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<h4><br>In our SynNotch system, we retained the functional sequence of the transmembrane domain and the cleavage site of the Notch 1 protein. At the N-terminus, we fused the extracellular domain of IL17RA with Notch 1 to specifically recognize IL-17A secreted by Th17 cells, so that our regulatory T cells to obtain the ability to response to IL-17A. We also connected the gene of Gal4-vp64 (a fusion protein) in the downstream of Notch 1. In the presence of inflammatory cytokines IL17A, SynNotch protein is cleaved, and thus Gal4-VP64 fusion protein is detached from the cell membrane.</h4><h4><br> | <h4><br>In our SynNotch system, we retained the functional sequence of the transmembrane domain and the cleavage site of the Notch 1 protein. At the N-terminus, we fused the extracellular domain of IL17RA with Notch 1 to specifically recognize IL-17A secreted by Th17 cells, so that our regulatory T cells to obtain the ability to response to IL-17A. We also connected the gene of Gal4-vp64 (a fusion protein) in the downstream of Notch 1. In the presence of inflammatory cytokines IL17A, SynNotch protein is cleaved, and thus Gal4-VP64 fusion protein is detached from the cell membrane.</h4><h4><br> | ||
− | The released Gal4-VP64 will recognize UAS sequence in the upstream of promoter USP7 (in our another part BBa_K2506004) and then these two proteins combine together, which enable USP7 gene express with high efficiency. USP7 proteins will deubiquitinate the ubiquitinated FOXP3, so that to enhance the stability of FOXP3 protein in the inflammation environment by protecting FOXP3 from degradation via ubiquitination. In this way, Treg cells can survive and play a role of immunosuppressor.</h4> | + | The released Gal4-VP64 will recognize UAS sequence in the upstream of promoter USP7 (in our another part <a href="http://parts.igem.org/Part:BBa_K2506004">BBa_K2506004</a>) and then these two proteins combine together, which enable USP7 gene express with high efficiency. USP7 proteins will deubiquitinate the ubiquitinated FOXP3, so that to enhance the stability of FOXP3 protein in the inflammation environment by protecting FOXP3 from degradation via ubiquitination. In this way, Treg cells can survive and play a role of immunosuppressor.</h4> |
<h3>Results</h3> | <h3>Results</h3> | ||
<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 through the lentivirus-mediated gene transfer system and electroporation respectively. The expression of the SynNotch system in Flag-FOXP3-Jurkat cells was confirmed by immunoblotting and real-time quantitative PCR (Figure 2 and Figure 3).</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 through the lentivirus-mediated gene transfer system and electroporation respectively. The expression of the SynNotch system in Flag-FOXP3-Jurkat cells was confirmed by immunoblotting and real-time quantitative PCR (Figure 2 and Figure 3).</h4> | ||
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<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 (BBa_K2506004)</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/2017/4/49/T--CPU_CHINA--part_image005.png" width = "700"></center> | <center><img src="https://static.igem.org/mediawiki/2017/4/49/T--CPU_CHINA--part_image005.png" width = "700"></center> | ||
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</div> | </div> | ||
<h3>Introduction</h3> | <h3>Introduction</h3> | ||
− | <h4><br>UUpromU is a system specailly designed for the SynNotch system (BBa_K2506001) that is capable of specifically expressing the target gene in the case of SynNotch. UUpromU consists of three parts: the promotor sequence of USP7 in the middle and three repeats of UAS gene in both 5’ and 3’ terminus. In order to activate the expression of USP7, we synthesized the UUpromU gene and the USP7 gene, and put it into pcDNA3.1 (+) plasmid (with the assistance from GenScript), where UUpromU replaced the CMV promoter in the pcDNA3.1(+). Therefore, in the case that the SynNotch system is stimulated by IL-17A, the Gal4-vp64 fusion protein that is detached from the cell membrane can specifically recognize and bind to the UAS sequence on UUpromU. Then, the transcription of USP7 gene can be motivated by VP-64, a transcription activator, which leads to the high expression of USP7. USP7 proteins can lead to de-ubiquitination of the FOXP3 protein, so that enhance the stability of FOXP3 protein in the inflammation environment by protecting FOXP3 from degradation via ubiquitination. Finally, the goal of regulating the stability of T cells can be achieved.</h4> | + | <h4><br>UUpromU is a system specailly designed for the SynNotch system (<a href="http://parts.igem.org/Part:BBa_K2506001">BBa_K2506001</a>) that is capable of specifically expressing the target gene in the case of SynNotch. UUpromU consists of three parts: the promotor sequence of USP7 in the middle and three repeats of UAS gene in both 5’ and 3’ terminus. In order to activate the expression of USP7, we synthesized the UUpromU gene and the USP7 gene, and put it into pcDNA3.1 (+) plasmid (with the assistance from GenScript), where UUpromU replaced the CMV promoter in the pcDNA3.1(+). Therefore, in the case that the SynNotch system is stimulated by IL-17A, the Gal4-vp64 fusion protein that is detached from the cell membrane can specifically recognize and bind to the UAS sequence on UUpromU. Then, the transcription of USP7 gene can be motivated by VP-64, a transcription activator, which leads to the high expression of USP7. USP7 proteins can lead to de-ubiquitination of the FOXP3 protein, so that enhance the stability of FOXP3 protein in the inflammation environment by protecting FOXP3 from degradation via ubiquitination. Finally, the goal of regulating the stability of T cells can be achieved.</h4> |
<h3>Result</h3> | <h3>Result</h3> | ||
− | <h4><br>UUpromU can synergize with SynNotch (BBa_K2506001), 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 (BBa_K2506001) 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/2017/9/9f/T--CPU_CHINA--part_image006.png" width = "700"></center> | <center><img src="https://static.igem.org/mediawiki/2017/9/9f/T--CPU_CHINA--part_image006.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> | ||
− | <h3>Part 3:CAR-CD20 (BBa_K2506002)</h3> | + | <h3>Part 3:CAR-CD20 (<a href="http://parts.igem.org/Part:BBa_K2506002">BBa_K2506002</a>)</h3> |
<div> | <div> | ||
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At the N terminus, we chose variable region of CD20 monoclonal antibody as the scFv fragment, so that it can accurately identify and bind to B lymphocyte surface antigen CD20. Then, we used two 4-1-BB sequences as a co-stimulatory signal and a CD3Z sequence as a stimulatory signal that allowed the signal to be delivered to the cell at a high level, thereby activating Treg cells in the presence of CD20 B lymphocytes. <br> | At the N terminus, we chose variable region of CD20 monoclonal antibody as the scFv fragment, so that it can accurately identify and bind to B lymphocyte surface antigen CD20. Then, we used two 4-1-BB sequences as a co-stimulatory signal and a CD3Z sequence as a stimulatory signal that allowed the signal to be delivered to the cell at a high level, thereby activating Treg cells in the presence of CD20 B lymphocytes. <br> | ||
− | In addition, we linked the fluorescent protein mCherry at the C-terminus of the CAR system to facilitate the detection of gene expression. In order to accomplish this goal, we optimized the codon on BBa_K106004 and connected it to the C-terminus of the CAR system.</h4> | + | In addition, we linked the fluorescent protein mCherry at the C-terminus of the CAR system to facilitate the detection of gene expression. In order to accomplish this goal, we optimized the codon on <a href="http://parts.igem.org/Part:BBa_K2506004">BBa_K106004</a> and connected it to the C-terminus of the CAR system.</h4> |
<h3>Result</h3> | <h3>Result</h3> | ||
<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 8 and 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 8 and Figure 9). We also observed a red fluorescence under a fluorescence microscope (Figure 10).</h4> |
Revision as of 08:23, 1 November 2017
PARTS
Parts Table
The parts we submitted are listed at the table below.
Part Name | Description | Design | Length |
---|---|---|---|
BBa_K2506001 | CDS of SynNotch fusion protein | Qihang Zhao | 2619bp |
BBa_K2506002 | CDS of CAR fusion protein | Qihang Zhao | 2313bp |
BBa_K2506004 | Human USP7 gene promoter and UAS sequence | Qihang Zhao | 2153bp |