DNA probes." size='[400px]'
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## Emory Testing
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@@ -62,41 +59,35 @@ We ran the reactions and control on a gel electrophoresis; only the well with 40
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 SYBR™ Safe dye (see [Experiments: SYBR™ Safe with RCP](https://2023.igem.wiki/lambert-ga/experiments/)), 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.
caption="Figure 5. Triplicate of RCA with SYBR™ Safe output done by independent hands" size='[400px]'
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## Ligation Time
Lambert iGEM’s 2022 RCA protocol (see [Experiments: RCA 2022 protocol](https://2023.igem.wiki/lambert-ga/experiments/)) 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 four different ligation times with miR-1: 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 [Experiments: Optimized RCA protocol](https://2023.igem.wiki/lambert-ga/experiments/)).
Lambert iGEM’s 2022 RCA protocol (see [Experiments: RCA 2022 protocol](https://2023.igem.wiki/lambert-ga/experiments/)) 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 ([BBa_K4245200](http://parts.igem.org/Part:BBa_K4245200)) with four different ligation times with miR-1 ([BBa_K4245006](http://parts.igem.org/Part:BBa_K4245006)): 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 [Experiments: Optimized RCA protocol](https://2023.igem.wiki/lambert-ga/experiments/)).
caption="Figure 6. 1: 15-minute ligation, 2: 30-minute ligation, 3: 1-hour ligation, 4: 2-hour ligation, A: control (no enzymes); visible bands could be seen for all triplicates except for the 15-minute ligation and control."
caption="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" size='[400px]'
/>
## Amplification Time
Lambert iGEM’s 2022 RCA protocol (see [Experiments: RCA 2022 protocol](https://2023.igem.wiki/lambert-ga/experiments/)) 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 [Experiments: SYBR™ Safe with RCP](https://2023.igem.wiki/lambert-ga/experiments/)). The reactions were run overnight and the subsequent fluorescence was quantified in 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.
Lambert iGEM’s 2022 RCA protocol (see [Experiments: RCA 2022 protocol](https://2023.igem.wiki/lambert-ga/experiments/)) 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 [Experiments: SYBR™ Safe with RCP](https://2023.igem.wiki/lambert-ga/experiments/)). 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.
caption="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 SYBR™ Safe."
caption="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 SYBR™ Safe." size='[400px]'
/>
Overall, we were able to reduce the RCA workflow from around 15 hours to 7 (see [Experiments: Optimized RCA protocol](https://2023.igem.wiki/lambert-ga/experiments/)).
## Phi29-XT
Lambert iGEM’s RCA protocol (see [Experiments: Optimized RCA protocol](https://2023.igem.wiki/lambert-ga/experiments/)) 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 [Experiments: Amplification with Phi29- XT](https://2023.igem.wiki/lambert-ga/experiments/)). 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).
caption="Figure 8. A: control (no enzymes), X: RCA with phi29-XT DNA polymerase; 2% agarose gel ran for 1 hour at 48V"
/>
# Exponential RCA (eRCA) Results
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@@ -116,9 +107,8 @@ We performed eRCA with 40.8 pM of miR-1 (see [Experiments: eRCA Protocol](https:
We then characterized and quantified the RCP from the eRCA reaction through the Lettuce reporting mechanism (see [Experiments: eRCA Readout](https://2023.igem.wiki/lambert-ga/experiments/)). 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).
caption="Figure 10. A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes); 2% agarose gel ran for 1 hour at 48V"
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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.
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@@ -127,7 +117,7 @@ In the future, we plan to test eRCA with the entire range of clinically relevant
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 [RCA: outputs](https://2023.igem.wiki/lambert-ga/rca/)).
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 40.8 pM of miRNA but were not able to visualize any dots of miRNA within the capillary tube (see Fig. 11) (see [Experiments: Capillary RCA](https://2023.igem.wiki/lambert-ga/experiments/)). 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.
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 ([BBa_K4245200](http://parts.igem.org/Part:BBa_K4245200)) and 40.8 pM of miR-1 ([BBa_K4245006](http://parts.igem.org/Part:BBa_K4245006)) but were not able to visualize any dots of miRNA within the capillary tube (see Fig. 11) (see [Experiments: Capillary RCA](https://2023.igem.wiki/lambert-ga/experiments/)). 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.
caption="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"
caption="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"
/>
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.
# Inclusivity Estrogen RCA Results
## Gel Experimentation
We performed RCA on these six miRNAs (miR-20b, miR-328,miR-146, miR-150, miR-122, miR-30c) and ran the subsequent rolling circle product (RCP) on a 1% agarose gel (see Fig. 13). 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.
We performed RCA on miR-20b, miR-328, miR-146a using our respective padlock probe designs ([BBa_K4683008](http://parts.igem.org/Part:BBa_K4683008); [BBa_K4683024](http://parts.igem.org/Part:BBa_K4683024); [BBa_K4683028](http://parts.igem.org/Part:BBa_K4683028)) 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.
