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<h3 class="ar-title">SynNotch (<a href="http://parts.igem.org/Part:BBa_K2506001">BBa_K2506001</a>) </h3> | <h3 class="ar-title">SynNotch (<a href="http://parts.igem.org/Part:BBa_K2506001">BBa_K2506001</a>) </h3> | ||
<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/4/46/T--CPU_CHINA--parts_11_1_figure_1.png |
" width = "700"></center> | " width = "700"></center> | ||
<h4 align=middle>Figure 1. The structure of SynNotch fusion protein.</h4> | <h4 align=middle>Figure 1. The structure of SynNotch fusion protein.</h4> | ||
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<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/2/2b/T--CPU_CHINA--parts_11_1_figure_2.png" width = "700"></center> |
<h4 align=middle>Figure 2. The plasmid of the SynNotch system</h4> | <h4 align=middle>Figure 2. The plasmid of the SynNotch system</h4> | ||
</div> | </div> | ||
<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/5/58/T--CPU_CHINA--parts_11_1_figure_3.png" width = "700"></center> |
<h4 align=middle>Figure 3. The expression of the SynNotch system in Flag-FOXP3-Jurkat cell transfected by lentivirus transfection and electroporation</h4> | <h4 align=middle>Figure 3. The expression of the SynNotch system in Flag-FOXP3-Jurkat cell transfected by lentivirus transfection and electroporation</h4> | ||
</div> | </div> | ||
<h4><br>In order to investigate the relationship between the concentration of IL-17A ad the level of activity of the SynNotch system in the presence of inflammatory cytokine IL-6, we administered different concentrations of IL-17A and detected the expression levels of USP7 and FOXP3 (Figure 4). The result showed that the expression of USP7 and FOXP3 protein increased with the increase of IL-17A, which indicates that IL-17A concentration is an important factor affecting the expression level of SynNotch system. This verifies that our SynNotch system has worked well.</h4> | <h4><br>In order to investigate the relationship between the concentration of IL-17A ad the level of activity of the SynNotch system in the presence of inflammatory cytokine IL-6, we administered different concentrations of IL-17A and detected the expression levels of USP7 and FOXP3 (Figure 4). The result showed that the expression of USP7 and FOXP3 protein increased with the increase of IL-17A, which indicates that IL-17A concentration is an important factor affecting the expression level of SynNotch system. This verifies that our SynNotch system has worked well.</h4> | ||
<div> | <div> | ||
− | <center><img src=" | + | <center><img src="hhttps://static.igem.org/mediawiki/2017/b/b4/T--CPU_CHINA--parts_11_1_figure_4.png" width"700"></center> |
<h4 align=middle>Figure 4. The expression of Flag-FOXP3 by activating the expression of the USP7 gene in SynNotch system in inflammatory condition</h4> | <h4 align=middle>Figure 4. The expression of Flag-FOXP3 by activating the expression of the USP7 gene in SynNotch system in inflammatory condition</h4> | ||
</div> | </div> | ||
<h3>Part 2:UUpromU (<a href="http://parts.igem.org/Part:BBa_K2506004">BBa_K2506004</a>)</h3> | <h3>Part 2:UUpromU (<a href="http://parts.igem.org/Part:BBa_K2506004">BBa_K2506004</a>)</h3> | ||
<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/1/13/T--CPU_CHINA--parts_11_1_figure_5.png" width = "700"></center> |
<h4 align=middle>Figure 5. The design of UUpromU and its application.</h4> | <h4 align=middle>Figure 5. The design of UUpromU and its application.</h4> | ||
</div> | </div> | ||
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<h4><br>UUpromU can synergize with SynNotch (<a href="http://parts.igem.org/Part:BBa_K2506001">BBa_K2506001</a>), activate the USP7 gene by the action of the inflammatory cytokine IL-17A and stabilize the expression of FOXP3. To verify that, we transfected three-plasmid expression system into Flag-FOXP3-Jurkat cells by lentivirus transfection and electroporation. In the presence of inflammatory cytokine IL-6, we administered IL-17A with different concentrations and detected the expression levels of USP7 and FOXP3 by immunoblotting (Figure 6). The result showed that the expression of USP7 and FOXP3 protein increased with the increase of IL-17A. It means that UUpromU can synergize with SynNotch (<a href="http://parts.igem.org/Part:BBa_K2506001">BBa_K2506001</a>) and activate USP7 gene under the action of inflammatory cytokine IL-17A, thus stabilizing the effect of FOXP3.</h4> | <h4><br>UUpromU can synergize with SynNotch (<a href="http://parts.igem.org/Part:BBa_K2506001">BBa_K2506001</a>), activate the USP7 gene by the action of the inflammatory cytokine IL-17A and stabilize the expression of FOXP3. To verify that, we transfected three-plasmid expression system into Flag-FOXP3-Jurkat cells by lentivirus transfection and electroporation. In the presence of inflammatory cytokine IL-6, we administered IL-17A with different concentrations and detected the expression levels of USP7 and FOXP3 by immunoblotting (Figure 6). The result showed that the expression of USP7 and FOXP3 protein increased with the increase of IL-17A. It means that UUpromU can synergize with SynNotch (<a href="http://parts.igem.org/Part:BBa_K2506001">BBa_K2506001</a>) and activate USP7 gene under the action of inflammatory cytokine IL-17A, thus stabilizing the effect of FOXP3.</h4> | ||
<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/9/ | + | <center><img src="https://static.igem.org/mediawiki/2017/9/9a/T--CPU_CHINA--parts_11_1_figure_6.png" width = "700"></center> |
<h4 align=middle>Figure 6. The expression of USP 7 under the synergy of UUpromU and SynNotch system in inflammatory conditions.</h4> | <h4 align=middle>Figure 6. The expression of USP 7 under the synergy of UUpromU and SynNotch system in inflammatory conditions.</h4> | ||
</div> | </div> | ||
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<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/2/24/T--CPU_CHINA--parts_11_1_figure_7.png" width = "700"></center> |
<h4 align=middle>Figure 7. The structure of CAR fusion protein.</h4> | <h4 align=middle>Figure 7. The structure of CAR fusion protein.</h4> | ||
</div> | </div> | ||
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<h4><br>Flag-FOXP3-Jurkat cell line is a stably transfected cell line that highly expresses Flag-FOXP3. It is established by transfecting Flag-FOXP3 fusion protein gene in Jurkat T cells, and is a good model to simulate the state of human regulatory T cells. We obtained it from the molecular immunology research group of Shanghai Institute of Immunology, School of Medicine in Shanghai Jiao Tong University. In our experiment, we transfected three- plasmid expression system into Flag-FOXP3-Jurkat cells by lentiviral transfection and electroporation respectively. The expression of the CAR system in Flag-FOXP3-Jurkat cells was confirmed by immunoblotting and real-time quantitative PCR (Figure 9). We also observed a red fluorescence under a fluorescence microscope (Figure 10).</h4> | <h4><br>Flag-FOXP3-Jurkat cell line is a stably transfected cell line that highly expresses Flag-FOXP3. It is established by transfecting Flag-FOXP3 fusion protein gene in Jurkat T cells, and is a good model to simulate the state of human regulatory T cells. We obtained it from the molecular immunology research group of Shanghai Institute of Immunology, School of Medicine in Shanghai Jiao Tong University. In our experiment, we transfected three- plasmid expression system into Flag-FOXP3-Jurkat cells by lentiviral transfection and electroporation respectively. The expression of the CAR system in Flag-FOXP3-Jurkat cells was confirmed by immunoblotting and real-time quantitative PCR (Figure 9). We also observed a red fluorescence under a fluorescence microscope (Figure 10).</h4> | ||
<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/d/d2/T--CPU_CHINA--parts_11_1_figure_8.png" width="700"></center> |
<h4 align=middle>Figure 8. The plasmid of the CAR system</h4> | <h4 align=middle>Figure 8. The plasmid of the CAR system</h4> | ||
</div> | </div> | ||
<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/c/c5/T--CPU_CHINA--parts_11_1_figure_9.png" width="700"></center> |
<h4 align=middle>Figure 9. The expression of the CAR system in Flag-FOXP3-Jurkat cell transfected by lentivirus transfection and electroporation</h4> | <h4 align=middle>Figure 9. The expression of the CAR system in Flag-FOXP3-Jurkat cell transfected by lentivirus transfection and electroporation</h4> | ||
</div> | </div> | ||
<div> | <div> | ||
− | <center><img src="https://static.igem.org/mediawiki/2017/ | + | <center><img src="https://static.igem.org/mediawiki/2017/4/4e/T--CPU_CHINA--parts_11_1_figure_10.png" width="700"></center> |
<h4 align=middle>Figure 10. The expression of fluorescence in CAR system observed by fluorescence microscope<br> | <h4 align=middle>Figure 10. The expression of fluorescence in CAR system observed by fluorescence microscope<br> | ||
<br>For more experimental data details, please refer to our <a href="https://2017.igem.org/Team:CPU_CHINA/Results">RESULTS</a> section.</h4> | <br>For more experimental data details, please refer to our <a href="https://2017.igem.org/Team:CPU_CHINA/Results">RESULTS</a> section.</h4> |
Revision as of 14:41, 1 November 2017