From ff428d6d8a9c8d0ba6ba4b7338b256545257c255 Mon Sep 17 00:00:00 2001
From: Dreta <32196824+Dreta@users.noreply.github.com>
Date: Wed, 2 Oct 2024 21:36:44 +0900
Subject: [PATCH] final content update
---
src/routes/description/+page.svelte | 12 +--
src/routes/engineering/+layout.svelte | 15 +--
src/routes/engineering/+page.svelte | 96 ++++++++++--------
src/routes/human-practices/+page.svelte | 2 +-
src/routes/model/+page.svelte | 123 ++++++------------------
src/routes/part/+layout.svelte | 36 ++++++-
src/routes/part/+page.svelte | 102 ++++++++++++++++++++
7 files changed, 237 insertions(+), 149 deletions(-)
diff --git a/src/routes/description/+page.svelte b/src/routes/description/+page.svelte
index 080a6ce..e11ee8b 100644
--- a/src/routes/description/+page.svelte
+++ b/src/routes/description/+page.svelte
@@ -173,9 +173,9 @@
biological systems.</p>
<div class="flex justify-center">
- <img alt="The photophysical properties of purified aquamarine" src={img8} />
+ <img alt="The photophysical properties of purified Aquamarine" src={img8} />
</div>
-<p class="slight">Figure 8. The photophysical properties of purified aquamarine are hereby presented. (A) Normalised
+<p class="slight">Figure 8. The photophysical properties of purified Aquamarine are hereby presented. (A) Normalised
absorption (dashed lines) and emission (solid lines) spectra, with the chromophore band set at maximum. The emission
spectra were recorded with excitation at a wavelength of 420 nm. (B) The fluorescence lifetime distributions at
neutral pH were obtained from the MEM analysis of the fluorescence decay curves. (C) The pH dependence of the
@@ -218,9 +218,9 @@
can be visually reflected by subtle changes in mBaoJin fluorescence intensity.</p>
<div class="flex justify-center lg:hidden">
- <img alt="A flowchart of our processes" src={img9} />
+ <img alt="The working mechanism of our sensor" src={img9} />
</div>
- <p class="slight lg:hidden">Figure 9. How mBaoJin takes effect</p>
+ <p class="slight lg:hidden">Figure 9. The working mechanism of our sensor</p>
<p>In the data analysis phase, we compared the differences in mBaoJin fluorescence intensity between the
treatment group (with drug intervention) and the control group (without drug treatment) to evaluate the
@@ -270,9 +270,9 @@
https://doi.org/10.1073/pnas.0904764106</p>
<p>Lambert, T. (n.d.). Aquamarine at FPbase. <i>FPbase</i>. Retrieved from
- https://www.fpbase.org/protein/aquamarine/</p>
+ https://www.fpbase.org/protein/Aquamarine/</p>
- <p>Lambert, T. (n.d.). Mbaojin at FPbase. <i>FPbase</i>. Retrieved from https://www.fpbase.org/protein/mBaoJin/</p>
+ <p>Lambert, T. (n.d.). mBaoJin at FPbase. <i>FPbase</i>. Retrieved from https://www.fpbase.org/protein/mBaoJin/</p>
<p>Miyawaki, A. (2003). Visualization of the spatial and temporal dynamics of intracellular signaling. <i>Developmental
Cell, 4</i>(3), 295–305. https://doi.org/10.1016/S1534-5807(03)00060-1</p>
diff --git a/src/routes/engineering/+layout.svelte b/src/routes/engineering/+layout.svelte
index 43422d8..641bc86 100644
--- a/src/routes/engineering/+layout.svelte
+++ b/src/routes/engineering/+layout.svelte
@@ -4,20 +4,15 @@
</script>
<ArticleLayout bg={engineering} sections={[
+ {
+ name: 'Overview',
+ id: 'overview',
+ subSections: []
+ },
{
name: 'Choosing a FRET Fluorescent Protein Pair',
id: 'choosing',
subSections: [
- {
- name: 'Design (Cycle 1)',
- id: 'design-1',
- subSections: []
- },
- {
- name: 'Build (Cycle 1)',
- id: 'build-1',
- subSections: []
- },
{
name: 'Test (Cycle 1)',
id: 'test-1',
diff --git a/src/routes/engineering/+page.svelte b/src/routes/engineering/+page.svelte
index 270f375..611b05a 100644
--- a/src/routes/engineering/+page.svelte
+++ b/src/routes/engineering/+page.svelte
@@ -1,5 +1,4 @@
<script>
- import { math } from 'mathlifier'
import img1 from '$lib/images/pages/results-img1.webp'
import img2 from '$lib/images/pages/results-img2.webp'
import img3 from '$lib/images/pages/results-img3.webp'
@@ -11,11 +10,34 @@
import References from '$lib/components/References.svelte'
</script>
+<h2 class="heading-2" id="overview">Overview</h2>
+
+<p>Breast cancer is the second leading cause of cancer deaths in women. According to data collected by the World Health
+ Organization in 2022, 2.3 million women worldwide were diagnosed with breast cancer, and 670,000 died from the
+ disease. In our program, we are going to find drugs that are highly targeted to the cancer cells, helping to improve
+ patient survival. For this reason, the team wanted to improve the screening methodology to efficiently screen
+ high-throughput drug libraries. Our team decided to use mBaoJin and Aquamarine as fluorescent pairs to design a
+ FRET-based biosensor for observing intracellular ATP concentration to reflect apoptosis and physiological
+ conditions. We successfully developed the tool named ScanCer and performed a high-throughput screening of 1220
+ compounds from the Novel Bioactive Compound Library. The Team screened the lead compounds and used protein docking
+ to discover downstream targets, which are expected to be new drugs for breast cancer. We adopted virtual screening
+ to further validate the results of the wet experiment. Moreover, we proposed recommendations for structural
+ modifications for the drugs.</p>
+
+<p>ScanCer can perform both high-throughput screening and cell-based validation, detecting cellular ATP levels through
+ optical signals to assess the effects of drugs on cell viability. This system is easy to operate, highly sensitive,
+ and has higher luminescence efficiency. In addition, we can verify cellular function, as the proteins emit
+ fluorescence under laser irradiation, enabling fluorescence microscopy to observe the subcellular distribution and
+ levels of ATP. This allows for further functional analysis within the cells. </p>
+
+<p>Through our high-throughput screening, we've screened two drugs that we found no previous reports on the anti-cancer
+ properties of these two compounds, suggesting that they may hold potential for further development in the future and
+ could offer new therapeutic targets for breast cancer treatment. </p>
+
<p>The following is a summary of the bioengineering process behind ScanCer.</p>
<h2 class="heading-2" id="choosing">Choosing a FRET Fluorescent Protein Pair</h2>
-<h3 class="heading-3" id="design-1">Design (Cycle 1)</h3>
<p>High-throughput screening (HTS) provides starting chemical matter in the adventure of developing a new drug. It
allows one to test thousands of samples of a very small volume in order to identify potential lead compounds in drug
discovery and it is one of the strategies used to discover starting compounds for small-molecule drug-design
@@ -39,8 +61,6 @@
opening up the possibility of a high-throughput screening system for drugs that inhibit these interactions in vitro.
Their team used ECFP-EYFP as a FRET fluorescent pair for the experiment.</p>
-<h3 class="heading-3" id="build-1">Build (Cycle 1)</h3>
-
<div class="flex justify-center">
<img alt="FRET protein pairs were screened through FPbase FRET Calculator" src={img1} />
</div>
@@ -53,9 +73,9 @@
<p class="slight">Figure 2. EYFP-EYFP donor-acceptor pair by FPbase FRET calculator</p>
<p>We used the FPbase FRET calculator to design the system of FRET. The spectral overlap between donor emission and
- acceptor absorption (the shaded part in Figure) provides the energetic resonance that is necessary for FRET. </p>
+ acceptor absorption (the shaded part in Figure 2) provides the energetic resonance that is necessary for FRET. </p>
-<p>We utilized a number of proteins, and eventually, we selected two newly developed proteins: aquamarine as the donor
+<p>We utilized a number of proteins, and eventually, we selected two newly developed proteins: Aquamarine as the donor
and
mBaoJin as the acceptor. Aquamarine is a basic (constitutively fluorescent) cyan-fluorescent protein published in
2013, derived from Aequorea Victoria. Its pKa is 3.3, indicating very low acid sensitivity and good stability in
@@ -72,10 +92,11 @@
prominent and helps to increase the sensitivity of the experiment, especially when detecting weak interactions
(Huang & Liu, 1994).</p>
-<p>The Förster radius （R₀） of the EYFP-EYFP donor-acceptor pair is 47.51 Å(Figure 2). However, the Förster radius of the
- Aquamarine-mBaoJin pair is 60.21 Å. Larger values of （R₀）usually mean that the donor and acceptor can be kept at a
+<p>The Förster radius （R₀） of the EYFP-EYFP donor-acceptor pair is 47.51 Å (Figure 2). However, the Förster radius of
+ the
+ Aquamarine-mBaoJin pair is 60.21 Ã…. Larger values of Râ‚€ usually mean that the donor and acceptor can be kept at a
greater distance from each other while still efficiently transferring energy (Figure 1). This directly affects the
- FRET efficiency. Specifically, larger（R₀）values help to increase the likelihood of energy transfer, which in turn
+ FRET efficiency. Specifically, larger Râ‚€ values help to increase the likelihood of energy transfer, which in turn
improves the strength and reliability of the signal (Huang & Liu, 1994).</p>
<p>As can be seen from the specifics in the figure, the Aquamarine-mBaoJin system has a clear advantage in quantum yield
@@ -83,7 +104,7 @@
signal-to-noise ratio due to smaller spectral overlap. These features allow the mBaoJin-Aquamarine system to obtain
clearer and more accurate signals in FRET experiments.</p>
-<p>Moreover, the emission spectrum of aquamarine significantly overlaps with the excitation spectrum of mBaoJin, as
+<p>Moreover, the emission spectrum of Aquamarine significantly overlaps with the excitation spectrum of mBaoJin, as
shown in the graph, the spectral overlap integral value is 2.85 × 10â»Â¹âµ cmⶠnmâ´, which is high, indicating that a
substantial portion of the donor’s emission falls within the acceptor’s range. This number is a key determinant of
FRET efficiency. Moreover, the Förster distance is 60.21Å, representing the distance at FRET efficiency is 50%.
@@ -93,18 +114,18 @@
interference and making it easy to distinguish the FRET signal from the donor and acceptor fluorescence. </p>
<div class="flex justify-center">
- <img alt="Visualization of the protein structures of mBaoJin (left) and aquamarine (right)" src={img2} />
+ <img alt="Visualization of the protein structures of mBaoJin (left) and Aquamarine (right)" src={img2} />
</div>
-<p class="slight">Figure 3. Visualization of the protein structures of mBaoJin (left) and aquamarine (right)</p>
-
-<h3 class="heading-3" id="test-1">Test (Cycle 1)</h3>
+<p class="slight">Figure 3. Visualization of the protein structures of mBaoJin (left) and Aquamarine (right)</p>
<div class="flex justify-center">
<img alt="Simulating FRET protein pairs" src={img3} />
</div>
-<p class="slight">Figure 3. Simulating FRET protein pairs</p>
+<p class="slight">Figure 4. Simulating FRET protein pairs</p>
+
+<p>We used AlphaFold to predict the function of the protein structure and predicted the structure we newly created, the
+ results are shown in Figure 4.</p>
-<h3 class="heading-3" id="learn-1">Learn (Cycle 1)</h3>
<p>Through this engineering cycle, we successfully designed a new FRET system using different FRET fluorophore pairs and
validated the newly constructed structure through AlphaFold simulations. Additionally, the corresponding part,
<a class="inline" href="https://parts.igem.org/Part:BBa_K5045005">BBa_K5045005</a>, has been created, providing
@@ -112,7 +133,6 @@
cost-effective, convenient, and easy-to-use tool for drug screening experiments.</p>
<h2 class="heading-2" id="acquiring">Acquiring Target Genes</h2>
-<h3 class="heading-3" id="design-2">Design (Cycle 2)</h3>
<p>Protein sequences of two fluorescents were taken from FPbase: Aquamarine (FPbase 963OH) and mBaoJin (FPbase 4XHVQ).
Sequence for epsilon subunit was chosen from modified Bacillus subtilis FoF1 ATP synthase in Nano-lantern_ATP1 from
462 to 597 (NCBI AFQ60642.1) slightly modified (597L replaced by F). The nucleotide sequence was designed based on
@@ -123,13 +143,20 @@
<object class="w-full aspect-square" data="https://static.igem.wiki/teams/5045/results-sequence.pdf"
title="ATP sensor sequence" type="application/pdf" />
-<p class="slight">Figure 4. ATP sensor sequence</p>
+<p class="slight">Figure 5. ATP sensor sequence</p>
<p class="slight">Certain browsers may not support embedded PDF.</p>
<p class="text-center"><a class="inline" href="https://static.igem.wiki/teams/5045/results-sequence.pdf">Open in new
tab</a>
+<div class="flex justify-center">
+ <img alt="The result of the PCR experiment" src="https://static.igem.wiki/teams/5045/final-img-2.webp" />
+</div>
+<p class="slight">Figure 6. The result of the PCR experiment</p>
+
+<p>We conducted polymerase chain reaction (PCR) of the plasmid we extracted. In the PCR image, marker is on the far
+ left, followed by pCDH-CMV-AεB and pGEX-4T-1-AεB to the right.</p>
+
<h2 class="heading-2" id="amplify">Amplifying and Extracting Vector Plasmids</h2>
-<h3 class="heading-3" id="build-2">Build (Cycle 2)</h3>
<p>Amplification vector plasmid is a key step in genetic engineering for inserting the target gene into the host cell
for expression. In this process, we chose the pCDH plasmid vector, which is a commonly used lentiviral vector
suitable for stable gene expression.</p>
@@ -186,23 +213,18 @@
cells. After 24 hours of puromycin treatment, infection success was confirmed under a microscope, and the cells were
further cultured. We successfully completed viral packaging and viral infection through our experiments.</p>
-<h3 class="heading-3" id="test-2">Test (Cycle 2)</h3>
-
<div class="flex justify-center">
- <img alt="Validating the effects of ScanCer wit a Revvity high-content analyzer" src={img6} />
+ <img alt="Detected sequence of Aquamarine in SnapGene" src="https://static.igem.wiki/teams/5045/final-img.webp" />
</div>
-<p class="slight">Figure 5. Validating the effects of ScanCer with a Revvity high-content analyzer</p>
+<p class="slight">Figure 7. Detected sequence of Aquamarine in SnapGene</p>
-<p>We conducted polymerase chain reaction (PCR) of the plasmid we extracted. In the PCR image, marker is on the far
- left, followed by pCDH-CMV-AεB and pGEX-4T-1-AεB to the right.</p>
+<p>We used SnapGene to detect an Aquamarine sequence, confirming the successful insertion of AεB into the plasmid
+ and validating the accuracy of the prior plasmid amplification.</p>
-<h3 class="heading-3" id="learn-2">Learn (Cycle 2)</h3>
-<p>We successfully completed the plasmid construction and viral transduction, achieving the expression of our ScanCer,
+<p>We successfully completed the plasmid construction and viral transduction, achieving the expression of ScanCer,
and are now ready to proceed with the next stage of experiments.</p>
<h2 class="heading-2" id="validation">Validating Drug Screening on Infected Cells</h2>
-<h3 class="heading-3" id="design-3">Design (Cycle 3)</h3>
-
<p>After successfully constructing ATP-probe-expressing cells, we validated them for drug screening and aimed to
identify active small molecule compounds through the drug screening system we developed, which could potentially be
further investigated in the future.</p>
@@ -218,23 +240,21 @@
efficiently narrow down a vast number of compounds to those most likely to have therapeutic potential, accelerating
the drug discovery process and reducing the need for extensive experimental testing.</p>
-<h3 class="heading-3" id="build-3">Build (Cycle 3)</h3>
<p>Our high-throughput drug screening experiments were conducted in the laboratory, with the assistance of researchers
and a Zhongxi Biological SC9210 fully automated pipetting workstation. By using the MCE compound library, all
compounds were dissolved in 100% DMSO or water. Prior to the experiment, we prepared the compounds to be pre-diluted
to 1 mM stock solutions.</p>
-<h3 class="heading-3" id="test-3">Test (Cycle 3)</h3>
<div class="flex justify-center">
<img alt="Verifying ScanCer's effect with a Revvity high-content analyzer" src={img6} />
</div>
-<p class="slight">Figure 6. Verifying ScanCer's effect with a Revvity high-content analyzer</p>
+<p class="slight">Figure 8. Verifying ScanCer's effect with a Revvity high-content analyzer</p>
<div class="flex justify-center">
<img alt="The relative fluorescence ratio of the cells with ScanCer"
src='https://static.igem.wiki/teams/5045/engineering-img2.webp' />
</div>
-<p class="slight">Figure 7. The relative fluorescence ratio of the cells with ScanCer</p>
+<p class="slight">Figure 9. The relative fluorescence ratio of the cells with ScanCer</p>
<p>We used the Revvity high-content analyzer to measure the fluorescence intensity of MDA-MB-231 cells under normal
conditions and after drug stimulation, utilizing the ScanCer technology we developed. It is evident that the
@@ -258,7 +278,7 @@
<p>During our high-throughput drug screening experiment, we conducted a standardized comprehensive screening (screening
all 1,220 compounds). From this experiment, we identified 15 small-molecule compounds. These small molecules did not
reduce the viability of normal mammary epithelial cells (MCF-10A) to below 80% (Above the red dotted line
- perpendicular to the Y-axis in the Figure7), while reducing the viability of breast cancer cells (MDA-MB-231) to
+ perpendicular to the Y-axis in the Figure 7), while reducing the viability of breast cancer cells (MDA-MB-231) to
below 30% (To the left of the red dotted line perpendicular to the X-axis in Figure 6). </p>
<ul class="list-disc list-outside">
@@ -282,7 +302,7 @@
<div class="flex justify-center">
<img alt="Scatter plot of drug screening data" src={imgSomething} />
</div>
-<p class="slight">Figure 8. Scatter plot of drug screening data</p>
+<p class="slight">Figure 10. Scatter plot of drug screening data</p>
<p>After conducting another round of standardized drug screening, we performed multiple validations from the MCE Novel
Known Bioactive Compound Library by subjecting them to high-throughput drug screening. This time, we identified two
@@ -293,12 +313,12 @@
<img alt="The structure of 6,7-Dihydro-3H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-4(5H)-one"
src={img7} />
</div>
-<p class="slight">Figure 9. The structure of 6,7-Dihydro-3H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-4(5H)-one</p>
+<p class="slight">Figure 11. The structure of 6,7-Dihydro-3H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-4(5H)-one</p>
<div class="flex justify-center">
<img alt="The structure of 1,10-Phenanthroline monohydrochloride monohydrate" src={img8} />
</div>
-<p class="slight">Figure 10. The structure of 1,10-Phenanthroline monohydrochloride monohydrate</p>
+<p class="slight">Figure 12. The structure of 1,10-Phenanthroline monohydrochloride monohydrate</p>
<p>Since 6,7-Dihydro-3H-cyclopenta[4,5]thieno[2,3-d]pyrimidin-4(5H)-one (12F4) has demonstrated stable anti-tumor
effects, our team conducted cellular functional validation using this drug. The result showed that 12F4 had
@@ -308,7 +328,7 @@
<div class="flex justify-center">
<img alt="The cell growth curve of 12F4" src={img9} />
</div>
-<p class="slight">Figure 11. The cell growth curve of 12F4</p>
+<p class="slight">Figure 13. The cell growth curve of 12F4</p>
<h2 class="heading-2" id="in-silico">Virtual Screening, Improving Drug Structures</h2>
<p>Through virtual screening, we can further validate the results of the wet experiment. We performed molecular docking
diff --git a/src/routes/human-practices/+page.svelte b/src/routes/human-practices/+page.svelte
index 7dae64a..41f16c8 100644
--- a/src/routes/human-practices/+page.svelte
+++ b/src/routes/human-practices/+page.svelte
@@ -256,7 +256,7 @@
<p>Initially, we reviewed several proteins through literature, but they were discarded due to issues like high acid
sensitivity, inability to form FRET pairs, and low stability. After further discussion with Dr. Guo, we identified
- two proteins suitable for constructing ScanCer: aquamarine and mBaoJin.
+ two proteins suitable for constructing ScanCer: Aquamarine and mBaoJin.
</p>
<div class="flex justify-center">
diff --git a/src/routes/model/+page.svelte b/src/routes/model/+page.svelte
index 1a4d449..7202d15 100644
--- a/src/routes/model/+page.svelte
+++ b/src/routes/model/+page.svelte
@@ -489,13 +489,10 @@
<h3 class="heading-3" id="double">Cross Validation</h3>
<p>It is quite reasonable to assume that if the results of the virtual screening of the drug library match, or
partially overlap, the results of the experimental screening are accurate and reliable. In other words, through
- virtual screening, we can further validate the results of the wet experiment. Similarly, if the results from the wet
+ virtual screening, we can further validate the results of the wet experiment. Conversely, if the results from the
+ wet
lab experiments deviate significantly from the virtual screening results, it indicates that there may be issues with
- the wet lab screening. We need to further examine the experimental procedures and operations in the wet lab. For
- example, the drugs selected in the first wet lab experiment had significant discrepancies from the results of the
- virtual screening. Therefore, the wet lab conducted a meticulous review and modification of the experimental
- procedures and operations, and in the second experiment, they obtained results that were closer to those from the
- virtual screening.</p>
+ the wet lab screening. We need to further examine the experimental procedures and operations in the wet lab.</p>
<p>Ultimately, the results of the experimental screening largely overlapped with those of the virtual screening,
indicating the scientific validity of the experimental setup, procedures, operations, and outcomes.</p>
@@ -630,101 +627,41 @@
<ol class="list-decimal list-outside">
<li>
- <p class="font-bold">Structural Optimization</p>
- <p>Using computational chemistry and molecular modeling techniques, one can simulate and predict the
- three-dimensional
- structure of drug molecules and their interactions with targets. Common methods include:</p>
- <ul class="list-disc list-inside">
- <li><span class="font-bold">Molecular Dynamics Simulation</span>: Helps understand the stability and dynamic
- behavior of drugs under various
- conditions.
- </li>
- <li><span class="font-bold">Quantum Chemistry Calculations</span>: Assist in optimizing electronic
- properties to enhance binding affinity.
- </li>
- <li><span class="font-bold">Virtual Screening</span>: Facilitates large-scale computational screening of
- potential active compounds, improving
- new drug discovery efficiency.
- </li>
- </ul>
+ <span class="font-bold">Structural Optimization.</span> Structural optimization is essential in drug
+ development, using computational chemistry and molecular modeling to predict how drug molecules will behave and
+ interact with biological targets. Techniques like molecular dynamics simulations help researchers understand the
+ stability and behavior of drugs under various conditions. Quantum chemistry calculations enhance the electronic
+ properties of drugs, improving their ability to bind to target proteins. Virtual screening allows for
+ large-scale analysis of potential drug candidates, making the discovery process more efficient.
</li>
<li>
- <p class="font-bold">Functional Group Replacement</p>
- <p>The biological activity of drugs is often linked to specific functional groups. Improvements can involve:</p>
- <ul class="list-disc list-inside">
- <li><span class="font-bold">Replacing Functional Groups</span>: For instance, substituting an ester group
- with an alcohol group may increase the
- drug's solubility or lipophilicity.
- </li>
- <li><span class="font-bold">Adding New Functional Groups</span>: Introducing groups that increase polarity
- or enhance targeting can improve
- bioavailability and selectivity.
- </li>
- </ul>
+ <span class="font-bold">Functional Group Replacement.</span> The activity of a drug often depends on specific
+ functional groups in its structure. Modifying these groups can enhance drug effectiveness. For example,
+ replacing an ester with an alcohol can improve solubility and absorption. Adding functional groups that increase
+ polarity can also enhance targeting of the drug to its biological target. This approach aims to optimize drug
+ performance while minimizing side effects by improving interactions with biological systems.
</li>
<li>
- <p class="font-bold">Stereochemical Modifications</p>
- <p>The stereochemical features of a drug significantly influence its biological activity. Approaches
- include:</p>
- <ul class="list-disc list-inside">
- <li><span class="font-bold">Creating Different Stereoisomers</span>: Designing various stereoisomers and
- testing their activity to identify the
- optimal structure.
- </li>
- <li><span class="font-bold">Optimizing Chiral Centers</span>: Selecting suitable chiral centers to improve
- target binding selectivity and reduce
- side effects.
- </li>
- </ul>
+ <span class="font-bold">Stereochemical Modifications.</span> Stereochemistry significantly affects how drugs
+ work. Different stereoisomers can vary in efficacy and safety, making it important to explore these variations
+ during drug development. Researchers create and test various stereoisomers to find the most effective ones.
+ Optimizing chiral centers in drug structures can improve binding selectivity and reduce side effects, leading to
+ safer and more effective compounds.
</li>
<li>
- <p class="font-bold">Pharmacokinetics Improvement</p>
- <p>ADME (Absorption, Distribution, Metabolism, Excretion) characteristics critically impact clinical efficacy.
- Improvement strategies include:</p>
- <ul class="list-disc list-inside">
- <li><span class="font-bold">Enhancing Absorption</span>: Modifying molecular structure to increase drug
- solubility in the gut.
- </li>
- <li><span class="font-bold">Prolonged Half-Life</span>: Introducing more stable structures or modifications
- to make drugs less prone to
- metabolism, thereby extending their duration of action.
- </li>
- <li><span class="font-bold">Improving Excretion Properties</span>: Designing drug structures to enhance
- their elimination efficiency in the body,
- reducing toxicity.
- </li>
- </ul>
+ <span class="font-bold">Pharmacokinetics Improvement.</span> The pharmacokinetic properties of a drug—how it's
+ absorbed, distributed, metabolized, and excreted—are crucial for its success. Enhancing absorption often
+ involves modifying the drug's structure to improve solubility, facilitating better uptake into the bloodstream.
+ Researchers also aim to prolong the drug's half-life by making it more stable and less susceptible to
+ metabolism. Improving excretion efficiency can reduce toxicity and enhance patient outcomes, allowing drugs to
+ provide maximum therapeutic benefits.
</li>
<li>
- <p class="font-bold">Targeting Enhancement</p>
- <p>Increasing the specificity of drugs can minimize side effects and enhance therapeutic effects. Methods
- include:</p>
- <ul class="list-disc list-inside">
- <li><span class="font-bold">Using Targeted Ligands</span>: Coupling antibodies, peptides, or small molecules
- to enable precise recognition and
- binding to specific cells or tissues.
- </li>
- <li><span class="font-bold">Nanocarrier Technology</span>: Utilizing nanoparticles as carriers to deliver
- drugs and release them selectively in
- tumor tissues.
- </li>
- </ul>
- </li>
- <li>
- <p class="font-bold">Multi-Target Design</p>
- <p>Multi-target drugs can act on multiple biological targets simultaneously, enhancing therapeutic effects.
- Approaches
- include:</p>
- <ul class="list-disc list-inside">
- <li><span class="font-bold">Designing Multifunctional Molecules</span>: Chemically synthesizing compounds
- that combine multiple active components
- targeting different sites.
- </li>
- <li><span class="font-bold">Drug Combination Strategies</span>: Using drugs with different mechanisms in
- combination to create synergistic
- effects.
- </li>
- </ul>
+ <span class="font-bold">Targeting Enhancement.</span> Increasing drug specificity helps minimize side effects
+ and maximize therapeutic effects. Targeted ligands, such as antibodies or small molecules, can be attached to
+ drugs for precise binding to specific cells or tissues, concentrating the drug's effects where needed.
+ Nanocarrier technology uses nanoparticles to deliver drugs selectively, enhancing delivery efficiency and
+ improving the drug's therapeutic index, making treatments more effective.
</li>
</ol>
diff --git a/src/routes/part/+layout.svelte b/src/routes/part/+layout.svelte
index f246e8f..aac6ff8 100644
--- a/src/routes/part/+layout.svelte
+++ b/src/routes/part/+layout.svelte
@@ -4,7 +4,41 @@
</script>
<ArticleLayout bg={part} sections={[
-
+ {
+ name: 'AεB ATP Sensor',
+ id: 'aepb',
+ subSections: []
+ },
+ {
+ name: 'Part Structure',
+ id: 'structure',
+ subSections: []
+ },
+ {
+ name: 'Working Mechanism',
+ id: 'working',
+ subSections: []
+ },
+ {
+ name: 'Validation of Functionality',
+ id: 'validation',
+ subSections: []
+ },
+ {
+ name: 'Usage',
+ id: 'usage',
+ subSections: []
+ },
+ {
+ name: 'Design Notes',
+ id: 'design',
+ subSections: []
+ },
+ {
+ name: 'Sources',
+ id: 'sources',
+ subSections: []
+ }
]}>
<span slot="title">New Composite Part</span>
<slot />
diff --git a/src/routes/part/+page.svelte b/src/routes/part/+page.svelte
index e69de29..9a5956c 100644
--- a/src/routes/part/+page.svelte
+++ b/src/routes/part/+page.svelte
@@ -0,0 +1,102 @@
+<script>
+ import img3 from '$lib/images/pages/results-img3.webp'
+ import img9 from '$lib/images/pages/overview-img9.webp'
+ import img6 from '$lib/images/pages/results-img5.webp'
+ import img1 from '$lib/images/pages/results-img1.webp'
+</script>
+
+<h2 class="heading-2" id="aepb">AεB ATP Sensor</h2>
+<p>AεB (Aquamarine-epsilon-mBaoJin), product name ScanCer, is a FRET-based ATP consisting of three major components: the
+ Aquamarine donor fluorescent protein (BBa_K5045002), the epsilon ATP-binding domain (BBa_K5045001), and the mBaoJin
+ acceptor fluorescent protein (BBa_K5045003). In our project, we constructed an AεB (Aquamarine-epsilon-mBaoJin) ATP
+ sensor plasmid. We synthesized the target fragment encoding the AεB ATP sensor with digesting sites of NheI and
+ BamHI, and the fragment was inserted in the pCDH-CMV vector by molecular cloning technique. The successfully
+ constructed plasmid needs to be amplified in prokaryotic E. coli cells and then integrated into eukaryotic cell
+ genomes through lentiviral vectors. We suggest people use A and pCDH-CMV(Addgene #72265) for two separate purposes.
+</p>
+
+<h2 class="heading-2" id="structure">Part Structure</h2>
+
+<p>In the sensor, Aquamarine and mBaoJin are attached to the N terminal and C terminal of the epsilon subunit
+ separately. The subunit changes its conformation when binding to ATP.
+</p>
+
+<div class="flex justify-center">
+ <img alt="Simulating FRET protein pairs" src={img3} />
+</div>
+<p class="slight">Figure 1. Simulating FRET protein pairs</p>
+
+<h2 class="heading-2" id="working">Working Mechanism</h2>
+<p>FRET (Förster resonance energy transfer) is the mechanism by which sensor AεB reflects ATP levels through color
+ changes. The Aquamarine donor, initially in its excited electronic state, may transfer energy to the mBaoJin
+ acceptor through nonradiative dipole-dipole coupling. The efficiency of this energy transfer is inversely
+ proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small
+ changes in distance. When ATP binds with the epsilon subunit, the subunit conformational changes occur, causing
+ mBaoJin and Aquamarine to approach each other, so the FRET efficiency increases significantly, and the green
+ fluorescence occurs. The strength of fluorescence changes as the concentration of ATP in the environment changes. By
+ detecting changes in the fluorescence signal, we can monitor the ATP levels in cells in real-time. Further, we can
+ measure the data quantitatively.
+</p>
+
+<div class="flex justify-center lg:hidden">
+ <img alt="The working mechanism of our sensor" src={img9} />
+</div>
+<p class="slight lg:hidden">Figure 2. The working mechanism of our sensor</p>
+
+<h2 class="heading-2" id="validation">Validation of Functionality</h2>
+<p>We tested the function of the AεB sensor by inserting it into MDA231 (MDA-MB-231) cells through lentivirus infection
+ and using a Revvity high-content analyzer to validate the effects in normal MDA231 cells and drug-stimulated MDA231
+ cells. The result shows that the sensor successfully reflected ATP change by decreasing fluorescence intensity.
+</p>
+
+<div class="flex justify-center">
+ <img alt="Verifying ScanCer's effect with a Revvity high-content analyzer" src={img6} />
+</div>
+<p class="slight">Figure 3. Verifying ScanCer's effect with a Revvity high-content analyzer</p>
+
+<h2 class="heading-2" id="usage">Usage</h2>
+<p>ATP could be a pertinent marker to determine cells' living status. As the major direct energy source of cells, ATP is
+ constantly repeatedly synthesized and used, with a concentration in the environment depending on the number of
+ living cells and their activities.
+</p>
+
+<h2 class="heading-2" id="design">Design Notes</h2>
+<p>To have the strongest ATP sensor's effect as possible, we screened a large number of potential protein pairs in the
+ FPbase to find the pair with maximum quantum yields and Förster radius two major factors deciding FRET efficiency-
+ and arrived at the Aquamarine-mBaoJin pair.
+</p>
+<p>We selected two newly developed proteins: Aquamarine as the donor and mBaoJin as the acceptor. Aquamarine is a
+ basic (constitutively fluorescent) cyan-fluorescent protein published in 2013, derived from Aequorea Victoria. Its
+ pKa is 3.3, indicating very low acid sensitivity and good stability in different pH environments. mBaoJin is a basic
+ (constitutively fluorescent) green fluorescent protein published in 2024, derived from Cytaeis Uchida. It is
+ reported to be a very rapidly maturing monomer with low acid sensitivity.
+</p>
+
+<div class="flex justify-center">
+ <img alt="FRET protein pairs were screened through FPbase FRET Calculator" src={img1} />
+</div>
+<p class="slight">Figure 4. FRET protein pairs were screened through FBbase FRET Calculator</p>
+
+<p>We made this choice because the emission spectrum of Aquamarine significantly overlaps with the excitation spectrum
+ of mBaoJin, as shown in the graph, the spectral overlap integral value is 2.85 × 10â»Â¹âµ cmⶠnmâ´, which is high,
+ indicating that a substantial portion of the donor’s emission falls within the acceptor’s range. This number is a
+ key determinant of FRET efficiency. Moreover, the Förster distance is 60.21Å, which represents the distance at FRET
+ efficiency is 50%. Additionally, compared to the traditional FRET pairs, such as CFP-YFP, the overlap for mBaoJin
+ and Aquamarine is more extensive, suggesting that this pair may produce stronger FRET signals. At the same time, the
+ emission spectra of the donor and acceptor remain distinct, minimizing spectral interference and making it easy to
+ distinguish the FRET signal from the donor and acceptor fluorescence.
+</p>
+
+<p>Sequences for the epsilon subunit were chosen from modified Bacillus subtilis FoF1 ATP synthase in Nano-lantern_ATP1
+ from 462 to 597 (NCBI AFQ60642.1) slightly modified (597L replaced by F). Epsilon was chosen because of its frequent
+ use in nano lanterns and ATP sensors, it also allows FRET to occur whilst ensuring the activity of the fluorescent
+ proteins. The nucleotide sequence was designed based on the three protein sequences in the order of
+ Aquamarine-epsilon-mBaoJin, ensuring that no restriction enzymes NheI and BamHI digestion sites are present.
+</p>
+
+<h2 class="heading-2" id="sources">Sources</h2>
+<p>The protein sequence of two fluorescents were taken from FPbase: Aquamarine (FPbase 963OH) which is an ECFP
+ derivative, and mBaoJin (FPbase 4XHVQ). Sequence for the epsilon subunit was chosen from modified Bacillus subtilis
+ FoF1 ATP synthase in Nano-lantern_ATP1 from 462 to 597 (NCBI AFQ60642.1) slightly modified (597L replaced by F).
+ Nucleotide sequence was the three protein sequences connected and reverse translated.
+</p>
--
GitLab