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| <li>Raise awareness of dengue fever in scientifically illiterate and high-risk groups.</li> | | <li>Raise awareness of dengue fever in scientifically illiterate and high-risk groups.</li> |
| </ol> | | </ol> |
− | <br>
| |
| <h4>Attributions</h4> | | <h4>Attributions</h4> |
| <ul> | | <ul> |
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| <div class="title">Parts</div> | | <div class="title">Parts</div> |
| <div class="text"> | | <div class="text"> |
− | Our peptides and proteins were expressed in E. Coli 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 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. |
| <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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| 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><br> |
− | <b>Method:</b>
| |
− | <br>
| |
− | 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.
| |
− | <br><br>
| |
− | <b>Results:</b>
| |
− | <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.
| |
− | <br>
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− | <img src="https://static.igem.org/mediawiki/2020/1/10/T--CCU_Taiwan--Poster_Detection_kit-2.png">
| |
− | <br>
| |
− | Figure 1. The Raman spectra of DCC/NHS modified AuNPs (yellow), and DCC/NHS modified AuNPs conjugated with 1 μM (blue) and 0.1 μM (red) DNA primers.
| |
− | <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.
| |
− | <br>
| |
− | <img src="https://static.igem.org/mediawiki/2020/b/bd/T--CCU_Taiwan--Poster_Detection_kit-3.png">
| |
− | <br><br>
| |
− | Figure 2. Fluorescence from modified glass fiber membranes reacting with 1x GFP stock, modified glass fiber membranes reacting with 0.5x GFP stock, and non-modified glass fiber membranes (control).
| |
− | <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. |
− | <b>Abbreviations:</b>
| |
− | MUA: 11-mercaptoundecanoic acid;
| |
− | MCH: 6-hydroxy-1-hexanethiol;
| |
− | EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;
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− | NHS: N-hydroxysuccinimide;
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− | DCC: N,N’-dicyclohexylcarbodiimide;
| |
− | MHA: 16-mercaptohexadecanoic acid;
| |
− | SB thiol: 1-(2-sulfosulfanylethylamino)tetradecane
| |
| </div> | | </div> |
| </div> | | </div> |
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| <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"> |
| + | <b>Detection Kit</b> |
| + | <br> |
| + | 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. |
| + | <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. |
| + | <br> |
| + | <img src="https://static.igem.org/mediawiki/2020/1/10/T--CCU_Taiwan--Poster_Detection_kit-2.png"> |
| + | <br> |
| + | Figure 1. The Raman spectra of DCC/NHS modified AuNPs (yellow), and DCC/NHS modified AuNPs conjugated with 1 μM (blue) and 0.1 μM (red) DNA primers. |
| + | <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. |
| + | <br> |
| + | <img src="https://static.igem.org/mediawiki/2020/b/bd/T--CCU_Taiwan--Poster_Detection_kit-3.png"> |
| + | <br><br> |
| + | Figure 2. Fluorescence from modified glass fiber membranes reacting with 1x GFP stock, modified glass fiber membranes reacting with 0.5x GFP stock, and non-modified glass fiber membranes (control). |
| + | <br> |
| + | <b>Abbreviations:</b> |
| + | 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 |
| </div> | | </div> |
| </div> | | </div> |