Difference between revisions of "Team:CCU Taiwan/Poster"

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<img src="https://static.igem.org/mediawiki/2020/9/99/T--CCU_Taiwan--Poster_Introduction.png">
 
<img src="https://static.igem.org/mediawiki/2020/9/99/T--CCU_Taiwan--Poster_Introduction.png">
 
<br><br>
 
<br><br>
−
Dengue fever can progress into more serious conditions like dengue hemorrhagic fever (DHF), which has a 20% mortality rate. If conditions continue to worsen, patients experience dengue shock syndrome (DSS), which has a mortality rate up to 40%. An estimated 500,000 patients develop DHF or DSS, resulting in about 25,000 deaths every year.
+
Dengue fever can progress into more severe conditions like dengue hemorrhagic fever (DHF), which has a 20% mortality rate. If conditions continue to worsen, patients experience dengue shock syndrome (DSS), which has a mortality rate of up to 40%. An estimated 500,000 patients develop DHF or DSS, resulting in about 25,000 deaths every year.
 
<br><br>
 
<br><br>
−
Taiwan is in the area under the threat of dengue virus. From 2014 to 2015, there were over 44,000 confirmed cases during a dengue fever outbreak that spread from Tainan to other cities, causing 228 deaths. Recently, there was local transmission of dengue fever in Taoyuan, showing that the potentially affected population keeps increasing.
+
Taiwan is in the area under the threat of the dengue virus. From 2014 to 2015, there were over 44,000 confirmed cases during a dengue fever outbreak that spread from Tainan to other cities, causing 228 deaths. Recently, there was a local transmission of dengue fever in Taoyuan, showing that the potentially affected population keeps increasing.
 
<br><br>
 
<br><br>
 
There is no specific treatment or vaccine for dengue, and health care systems are still facing a challenge from dengue fever.
 
There is no specific treatment or vaccine for dengue, and health care systems are still facing a challenge from dengue fever.
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After infection by the dengue virus, a nonstructural protein, NS1, appears in blood serum early in the infection. The current method of detecting dengue fever uses antibodies to recognize NS1 antigens. Nevertheless, the cost of the antibodies is high, the antibodies are hard to purify, and the procedure of making antibodies is time-consuming.
 
After infection by the dengue virus, a nonstructural protein, NS1, appears in blood serum early in the infection. The current method of detecting dengue fever uses antibodies to recognize NS1 antigens. Nevertheless, the cost of the antibodies is high, the antibodies are hard to purify, and the procedure of making antibodies is time-consuming.
 
<br><br>
 
<br><br>
−
We took a step back to seek an alternative. We aimed to find something smaller and easier to produce to detect dengue virus instead of using antibodies. Symptoms of DHF and DSS result from interaction between C-type lectin domain, family 5, member A (CLEC5A) on macrophages and the envelope protein (E proteins) of dengue virus.
+
We took a step back to seek an alternative. We aimed to find something smaller and easier to produce to detect the dengue virus instead of antibodies. Symptoms of DHF and DSS result from the interaction between C-type lectin domain, family 5, member A (CLEC5A) on macrophages and the envelope protein (E proteins) of dengue virus.
 
<br><br>
 
<br><br>
 
<div id="imginfo">
 
<div id="imginfo">
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</div>
 
</div>
 
<br>
 
<br>
−
We found the sequences from CLEC5A most likely to bind the dengue virus and confirmed this using Rosetta simulations. We believe these peptides have potential to detect the dengue virus.
+
We found the sequences from CLEC5A most likely to bind the dengue virus and confirmed these using Rosetta simulations. We believe these peptides have the potential to detect the dengue virus.
 
<br><br>
 
<br><br>
−
We designed a detection kit with these peptides that can bind to the E protein of dengue virus. This would allow people who may have dengue fever or live in an outbreak region of dengue fever to get treatment or protection and control the spread early.
+
We designed a detection kit with these peptides that can bind to the E protein of the dengue virus. This would allow people who may have dengue fever or live in an outbreak region of dengue fever to get treatment or protection and control the spread early.
 
</div>
 
</div>
 
</div>
 
</div>
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<div class="title">Linear Array Epitope</div>
 
<div class="title">Linear Array Epitope</div>
 
<div class="text">
 
<div class="text">
−
Linear array epitope (LAE) is a technique to produce tandem-repeated sequences (TRSs). After the procedure, the TRSs would be expressed as a long-chain peptide which can be cleaved by an enzyme, resulting in many identical peptides.
+
Linear array epitope (LAE) is a technique to produce tandem-repeated sequences (TRSs). The TRSs would be expressed as a long-chain peptide which can be cleaved by an enzyme, resulting in many identical peptides.
 
<br><br>
 
<br><br>
−
<b>The Procedure of LAE: </b>
+
<ul>
 +
<li><b>The Procedure of LAE: </b></li>
 +
</ul>
 
<ol>
 
<ol>
−
<li>Design DNA oligo primers.</li>
+
<li>The design of DNA oligo primers</li>
−
<li>Template-repeated PCR (TR-PCR).</li>
+
<li>Template-repeated PCR (TR-PCR)</li>
−
<li>Adaptor PCR (AD-PCR).</li>
+
<li>Adaptor PCR (AD-PCR)</li>
 
</ol>
 
</ol>
 
<br>
 
<br>
−
For the primer design, TR-PCR primers need to be partially complementary to each other and AD-PCR primers need to have restriction sites.
+
For the primer design, TR-PCR primers need to be partially complementary to each other, and AD-PCR primers need to have restriction sites.
 
<br>
 
<br>
 
<img src="https://static.igem.org/mediawiki/2020/2/2d/T--CCU_Taiwan--Poster_LAE-1.gif">
 
<img src="https://static.igem.org/mediawiki/2020/2/2d/T--CCU_Taiwan--Poster_LAE-1.gif">
 
<br><br>
 
<br><br>
−
In TR-PCR, those primers act as both primer and template and they are partially complementary. The TRSs can be produced during the PCR cycling process. In AD-PCR, restriction sites need be introduced into the products of TR-PCR by adding the AD-PCR primers in PCR.
+
In TR-PCR, those primers act as both primer and template, and they are partially complementary. The TRSs can be produced during the PCR cycling process. In AD-PCR, restriction sites need be be introduced into the products of TR-PCR by adding the AD-PCR primers in PCR.
 
<br>
 
<br>
 
<img src="https://static.igem.org/mediawiki/2020/4/4c/T--CCU_Taiwan--Poster_LAE-2.gif">
 
<img src="https://static.igem.org/mediawiki/2020/4/4c/T--CCU_Taiwan--Poster_LAE-2.gif">
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<div class="title">BioBricks</div>
 
<div class="title">BioBricks</div>
 
<div class="text">
 
<div class="text">
−
Our peptides and proteins were expressed in <i>E. Coli</i> with T7 promotor and lac operator, which can be induced by IPTG. The thrombin site was included to remove unnecessary sections. The His tag was used in Western blot to confirm the expression of protein, and the HA tag and myc tag were utilized for purification.
+
Our peptides and proteins were expressed in <i>E. Coli</i> with a T7 promoter and a lac operator, which can be induced by IPTG. The thrombin site was included to remove unnecessary sections. The His tag was used in Western blot to confirm the expression of the protein, and the HA tag and myc tag were utilized for purification.
 
<br><br>
 
<br><br>
 
<img src="https://static.igem.org/mediawiki/2020/8/85/T--CCU_Taiwan--Poster_Parts-1.png">
 
<img src="https://static.igem.org/mediawiki/2020/8/85/T--CCU_Taiwan--Poster_Parts-1.png">
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<div class="title">Interaction between the Peptide of Tandem-repeated Sequence and E Protein</div>
 
<div class="title">Interaction between the Peptide of Tandem-repeated Sequence and E Protein</div>
 
<div class="text">
 
<div class="text">
−
<b>Purpose:</b>
+
<ul>
−
<br>
+
<li><b>Purpose:</b></li>
−
Our goal is to design a peptide of tandem-repeated sequence (PTRSs) to imitate the binding of CLEC5A to the E protein from the dengue virus. In order to ensure the PTRSs and the E protein interact, all the structure of PTRSs and proteins and their interactions were modeled using Rosetta.
+
Our goal is to design the peptide of tandem-repeated sequences (PTRSs) to imitate the binding of CLEC5A to the E protein from the dengue virus. In order to ensure the PTRSs and the E protein interact, all the structure of PTRSs and proteins and their interactions were modeled using Rosetta.
 +
<br><br>
 +
<li><b>Method and Results:</b></li>
 +
<ol>
 +
<li>The structure of E protein from a local strain (PL046) was generated using RosettaCM (comparative modeling with Rosetta) based on the crystal structure (PDB: 1OAN).</li>
 +
<li>The structures of PTRSs were predicted purely based on their sequences using the <i>ab initio</i> method in Rosetta.</li>
 +
<li>The most probable structure of the PTRS was identified by clustering.</li>
 +
<li>The interactions between the PTRSs and the E protein were evaluated using global protein-protein docking. The 100 most frequent docking sites are shown below.</li>
 +
</ol>
 +
<img src="https://static.igem.org/mediawiki/2020/c/cd/T--CCU_Taiwan--Poster_Modeling1.png">
 +
<img src="https://static.igem.org/mediawiki/2020/0/0d/T--CCU_Taiwan--Poster_Modeling2.png">
 +
</ul>
 
<br><br>
 
<br><br>
−
<b>Method and Results:</b>
 
−
<ol>
 
−
<li>The structure of E protein from a local strain (PL046) was generated using RosettaCM (comparative modeling with Rosetta) based on the crystal structure (PDB: 1OAN).</li>
 
−
<li>The structures of PTRSs were predicted purely based on their sequences using the <i>ab initio</i> method in Rosetta.</li>
 
−
<li>The most probable structure of the PTRS were identified by clustering.</li>
 
−
<li>The interactions between the PTRSs and the E protein were evaluated using global protein-protein docking. The 100 most frequent docking sites are shown below.</li>
 
−
</ol>
 
−
<img src="https://static.igem.org/mediawiki/2020/c/cd/T--CCU_Taiwan--Poster_Modeling1.png">
 
−
<img src="https://static.igem.org/mediawiki/2020/0/0d/T--CCU_Taiwan--Poster_Modeling2.png">
 
−
<br>
 
 
<h3>Interaction between Gold Nanoparticles</h3>
 
<h3>Interaction between Gold Nanoparticles</h3>
−
<b>Purpose:</b>
+
<ul>
−
<br>
+
<li><b>Purpose:</b></li>
−
A potential weakness of our design is that peptides on the gold nanoparticles and on the glass fiber compete for the same binding sites on the E protein. If the E proteins on the virus particles are fully covered by gold nanoparticles, there would be no sites available to interact with the peptides attached to the glass fiber. Hence, we need to understand if this situation would hinder development of our kit.
+
We use gold nanoparticles (AuNPs) as an indicator in the detection kit. A potential weakness of our design is that peptides on the AuNPs and those on the glass fiber membranes compete for the same binding sites on the E protein. If the E proteins on the virus particles are fully covered by AuNPs, there would be no sites available to interact with the peptides attached to the glass fiber membranes. Hence, we need to understand if this situation would hinder the development of our kit.
−
<br><br>
+
<br><br>
−
<b>Method:</b>
+
<li><b>Method:</b></li>
−
<br>
+
We used the DLVO theory to calculate the repulsion between AuNPs to estimate the number of AuNPs that would bind to a virus particle. The structure of the dengue virus is icosahedral, and the distances between the potential binding sites can be obtained from the structure in the protein data bank (1K4R). We took several representative positions on adjoining faces of the icosahedron to calculate the interactions between the AuNPs based on the DLVO theory.
−
We used DLVO theory to calculate the repulsion between gold nanoparticles to estimate the number of gold nanoparticles that would bind to a virus particle. The structure of dengue virus is icosahedral, and there are nine E proteins on each surface. The distances between the potential binding sites can be obtained from the structure in the protein data bank (1K4R). We took several representative positions on adjoining faces of the icosahedron to calculate the interactions between the gold nanoparticles based on DLVO theory.
+
<br><br>
−
<br><br>
+
<li><b>Results:</b></li>
−
<b>Results:</b>
+
The total energies of the interactions between AuNPs are all positive, indicating repulsive forces, and these total energies are also larger than the typical biological interactions (~0.5 kcal/mol or 3.49 x10-21 J). The results suggest that there will always be free faces on the virus particles to interact with the peptides conjugated on the test line.
−
<br>
+
<br>
−
The total energies are all positive, indicating repulsive forces, and these total energies are also larger than the typical biological interactions (~0.5 kcal/mol or 3.49 x10-21 J). The results suggest that there will always be free faces on the virus particles to interact with the peptides conjugated on the test line.
+
<table>
−
<br>
+
<tbody>
−
<img src="https://static.igem.org/mediawiki/2020/f/f4/T--CCU_Taiwan--Poster_Modeling-4.png">
+
<tr>
 +
<th></th>
 +
<th><img id="imginfo2" src="https://static.igem.org/mediawiki/2020/5/57/T--CCU_Taiwan--Poster_model01.png"></th>
 +
<th><img id="imginfo2" src="https://static.igem.org/mediawiki/2020/8/81/T--CCU_Taiwan--Poster_model02.png"></th>
 +
<th><img id="imginfo2" src="https://static.igem.org/mediawiki/2020/3/3e/T--CCU_Taiwan--Poster_model03.png"></th>
 +
<th><img id="imginfo2" src="https://static.igem.org/mediawiki/2020/0/03/T--CCU_Taiwan--Poster_model04.png"></th>
 +
</tr>
 +
<tr>
 +
<td>Center to center distance (nm)</td>
 +
<td>54.58</td>
 +
<td>42.71</td>
 +
<td>31.71</td>
 +
<td>43.24</td>
 +
</tr>
 +
<tr>
 +
<td>Attractive energy (10<sup>-21</sup>J)</td>
 +
<td>-3.3</td>
 +
<td>-4.6</td>
 +
<td>-7.2</td>
 +
<td>-4.5</td>
 +
</tr>
 +
<tr>
 +
<td>Repulsive energy (10<sup>-21</sup>J)</td>
 +
<td>51.0</td>
 +
<td>51.3</td>
 +
<td>51.6</td>
 +
<td>51.3</td>
 +
</tr>
 +
<tr>
 +
<td>Total energy (10<sup>-21</sup>J)</td>
 +
<td>47.7</td>
 +
<td>46.7</td>
 +
<td>44.4</td>
 +
<td>46.8</td>
 +
</tr>
 +
<tr>
 +
<th></th>
 +
<th><img id="imginfo2" src="https://static.igem.org/mediawiki/2020/1/15/T--CCU_Taiwan--Poster_model05.png"></th>
 +
<th><img id="imginfo2" src="https://static.igem.org/mediawiki/2020/4/41/T--CCU_Taiwan--Poster_model06.png"></th>
 +
<th><img id="imginfo2" src="https://static.igem.org/mediawiki/2020/c/c3/T--CCU_Taiwan--Poster_model07.png"></th>
 +
<th><img id="imginfo2" src="https://static.igem.org/mediawiki/2020/1/14/T--CCU_Taiwan--Poster_model08.png"></th>
 +
</tr>
 +
<tr>
 +
<td>Center to center distance (nm)</td>
 +
<td>30.32</td>
 +
<td>31.06</td>
 +
<td>33.19</td>
 +
<td>30.32</td>
 +
</tr>
 +
<tr>
 +
<td>Attractive energy (10<sup>-21</sup>J)</td>
 +
<td>-7.8</td>
 +
<td>-7.5</td>
 +
<td>-6.7</td>
 +
<td>-7.8</td>
 +
</tr>
 +
<tr>
 +
<td>Repulsive energy (10<sup>-21</sup>J)</td>
 +
<td>51.7</td>
 +
<td>51.7</td>
 +
<td>51.6</td>
 +
<td>51.7</td>
 +
</tr>
 +
<tr>
 +
<td>Total energy (10<sup>-21</sup>J)</td>
 +
<td>43.9</td>
 +
<td>44.2</td>
 +
<td>44.9</td>
 +
<td>43.9</td>
 +
</tr>
 +
  </tbody>
 +
  </table>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>
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<div class="title">The Detection Kit</div>
 
<div class="title">The Detection Kit</div>
 
<div class="text">
 
<div class="text">
−
<b>Content of DENDETX and Materials Used:</b>
+
<ul>
 +
<li><b>Content of DENDETX and Materials Used:</b></li>
 +
</ul>
 
<ol>
 
<ol>
 
<li>Sample pad (cellulose fibers)</li>
 
<li>Sample pad (cellulose fibers)</li>
−
<li>Conjugate pad (glass fibers)</li>
+
<li>Conjugate pad (glass fiber membranes)</li>
−
<li>Test line (glass fibers)</li>
+
<li>Test line (glass fiber membranes)</li>
−
<li>Control line (glass fibers)</li>
+
<li>Control line (glass fiber membranes)</li>
 
<li>Absorbent pad (cellulose fibers)</li>
 
<li>Absorbent pad (cellulose fibers)</li>
 
</ol>
 
</ol>
−
The conjugate pad contains the gold nanoparticles (AuNPs) coupled with the first PTRS (PTRS-1), in which PTRS-1 is designed to interact with the virions. The second PTRS (PTRS-2) is mounted on the test line to capture the virion. E proteins are conjugated on the control line, which should always interact with PTRS-1, to ensure the kit functions normally.
+
<br>
 +
The conjugate pad contains the AuNPs coupled with the first PTRS (PTRS-1), in which PTRS-1 is designed to interact with the virions. The second PTRS (PTRS-2) is mounted on the test line to capture the virion. E proteins are conjugated on the control line, which should always interact with PTRS-1, to ensure the kit functions normally.
 
<br>
 
<br>
 
<img src="https://static.igem.org/mediawiki/2020/1/13/T--CCU_Taiwan--Poster_Detection_kit-1.gif">
 
<img src="https://static.igem.org/mediawiki/2020/1/13/T--CCU_Taiwan--Poster_Detection_kit-1.gif">
−
<br>
+
<br><br>
 
<h4>Sample without Dengue Virions:</h4>
 
<h4>Sample without Dengue Virions:</h4>
−
The AuNPs will not accumulate on the test line, and no color change will be found. The AuNPs will aggregate on control line via the interaction of PTRS-1 and E protein, so the control line turns red.
+
The AuNPs will not accumulate on the test line, and no color change will be found. The AuNPs will aggregate on the control line via the interaction of PTRS-1 and E protein, so the control line turns red.
−
<br>
+
<br><br>
 
<h4>Sample with Dengue Virions:</h4>
 
<h4>Sample with Dengue Virions:</h4>
 
The AuNPs bound to the virions via PTRS-1 will accumulate on the test line, and the residual AuNPs will aggregate on the control line. Both the test and control lines will be red.
 
The AuNPs bound to the virions via PTRS-1 will accumulate on the test line, and the residual AuNPs will aggregate on the control line. Both the test and control lines will be red.
−
<br>
+
<br><br>
 
<h4>Current Progress of DENDETX:</h4>
 
<h4>Current Progress of DENDETX:</h4>
 
We have shown that we are able to conjugate peptides/proteins to the AuNP, control line, and test line.
 
We have shown that we are able to conjugate peptides/proteins to the AuNP, control line, and test line.
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<img src="https://static.igem.org/mediawiki/2020/f/f2/T--CCU_Taiwan--Poster_Results1.png">
 
<img src="https://static.igem.org/mediawiki/2020/f/f2/T--CCU_Taiwan--Poster_Results1.png">
 
<br><br>
 
<br><br>
−
The TR-PCR products were taken to AD-PCR. The AD-PCR product of TRS-110*3 TRS-151*7 with sizes ranging from 200-500 bp were extracted and ligated to TA vectors.
+
The TR-PCR products were taken to AD-PCR. The AD-PCR products of TRS-110*3 and TRS-151*7 with sizes ranging from 200-500 bp were extracted and ligated to TA vectors.
 
<br>
 
<br>
 
<img src="https://static.igem.org/mediawiki/2020/6/6b/T--CCU_Taiwan--Poster_Results2.png">
 
<img src="https://static.igem.org/mediawiki/2020/6/6b/T--CCU_Taiwan--Poster_Results2.png">
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<h4>E Protein</h4>
 
<h4>E Protein</h4>
 
We expressed E protein to use it in the control line.
 
We expressed E protein to use it in the control line.
−
<br><br>
+
<br>
−
The weak bands from 63 to 48 kDa indicate expression of the E protein with the HA tag, which has a size of 61.2 kDa. We further confirmed using Western blot with the anti-His tag antibodies.
+
The weak bands from 63 to 48 kDa indicate the E protein expression with the HA tag, which has a size of 61.2 kDa. We further confirmed using Western blot with the anti-His tag antibodies.
 
<br>
 
<br>
 
<img src="https://static.igem.org/mediawiki/2020/f/f3/T--CCU_Taiwan--Poster_Results3.png">
 
<img src="https://static.igem.org/mediawiki/2020/f/f3/T--CCU_Taiwan--Poster_Results3.png">
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<h4>CLEC5A</h4>
 
<h4>CLEC5A</h4>
 
In addition to the required E protein used in our kit, we attempted to express CLEC5A as a model protein to interact with E protein.
 
In addition to the required E protein used in our kit, we attempted to express CLEC5A as a model protein to interact with E protein.
−
<br><br>
+
<br>
−
The strong bands on 26.6 kDa indicate expression of the CLEC5A extracellular domain with the Myc tag, which is also confirmed using western blot with the anti-His tag antibodies.
+
The strong bands on 26.6 kDa indicate the CLEC5A extracellular domain with the Myc tag, which is also confirmed using western blot with the anti-His tag antibodies.
 
<br>
 
<br>
 
<img src="https://static.igem.org/mediawiki/2020/0/0c/T--CCU_Taiwan--Poster_Results4.png">
 
<img src="https://static.igem.org/mediawiki/2020/0/0c/T--CCU_Taiwan--Poster_Results4.png">
 
<br><br>
 
<br><br>
 
<h4>Detection Kit</h4>
 
<h4>Detection Kit</h4>
−
The AuNPs were first modified with MUA/MCH so that carboxyl groups are exposed on the surface of the particles. Then, peptides with the amine sidechains could be conjugated to the AuNPs using the EDC/NHS approach. Similarly, the glass fiber membranes, the substrate of the test and control lines, were modified with CES to form a carboxyl surface, which allows us to employ the EDC/NHS approach to conjugate with peptides or proteins.
+
The AuNPs were first modified with MUA/MCH, and then, peptides with the amine sidechains could be conjugated to the AuNPs using the EDC/NHS approach. Similarly, the glass fiber membranes, the substrate of the test and control lines, were modified with CES, which allows us to employ the EDC/NHS approach to conjugate with peptides or proteins.
 
<br><br>
 
<br><br>
−
As a proof of concept that we are able to form the covalent bonds between the primary amines (from the PTRS) and AuNPs, we tried to conjugate the DNA primers with the modified AuNPs. We found that with the DNA conjugation, the Raman signals have a significant decrease (Figure 1). Although we have no model to explain this effect, we believe it resulted from interactions with the DNA, suggesting DNA can bind to AuNPs.
+
As a proof of concept that we are able to form the covalent bonds between the primary amines (from the PTRS) and AuNPs, we tried to conjugate the DNA primers with the modified AuNPs. We found that with the DNA conjugation, the Raman signals have a significant decrease. Although we have no model to explain this effect, we believe it resulted from interactions with the DNA, suggesting DNA can bind to AuNPs.
 
<br><br>
 
<br><br>
−
We expressed green fluorescent protein (GFP) as a mock E protein to show that after modification the glass fiber membrane can bind to the primary amines (side-chain amines) from a peptide or protein. The intensity of membrane reaction with 0.5x and 1x GFP supernatant are shown in Figure 2. The intensity was about twice as strong in the 1x supernatant as the 0.5x one, suggesting the conjugation experiments were successful.
+
We expressed green fluorescent protein (GFP) as a mock E protein to show that the modified glass fiber membranes can bind to the primary amines (sidechain amines) from a peptide or protein after modification. The intensities of emission correlate to GFP concentration using in the reactions, suggesting the conjugation experiments were successful.
 
<br>
 
<br>
 
<img src="https://static.igem.org/mediawiki/2020/2/2a/T--CCU_Taiwan--Poster_Results5.png">
 
<img src="https://static.igem.org/mediawiki/2020/2/2a/T--CCU_Taiwan--Poster_Results5.png">
 +
<br>
 
<b>Abbreviations:</b>
 
<b>Abbreviations:</b>
 
MUA: 11-mercaptoundecanoic acid;
 
MUA: 11-mercaptoundecanoic acid;
Line 256: Line 351:
 
<div class="text">
 
<div class="text">
 
<h4>Interviews</h4>
 
<h4>Interviews</h4>
−
We wanted to shape our project from different perspectives to achieve socially responsible research. Thus, we consulted with government officials, experts, and academic specialists to consider the application of our project in society. From the interviews, we understood the process and the standards for verification for medical devices from the Taiwan Food and Drug Administration (TFDA). Moreover, we learned about pretreatment of blood sample from The Dengue Prevention and Control Center. We also comprehended the modification of gold nanoparticles after consulting with experts in the Department of Chemistry and Biochemistry at National Chung Cheng University.
+
We wanted to shape our project from different perspectives to achieve socially responsible research. Thus, we consulted with government officials, experts, and academic specialists to consider the application of our project in society. From the interviews, we understood the process and the standards for verification for medical devices from the Taiwan Food and Drug Administration. Moreover, we learned about the pretreatment of the blood sample from The Dengue Prevention and Control Center. We also comprehended the modification of gold nanoparticles after consulting with experts in the Department of Chemistry and Biochemistry at National Chung Cheng University.
−
<br>
+
<br><br>
 
<h4>Public Survey</h4>
 
<h4>Public Survey</h4>
−
We wanted to find out how much the public knows about dengue and their demands for a dengue virus detection kit. Thus, we created a questionnaire and received 248 questionnaires by sharing the it online and distributing it in the community.
+
We want to find out how much the public knows about dengue and their demands for a dengue virus detection kit. Thus, we created a questionnaire and received 248 questionnaires by sharing it online and distributing it in the community.
 
<br><br>
 
<br><br>
−
The results showed nearly 70% of people did not know about severe dengue and nearly 70% did not know what kind of treatment is required for dengue fever. This shows that the public in Taiwan lack common understanding about dengue, which leads to low awareness about the severity of this disease in Taiwan. This urged us to conduct outreach and education programs to popularize knowledge of dengue fever.
+
The results showed that nearly 70% of people did not know about severe dengue, and nearly 70% did not know what kind of treatment is required for dengue fever. This shows that the public in Taiwan lacks a common understanding of dengue, which leads to low awareness about the severity of this disease in Taiwan. This urged us to conduct outreach and education programs to popularize knowledge of dengue fever.
 +
<br><br>
 +
From the survey, we also determined that our detection kit meets their needs, as it should have high sensitivity during the first five days of symptoms, which is when people would consult a doctor.
 
<br><br>
 
<br><br>
−
From the survey we also determined that our detection kit meets their needs, as it should have high sensitivity during the first five days of symptoms, which is when people would consult a doctor.
 
−
<br>
 
 
<h4>Education</h4>
 
<h4>Education</h4>
−
<b>Podcast</b>
+
<ul>
−
<br>
+
<li><b>Podcast</b></li>
−
Podcast has become a popular platform to convey knowledge in Taiwan. In order to reach the general public, we created a podcast program with 7 episodes talking about synthetic biology.
+
Podcast has become a popular platform to convey knowledge in Taiwan. In order to reach the general public, we created a podcast program with 7 episodes talking about synthetic biology.
−
<br><br>
+
<br><br>
−
<b>Dengue Fever Information in Taiwanese Hokkien</b>
+
<li><b>Dengue Fever Information in Taiwanese Hokkien</b></li>
−
<div id="imginfo1">
+
<div id="imginfo1">
−
<img src="https://static.igem.org/mediawiki/2020/1/1b/T--CCU_Taiwan--Poster_HP-6.jpeg">
+
<img src="https://static.igem.org/mediawiki/2020/1/1b/T--CCU_Taiwan--Poster_HP-6.jpeg">
−
</div>
+
</div>
−
Taiwan is considered an aged society, as the aged population reached 16% in 2018. We invited the elderly in our community help us record a dengue fever information audio in Taiwanese Hokkien, targeting senior citizens, who mostly use this local dialect.
+
Taiwan is considered an ageing society, as the aged population reached 16% in 2018. We invited the elderly in our community to help us record a dengue fever information audio in Taiwanese Hokkien, targeting senior citizens, who mostly use this local dialect.
−
<br><br>
+
<br><br>
−
<b>Children’s Storybook</b>
+
<li><b>Children’s Storybook</b></li>
−
<div id="imginfo1">
+
<div id="imginfo1">
−
<img src="https://static.igem.org/mediawiki/2020/4/43/T--CCU_Taiwan--Poster_HP-7.jpeg">
+
<img src="https://static.igem.org/mediawiki/2020/4/43/T--CCU_Taiwan--Poster_HP-7.jpeg">
−
</div>
+
</div>
−
We created a children’s storybook to spark children’s interest in science. To reach to more children and increase the impact of our storybook, we translated it into English, Chinese, and Hindi.
+
We created a children’s storybook to spark children’s interest in science. To reach more children and increase the impact of our storybook, we translated it into English, Chinese, and Hindi.
−
<br><br>
+
<br><br>
−
<b>Educational Activity</b>
+
<li><b>Educational Activity</b></li>
−
<br>
+
We went to the Cheng-He Confucian academic center, a religious center where children study Confucianism, teaching them basic ideas about biology.
−
We went to the Cheng-He Confucian academic center, a religious center where children study Confucianism, teaching them basic ideas about biology.
+
<br><br>
−
<br><br>
+
<li><b>Radio Interview</b></li>
−
<b>Radio Interview</b>
+
We were invited by National Education Radio to introduce synthetic biology, iGEM competition, and also our project.
−
<br>
+
<br><br>
−
We were invited by National Education Radio to introduce synthetic biology, iGEM competition and also our project.
+
<li><b>Communication</b></li>
−
<br><br>
+
We joined the 2020 Taiwan iGEM Conference, which provided us a chance to share our project, and received lots of valuable advice from other teams and professors. We also participated in a panel discussion focusing on sustainable development goals (SDGs). This leads us to have a deeper understanding of SDGs.
−
<b>Communication</b>
+
</ul>
−
<br>
+
−
We joined the 2020 Taiwan iGEM Conference, which provided us a chance to share our project and received lots of valuable advice from other teams and professors. We also participated in a panel discussion focusing on sustainable development goals (SDGs). This lead us to have a deeper understanding of SDGs.
+
 
</div>
 
</div>
 
</div>
 
</div>
Line 305: Line 398:
 
<img src="https://static.igem.org/mediawiki/2020/4/47/T--CCU_Taiwan--Poster_Inclusion-1.jpg">
 
<img src="https://static.igem.org/mediawiki/2020/4/47/T--CCU_Taiwan--Poster_Inclusion-1.jpg">
 
</div>
 
</div>
−
We wanted to reach out to communities that are underrepresented in science to allow them to participate in science. Thus, we went to a juvenile correction school to popularize science, as juvenile inmates lack opportunities to learn science.
+
We want to reach out to communities underrepresented in science to allow them to participate in science. Thus, we went to a juvenile correction school to popularize science, as juvenile inmates lack opportunities to learn science.
 +
<br><br>
 +
From this activity, we found that inclusion in science communication is a two-way street. Science should include communities that lack access to science by eliminating the access barriers with diverse and engaging ways to bring science closer to them. In order to experience the beauty of science, underrepresented communities must overcome conventional thinking that science is too difficult.
 
<br><br>
 
<br><br>
−
From this activity, we found that inclusion in science communication is a two-way street. Science should include communities who lack access to science by eliminating the access barriers with diverse and engaging ways to bring science closer to them. In order to experience the beauty of science, underrepresented communities must overcome conventional thinking that science is too difficult.
 
−
<br>
 
 
<h4>Glove Puppetry</h4>
 
<h4>Glove Puppetry</h4>
 
<img src="https://static.igem.org/mediawiki/2020/0/0c/T--CCU_Taiwan--Poster_Inclusion-2.jpeg">
 
<img src="https://static.igem.org/mediawiki/2020/0/0c/T--CCU_Taiwan--Poster_Inclusion-2.jpeg">
−
Glove puppetry is a traditional performing art in Taiwan with its own unique elements such as using the language most spoken by Taiwanese elderly, Taiwanese Hokkien, for narration. This inspired us to integrate this folk art into science communication. We thought that a non-scientific artist could also provide a different perspective on science.
+
Glove puppetry is a traditional performing art in Taiwan with its own unique elements, such as using the language most spoken by Taiwanese elderly, Taiwanese Hokkien, for narration. This inspired us to integrate this folk art into science communication. We thought that a non-scientific artist could also provide a different perspective on science.
 
<br><br>
 
<br><br>
 
Thus, we collaborated with a glove puppetry troupe to transmit knowledge of dengue by narrating and filming a story as a 13-minute video.
 
Thus, we collaborated with a glove puppetry troupe to transmit knowledge of dengue by narrating and filming a story as a 13-minute video.
 
<br><br>
 
<br><br>
−
We realized that continuous development of science requires adding diverse fields and perspective to make it more widely recognized, just as glove puppetry integrates modern elements to keep up with the times.
+
We realized that the continuous development of science requires adding diverse fields and perspectives to make it more widely recognized, just as glove puppetry integrates modern elements to keep up with the times.
 
</div>
 
</div>
 
</div>
 
</div>
Line 324: Line 417:
 
<div class="title">References</div>
 
<div class="title">References</div>
 
<div class="text">
 
<div class="text">
−
<b>Introduction</b>
+
<ul>
−
<ol>
+
<li><b>Introduction</b></li>
−
<li>Taiwan National Infectious Disease Statistics System - Taiwan Centers of Disease Control (TCDC), https://reurl.cc/EzLvE1</li>
+
<ol>
−
<li>Dengue and severe dengue - World Health Organization, https://reurl.cc/q84qQp</li>
+
<li>Dengue and severe dengue - World Health Organization.</li>
−
<li>Dengue Fever – Taiwan Centers of Disease Control (TCDC), https://reurl.cc/q84qQR</li>
+
                                                                        <li>Taiwan National Infectious Disease Statistics System - Taiwan Centers of Disease Control.</li>
−
</ol>
+
<li>Dengue Fever – Taiwan Centers of Disease Control.</li>
−
<br>
+
</ol>
−
<b>Inspiration</b>
+
<br>
−
<ol>
+
<li><b>Inspiration</b></li>
−
<li>Szu-Ting Chen, Fei-Ju Li, Tzy-yun Hsu, Shu-Mei Liang, Yi-Chen Yeh, Wen-Yu Liao, Teh-Ying Chou, Nien-Jun Chen, Michael Hsiao, Wen-Bin Yang, and Shie-Liang Hsieh. CLEC5A is a critical receptor in innate immunity against Listeria infection. Nature Communications. 2017; 8(1): 299. doi:10.1038/s41467-017-00356-3.</li>
+
<ol>
−
<li>Aleksandra A Watson, Andrey A Lebedev, Benjamin A Hall, Angharad E Fenton-May, Alexei A Vagin, Wanwisa Dejnirattisai, James Felce, Juthathip Mongkolsapaya, Angelina S Palma, Yan Liu, Ten Feizi, Gavin R Screaton, Garib N Murshudov, and Christopher A O'Callaghan. Structural flexibility of the macrophage dengue virus receptor CLEC5A: implications for ligand binding and signaling. Journal of Biological Chemistry. 2011; 286(27): 24208-24218. doi: 10.1074/jbc.M111.226142.</li>
+
<li>Szu-Ting Chen, Fei-Ju Li, Tzy-yun Hsu, Shu-Mei Liang, Yi-Chen Yeh, Wen-Yu Liao, Teh-Ying Chou, Nien-Jun Chen, Michael Hsiao, Wen-Bin Yang, and Shie-Liang Hsieh. CLEC5A is a critical receptor in innate immunity against Listeria infection. Nature Communications. 2017; 8(1): 299. doi:10.1038/s41467-017-00356-3.</li>
−
</ol>
+
<li>Aleksandra A Watson, Andrey A Lebedev, Benjamin A Hall, Angharad E Fenton-May, Alexei A Vagin, Wanwisa Dejnirattisai, James Felce, Juthathip Mongkolsapaya, Angelina S Palma, Yan Liu, Ten Feizi, Gavin R Screaton, Garib N Murshudov, and Christopher A O'Callaghan. Structural flexibility of the macrophage dengue virus receptor CLEC5A: implications for ligand binding and signaling. Journal of Biological Chemistry. 2011; 286(27): 24208-24218. doi:10.1074/jbc.M111.226142.</li>
−
<br>
+
</ol>
−
<b>LAE</b>
+
<br>
−
<br>
+
<li><b>LAE</b></li>
−
<ol>
+
<ol>
−
<li>Peng-Yeh Lai, Chia-Tse Hsu, Shao-Hung Wang, Jin-Ching Lee, Min-Jen Tseng, Jaulang Hwang, Wen-Tsai Ji, and Hau-Ren Chen. Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique. Journal of General Virology. 2014; 95(10): 2155-2165. doi: 10.1099/vir.0.062562-0.</li>
+
<li>Peng-Yeh Lai, Chia-Tse Hsu, Shao-Hung Wang, Jin-Ching Lee, Min-Jen Tseng, Jaulang Hwang, Wen-Tsai Ji, and Hau-Ren Chen. Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique. Journal of General Virology. 2014; 95(10): 2155-2165. doi:10.1099/vir.0.062562-0.</li>
−
</ol>
+
</ol>
−
<br><br>
+
<br><br>
−
<b>Modeling: Interaction between the Tandem-repeated Sequence Peptide and E Protein</b>
+
<li><b>Modeling: Interaction between the Tandem-repeated Sequence Peptide and E Protein</b></li>
−
<ol>
+
<ol>
−
<li>Maciej Ciemny, Mateusz Kurcinski, Karol Kamel, Andrzej Kolinski, Nawsad Alam, Ora Schueler-Furman, and Sebastian Kmiecik. Protein–peptide docking: opportunities and challenges. Drug Discovery Today. 2018; 23(8): 1530-1537. doi: 10.1016/j.drudis.2018.05.006.</li>
+
<li>Maciej Ciemny, Mateusz Kurcinski, Karol Kamel, Andrzej Kolinski, Nawsad Alam, Ora Schueler-Furman, and Sebastian Kmiecik. Protein–peptide docking: opportunities and challenges. Drug Discovery Today. 2018; 23(8): 1530-1537. doi:10.1016/j.drudis.2018.05.006.</li>
−
<li>Steven A Combs, Samuel L Deluca, Stephanie H Deluca, Gordon H Lemmon, David P Nannemann, Elizabeth D Nguyen, Jordan R Willis, Jonathan H Sheehan, and Jens Meiler. Small-molecule ligand docking into comparative models with Rosetta. Nature Protocols. 2013; 8(7): 1277-1298. doi: 10.1038/nprot.2013.074.</li>
+
<li>Steven A Combs, Samuel L Deluca, Stephanie H Deluca, Gordon H Lemmon, David P Nannemann, Elizabeth D Nguyen, Jordan R Willis, Jonathan H Sheehan, and Jens Meiler. Small-molecule ligand docking into comparative models with Rosetta. Nature Protocols. 2013; 8(7): 1277-1298. doi:10.1038/nprot.2013.074.</li>
−
</ol>
+
</ol>
−
<br>
+
<br>
−
<b>Modeling: Interaction between Gold Nanoparticles</b>
+
<li><b>Modeling: Interaction between Gold Nanoparticles</b></li>
−
<ol>
+
<ol>
−
<li>Elena Pokidysheva, Ying Zhang, Anthony J Battisti, Carol M Bator-Kelly, Paul R Chipman, Chuan Xiao, G Glenn Gregorio, Wayne A Hendrickson, Richard J Kuhn, and Michael G Rossmann. Cryo-EM Reconstruction of Dengue Virus in Complex with the Carbohydrate Recognition Domain of DC-SIGN. Cell. 2006; 124(3): 485-93. doi: 10.1016/j.cell.2005.11.042.</li>
+
<li>Elena Pokidysheva, Ying Zhang, Anthony J Battisti, Carol M Bator-Kelly, Paul R Chipman, Chuan Xiao, G Glenn Gregorio, Wayne A Hendrickson, Richard J Kuhn, and Michael G Rossmann. Cryo-EM Reconstruction of Dengue Virus in Complex with the Carbohydrate Recognition Domain of DC-SIGN. Cell. 2006; 124(3): 485-493. doi:10.1016/j.cell.2005.11.042.</li>
−
<li>Jörg Polte. Fundamental growth principles of colloidal metal nanoparticles – a new perspective. CrystEngComm. 2015, 17(36): 6809-6830. doi:10.1039/C5CE01014D.</li>
+
<li>Jörg Polte. Fundamental growth principles of colloidal metal nanoparticles – a new perspective. CrystEngComm. 2015, 17(36): 6809-6830. doi:10.1039/C5CE01014D.</li>
−
<li>Phillip E Mason, Adrien Lerbret, Marie-Louise Saboungi, George W Neilson, Christopher E Dempsey, and John W Brady. Glucose Interactions with a Model Peptide. Proteins. 2011; 79(7): 2224-2232. doi: 10.1002/prot.23047.</li>
+
<li>Phillip E Mason, Adrien Lerbret, Marie-Louise Saboungi, George W Neilson, Christopher E Dempsey, and John W Brady. Glucose Interactions with a Model Peptide. Proteins. 2011; 79(7): 2224-2232. doi:10.1002/prot.23047.</li>
−
<li>Taehoon Kim, Kangtaek Lee, Myoung-seon Gong, and Sang-Woo Joo. Control of Gold Nanoparticle Aggregates by Manipulation of Interparticle Interaction. Langmuir . 2005; 21(21): 9524-9528. doi: 10.1021/la0504560.</li>
+
<li>Taehoon Kim, Kangtaek Lee, Myoung-seon Gong, and Sang-Woo Joo. Control of Gold Nanoparticle Aggregates by Manipulation of Interparticle Interaction. Langmuir. 2005; 21(21): 9524-9528. doi:10.1021/la0504560.</li>
−
</ol>
+
</ol>
 +
</ul>
 
</div>
 
</div>
 
</div>
 
</div>

Latest revision as of 01:43, 11 November 2020

DENDETX: A Dengue Virus Detection Kit

What We Want to Achieve

  1. Develop a rapid test for dengue fever to improve treatment outcomes.
  2. Develop a method to mass-produce peptides for virus recognition, reducing cost.
  3. Raise awareness of dengue fever in scientifically illiterate and high-risk groups.

Who We Are

  • Team member:
    Chia-Wei Shen, Jyue-Han Chen, Xiang-Yen Wong, Chiau-Shu Pan, Hong-Fu Liao, Yuan-Zhi Li, Young-Cheng Lin, Chen-Shing Tseng, Li-Yun Cheng, Ching-Chia Kuo, Yu-Shiuan Chang, Wen-Han Chang, Xin-Yu Chang, En-Chi Chang
  • Instructors:
    Eugene C. Lin, Hau-Ren Chen, Chun-Ying Yu, Cheng-I Lee, Victoria Rau, Gerald Rau, Yen-Ta Tseng
  • Advisors:
    Cheng-Yu Lee, Wei-Cheng Wang, Hooi-Hui Khor, Tzu-Cheng Chuang, Meng-Jing Lee, Yu-Xian Cheng
  • Sponsors:
    National Chung Cheng University, Academic Foundation Chung Cheng University, Association of CCU Alumni, 國立中正大學之友會, Integrated DNA Technologies, Software for molecular biology, Benching, MathWorks
Dengue Fever is Threatening Taiwan and the World
Dengue fever occurs in tropical and subtropical regions. Half of the world population is now at risk, with 390 million infections every year, causing dengue fever to be listed by WHO as a top 10 health threat.



Dengue fever can progress into more severe conditions like dengue hemorrhagic fever (DHF), which has a 20% mortality rate. If conditions continue to worsen, patients experience dengue shock syndrome (DSS), which has a mortality rate of up to 40%. An estimated 500,000 patients develop DHF or DSS, resulting in about 25,000 deaths every year.

Taiwan is in the area under the threat of the dengue virus. From 2014 to 2015, there were over 44,000 confirmed cases during a dengue fever outbreak that spread from Tainan to other cities, causing 228 deaths. Recently, there was a local transmission of dengue fever in Taoyuan, showing that the potentially affected population keeps increasing.

There is no specific treatment or vaccine for dengue, and health care systems are still facing a challenge from dengue fever.
Interaction between the CLEC5A and E Protein Suggests Proteins/Peptides Might be Effective in Detection
After infection by the dengue virus, a nonstructural protein, NS1, appears in blood serum early in the infection. The current method of detecting dengue fever uses antibodies to recognize NS1 antigens. Nevertheless, the cost of the antibodies is high, the antibodies are hard to purify, and the procedure of making antibodies is time-consuming.

We took a step back to seek an alternative. We aimed to find something smaller and easier to produce to detect the dengue virus instead of antibodies. Symptoms of DHF and DSS result from the interaction between C-type lectin domain, family 5, member A (CLEC5A) on macrophages and the envelope protein (E proteins) of dengue virus.


We found the sequences from CLEC5A most likely to bind the dengue virus and confirmed these using Rosetta simulations. We believe these peptides have the potential to detect the dengue virus.

We designed a detection kit with these peptides that can bind to the E protein of the dengue virus. This would allow people who may have dengue fever or live in an outbreak region of dengue fever to get treatment or protection and control the spread early.
Linear Array Epitope
Linear array epitope (LAE) is a technique to produce tandem-repeated sequences (TRSs). The TRSs would be expressed as a long-chain peptide which can be cleaved by an enzyme, resulting in many identical peptides.

  • The Procedure of LAE:
  1. The design of DNA oligo primers
  2. Template-repeated PCR (TR-PCR)
  3. Adaptor PCR (AD-PCR)

For the primer design, TR-PCR primers need to be partially complementary to each other, and AD-PCR primers need to have restriction sites.


In TR-PCR, those primers act as both primer and template, and they are partially complementary. The TRSs can be produced during the PCR cycling process. In AD-PCR, restriction sites need be be introduced into the products of TR-PCR by adding the AD-PCR primers in PCR.
BioBricks
Our peptides and proteins were expressed in E. Coli with a T7 promoter and a lac operator, which can be induced by IPTG. The thrombin site was included to remove unnecessary sections. The His tag was used in Western blot to confirm the expression of the protein, and the HA tag and myc tag were utilized for purification.





Interaction between the Peptide of Tandem-repeated Sequence and E Protein
  • Purpose:
    • Our goal is to design the peptide of tandem-repeated sequences (PTRSs) to imitate the binding of CLEC5A to the E protein from the dengue virus. In order to ensure the PTRSs and the E protein interact, all the structure of PTRSs and proteins and their interactions were modeled using Rosetta.

  • Method and Results:
    1. The structure of E protein from a local strain (PL046) was generated using RosettaCM (comparative modeling with Rosetta) based on the crystal structure (PDB: 1OAN).
    2. The structures of PTRSs were predicted purely based on their sequences using the ab initio method in Rosetta.
    3. The most probable structure of the PTRS was identified by clustering.
    4. The interactions between the PTRSs and the E protein were evaluated using global protein-protein docking. The 100 most frequent docking sites are shown below.


Interaction between Gold Nanoparticles

  • Purpose:
    • We use gold nanoparticles (AuNPs) as an indicator in the detection kit. A potential weakness of our design is that peptides on the AuNPs and those on the glass fiber membranes compete for the same binding sites on the E protein. If the E proteins on the virus particles are fully covered by AuNPs, there would be no sites available to interact with the peptides attached to the glass fiber membranes. Hence, we need to understand if this situation would hinder the development of our kit.

  • Method:
    • We used the DLVO theory to calculate the repulsion between AuNPs to estimate the number of AuNPs that would bind to a virus particle. The structure of the dengue virus is icosahedral, and the distances between the potential binding sites can be obtained from the structure in the protein data bank (1K4R). We took several representative positions on adjoining faces of the icosahedron to calculate the interactions between the AuNPs based on the DLVO theory.

  • Results:
    • The total energies of the interactions between AuNPs are all positive, indicating repulsive forces, and these total energies are also larger than the typical biological interactions (~0.5 kcal/mol or 3.49 x10-21 J). The results suggest that there will always be free faces on the virus particles to interact with the peptides conjugated on the test line.
    Center to center distance (nm) 54.58 42.71 31.71 43.24
    Attractive energy (10-21J) -3.3 -4.6 -7.2 -4.5
    Repulsive energy (10-21J) 51.0 51.3 51.6 51.3
    Total energy (10-21J) 47.7 46.7 44.4 46.8
    Center to center distance (nm) 30.32 31.06 33.19 30.32
    Attractive energy (10-21J) -7.8 -7.5 -6.7 -7.8
    Repulsive energy (10-21J) 51.7 51.7 51.6 51.7
    Total energy (10-21J) 43.9 44.2 44.9 43.9
The Detection Kit
  • Content of DENDETX and Materials Used:
  1. Sample pad (cellulose fibers)
  2. Conjugate pad (glass fiber membranes)
  3. Test line (glass fiber membranes)
  4. Control line (glass fiber membranes)
  5. Absorbent pad (cellulose fibers)

The conjugate pad contains the AuNPs coupled with the first PTRS (PTRS-1), in which PTRS-1 is designed to interact with the virions. The second PTRS (PTRS-2) is mounted on the test line to capture the virion. E proteins are conjugated on the control line, which should always interact with PTRS-1, to ensure the kit functions normally.


Sample without Dengue Virions:

The AuNPs will not accumulate on the test line, and no color change will be found. The AuNPs will aggregate on the control line via the interaction of PTRS-1 and E protein, so the control line turns red.

Sample with Dengue Virions:

The AuNPs bound to the virions via PTRS-1 will accumulate on the test line, and the residual AuNPs will aggregate on the control line. Both the test and control lines will be red.

Current Progress of DENDETX:

We have shown that we are able to conjugate peptides/proteins to the AuNP, control line, and test line.
Results

LAE

The results of TR-PCR indicate the length of TRS varies with the concentration of the primers. We found the typical trend that the lower the concentration, the higher the repeats of the TR-PCR products. However, the product concentration decreases when the primer concentration increases. TRS-110*3 and TRS-151*7, with sizes ranging from 200-500 bp.


The TR-PCR products were taken to AD-PCR. The AD-PCR products of TRS-110*3 and TRS-151*7 with sizes ranging from 200-500 bp were extracted and ligated to TA vectors.


E Protein

We expressed E protein to use it in the control line.
The weak bands from 63 to 48 kDa indicate the E protein expression with the HA tag, which has a size of 61.2 kDa. We further confirmed using Western blot with the anti-His tag antibodies.


CLEC5A

In addition to the required E protein used in our kit, we attempted to express CLEC5A as a model protein to interact with E protein.
The strong bands on 26.6 kDa indicate the CLEC5A extracellular domain with the Myc tag, which is also confirmed using western blot with the anti-His tag antibodies.


Detection Kit

The AuNPs were first modified with MUA/MCH, and then, peptides with the amine sidechains could be conjugated to the AuNPs using the EDC/NHS approach. Similarly, the glass fiber membranes, the substrate of the test and control lines, were modified with CES, which allows us to employ the EDC/NHS approach to conjugate with peptides or proteins.

As a proof of concept that we are able to form the covalent bonds between the primary amines (from the PTRS) and AuNPs, we tried to conjugate the DNA primers with the modified AuNPs. We found that with the DNA conjugation, the Raman signals have a significant decrease. Although we have no model to explain this effect, we believe it resulted from interactions with the DNA, suggesting DNA can bind to AuNPs.

We expressed green fluorescent protein (GFP) as a mock E protein to show that the modified glass fiber membranes can bind to the primary amines (sidechain amines) from a peptide or protein after modification. The intensities of emission correlate to GFP concentration using in the reactions, suggesting the conjugation experiments were successful.

Abbreviations: MUA: 11-mercaptoundecanoic acid; MCH: 6-hydroxy-1-hexanethiol; EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS: N-hydroxysuccinimide; DCC: N,N’-dicyclohexylcarbodiimide; MHA: 16-mercaptohexadecanoic acid; SB thiol: 1-(2-sulfosulfanylethylamino)tetradecane
Human Practices

Interviews

We wanted to shape our project from different perspectives to achieve socially responsible research. Thus, we consulted with government officials, experts, and academic specialists to consider the application of our project in society. From the interviews, we understood the process and the standards for verification for medical devices from the Taiwan Food and Drug Administration. Moreover, we learned about the pretreatment of the blood sample from The Dengue Prevention and Control Center. We also comprehended the modification of gold nanoparticles after consulting with experts in the Department of Chemistry and Biochemistry at National Chung Cheng University.

Public Survey

We want to find out how much the public knows about dengue and their demands for a dengue virus detection kit. Thus, we created a questionnaire and received 248 questionnaires by sharing it online and distributing it in the community.

The results showed that nearly 70% of people did not know about severe dengue, and nearly 70% did not know what kind of treatment is required for dengue fever. This shows that the public in Taiwan lacks a common understanding of dengue, which leads to low awareness about the severity of this disease in Taiwan. This urged us to conduct outreach and education programs to popularize knowledge of dengue fever.

From the survey, we also determined that our detection kit meets their needs, as it should have high sensitivity during the first five days of symptoms, which is when people would consult a doctor.

Education

  • Podcast
    • Podcast has become a popular platform to convey knowledge in Taiwan. In order to reach the general public, we created a podcast program with 7 episodes talking about synthetic biology.

  • Dengue Fever Information in Taiwanese Hokkien
    • Taiwan is considered an ageing society, as the aged population reached 16% in 2018. We invited the elderly in our community to help us record a dengue fever information audio in Taiwanese Hokkien, targeting senior citizens, who mostly use this local dialect.

  • Children’s Storybook
    • We created a children’s storybook to spark children’s interest in science. To reach more children and increase the impact of our storybook, we translated it into English, Chinese, and Hindi.

  • Educational Activity
    • We went to the Cheng-He Confucian academic center, a religious center where children study Confucianism, teaching them basic ideas about biology.

  • Radio Interview
    • We were invited by National Education Radio to introduce synthetic biology, iGEM competition, and also our project.

  • Communication
    • We joined the 2020 Taiwan iGEM Conference, which provided us a chance to share our project, and received lots of valuable advice from other teams and professors. We also participated in a panel discussion focusing on sustainable development goals (SDGs). This leads us to have a deeper understanding of SDGs.
Inclusion

Inmates

We want to reach out to communities underrepresented in science to allow them to participate in science. Thus, we went to a juvenile correction school to popularize science, as juvenile inmates lack opportunities to learn science.

From this activity, we found that inclusion in science communication is a two-way street. Science should include communities that lack access to science by eliminating the access barriers with diverse and engaging ways to bring science closer to them. In order to experience the beauty of science, underrepresented communities must overcome conventional thinking that science is too difficult.

Glove Puppetry

Glove puppetry is a traditional performing art in Taiwan with its own unique elements, such as using the language most spoken by Taiwanese elderly, Taiwanese Hokkien, for narration. This inspired us to integrate this folk art into science communication. We thought that a non-scientific artist could also provide a different perspective on science.

Thus, we collaborated with a glove puppetry troupe to transmit knowledge of dengue by narrating and filming a story as a 13-minute video.

We realized that the continuous development of science requires adding diverse fields and perspectives to make it more widely recognized, just as glove puppetry integrates modern elements to keep up with the times.
References
  • Introduction
    1. Dengue and severe dengue - World Health Organization.
    2. Taiwan National Infectious Disease Statistics System - Taiwan Centers of Disease Control.
    3. Dengue Fever – Taiwan Centers of Disease Control.

  • Inspiration
    1. Szu-Ting Chen, Fei-Ju Li, Tzy-yun Hsu, Shu-Mei Liang, Yi-Chen Yeh, Wen-Yu Liao, Teh-Ying Chou, Nien-Jun Chen, Michael Hsiao, Wen-Bin Yang, and Shie-Liang Hsieh. CLEC5A is a critical receptor in innate immunity against Listeria infection. Nature Communications. 2017; 8(1): 299. doi:10.1038/s41467-017-00356-3.
    2. Aleksandra A Watson, Andrey A Lebedev, Benjamin A Hall, Angharad E Fenton-May, Alexei A Vagin, Wanwisa Dejnirattisai, James Felce, Juthathip Mongkolsapaya, Angelina S Palma, Yan Liu, Ten Feizi, Gavin R Screaton, Garib N Murshudov, and Christopher A O'Callaghan. Structural flexibility of the macrophage dengue virus receptor CLEC5A: implications for ligand binding and signaling. Journal of Biological Chemistry. 2011; 286(27): 24208-24218. doi:10.1074/jbc.M111.226142.

  • LAE
    1. Peng-Yeh Lai, Chia-Tse Hsu, Shao-Hung Wang, Jin-Ching Lee, Min-Jen Tseng, Jaulang Hwang, Wen-Tsai Ji, and Hau-Ren Chen. Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique. Journal of General Virology. 2014; 95(10): 2155-2165. doi:10.1099/vir.0.062562-0.


  • Modeling: Interaction between the Tandem-repeated Sequence Peptide and E Protein
    1. Maciej Ciemny, Mateusz Kurcinski, Karol Kamel, Andrzej Kolinski, Nawsad Alam, Ora Schueler-Furman, and Sebastian Kmiecik. Protein–peptide docking: opportunities and challenges. Drug Discovery Today. 2018; 23(8): 1530-1537. doi:10.1016/j.drudis.2018.05.006.
    2. Steven A Combs, Samuel L Deluca, Stephanie H Deluca, Gordon H Lemmon, David P Nannemann, Elizabeth D Nguyen, Jordan R Willis, Jonathan H Sheehan, and Jens Meiler. Small-molecule ligand docking into comparative models with Rosetta. Nature Protocols. 2013; 8(7): 1277-1298. doi:10.1038/nprot.2013.074.

  • Modeling: Interaction between Gold Nanoparticles
    1. Elena Pokidysheva, Ying Zhang, Anthony J Battisti, Carol M Bator-Kelly, Paul R Chipman, Chuan Xiao, G Glenn Gregorio, Wayne A Hendrickson, Richard J Kuhn, and Michael G Rossmann. Cryo-EM Reconstruction of Dengue Virus in Complex with the Carbohydrate Recognition Domain of DC-SIGN. Cell. 2006; 124(3): 485-493. doi:10.1016/j.cell.2005.11.042.
    2. Jörg Polte. Fundamental growth principles of colloidal metal nanoparticles – a new perspective. CrystEngComm. 2015, 17(36): 6809-6830. doi:10.1039/C5CE01014D.
    3. Phillip E Mason, Adrien Lerbret, Marie-Louise Saboungi, George W Neilson, Christopher E Dempsey, and John W Brady. Glucose Interactions with a Model Peptide. Proteins. 2011; 79(7): 2224-2232. doi:10.1002/prot.23047.
    4. Taehoon Kim, Kangtaek Lee, Myoung-seon Gong, and Sang-Woo Joo. Control of Gold Nanoparticle Aggregates by Manipulation of Interparticle Interaction. Langmuir. 2005; 21(21): 9524-9528. doi:10.1021/la0504560.