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| <br> <img src="https://static.igem.org/mediawiki/2020/0/03/T--NUDT_CHINA--Poster_design1.png"> | | <br> <img src="https://static.igem.org/mediawiki/2020/0/03/T--NUDT_CHINA--Poster_design1.png"> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure. the design of our PREDATOR Pro system.</b></p><br> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 3. the design of our PREDATOR Pro system.</b></p><br> |
| <p style="font-size:20px">The Structure of Trim21</p> > RING domain: N-terminal, with E3 ligase activity;<br> > B-box domain, a coiled-coil dimerization domain;<br> > Coiled-coil domain; <br> > PRYSPRY domain, high affinity with constant Fc domain of the antibody. </p> | | <p style="font-size:20px">The Structure of Trim21</p> > RING domain: N-terminal, with E3 ligase activity;<br> > B-box domain, a coiled-coil dimerization domain;<br> > Coiled-coil domain; <br> > PRYSPRY domain, high affinity with constant Fc domain of the antibody. </p> |
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| <p style="font-size:16px;text-align:justify"> Trim21 binds the Fc domain of the antibodies and form a Trim21-antibody-antigen trimer, and thus mediate proteasomal degradation of the antigen. </p> | | <p style="font-size:16px;text-align:justify"> Trim21 binds the Fc domain of the antibodies and form a Trim21-antibody-antigen trimer, and thus mediate proteasomal degradation of the antigen. </p> |
| <br> | | <br> |
− | <p style="font-size:16px;text-align:justify">Since it has demonstrated in previous research and our PREDATOR system that the interaction between the PRYSPRY domain and Fc is functionally independent to of other domains in Trim21, we <span style="color:#0069A6;font-size:16px;font-weight: bold;">modularized the PREDATOR system</span> into two modules (a.k.a. Targeting module and Functional module). These two modules were connected by a protein-protein interaction-based interface (see Figure).</p> <br> | + | <p style="font-size:16px;text-align:justify">Since it has demonstrated in previous research and our PREDATOR system that the interaction between the PRYSPRY domain and Fc is functionally independent to of other domains in Trim21, we <span style="color:#0069A6;font-size:16px;font-weight: bold;">modularized the PREDATOR system</span> into two modules (a.k.a. Targeting module and Functional module). These two modules were connected by a protein-protein interaction-based interface (see Figure 3).</p> <br> |
| <p style="font-size:20px">We rationally hypothesized that:</p> | | <p style="font-size:20px">We rationally hypothesized that:</p> |
| <span style="color:#0069A6;font-size:16px;font-weight: bold;text-align:justify">the interface of the Predator system can be replaced with other well-characterized protein-protein interactions (Replaceability) </span>.</p> | | <span style="color:#0069A6;font-size:16px;font-weight: bold;text-align:justify">the interface of the Predator system can be replaced with other well-characterized protein-protein interactions (Replaceability) </span>.</p> |
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| <div style="text-align:center;"><img style="width:100%;" src="https://static.igem.org/mediawiki/2020/f/f1/T--NUDT_CHINA--Poster_resultphase1-1.png"></div> | | <div style="text-align:center;"><img style="width:100%;" src="https://static.igem.org/mediawiki/2020/f/f1/T--NUDT_CHINA--Poster_resultphase1-1.png"></div> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 1. Characterization of GFP PREDATOR Pro system based on the DocS-Coh2 interaction.</b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 4. Characterization of GFP PREDATOR Pro system based on the DocS-Coh2 interaction.</b></p> |
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− | <p style="font-size:16px;text-align:justify">● Schematic representation showing the design and function of GFP PrePro(Fig 1A). </p> | + | <p style="font-size:16px;text-align:justify">● Schematic representation showing the design and function of GFP PrePro(Fig 4A). </p> |
− | <p style="font-size:16px;text-align:justify">● Schematic representation showing the experimental workflow (Fig 1B.left panel) and quantified GFP fluorescence heatmap 48 h post transfection of corresponding amount of plasmid (Fig 1B.right panel). </p> | + | <p style="font-size:16px;text-align:justify">● Schematic representation showing the experimental workflow (Fig 4B.left panel) and quantified GFP fluorescence heatmap 48 h post transfection of corresponding amount of plasmid (Fig 4B.right panel). </p> |
− | <p style="font-size:16px;text-align:justify">● Fluorescence images and quantified fluorescent intensity of HEK-293T cells 48 h post co-transfection with pEGFP plasmid and GFP PrePro/control plasmid(Fig 1C). </p> | + | <p style="font-size:16px;text-align:justify">● Fluorescence images and quantified fluorescent intensity of HEK-293T cells 48 h post co-transfection with pEGFP plasmid and GFP PrePro/control plasmid(Fig 4C). </p> |
− | <p style="font-size:16px;text-align:justify">● Representative Western blotting was performed to determine the GFP plasmid protein abundance in HEK-293T cells 48 h post co-transfected with pEGFP plasmid and GFP PrePro/control plasmid (Fig 1D.left panel), and quantified GFP protein level in three biological replicates (Fig 1D.right panel). </p> | + | <p style="font-size:16px;text-align:justify">● Representative Western blotting was performed to determine the GFP plasmid protein abundance in HEK-293T cells 48 h post co-transfected with pEGFP plasmid and GFP PrePro/control plasmid (Fig 4D.left panel), and quantified GFP protein level in three biological replicates (Fig 4D.right panel). </p> |
− | <p style="font-size:16px;text-align:justify">● Representative fluorescence images were captured in HEK-293T cell imaged 12-144 h post co-transfection with pEGFP plasmid and GFP PrePro /control plasmid(Fig 1E).</p> | + | <p style="font-size:16px;text-align:justify">● Representative fluorescence images were captured in HEK-293T cell imaged 12-144 h post co-transfection with pEGFP plasmid and GFP PrePro /control plasmid(Fig 4E).</p> |
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| <p style="font-size:16px;text-align:justify">These results suggested that | | <p style="font-size:16px;text-align:justify">These results suggested that |
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| <div style="text-align:center;"><img style="width:100%;" src="https://static.igem.org/mediawiki/2020/2/22/T--NUDT_CHINA--Poster_phase2.png"></div> | | <div style="text-align:center;"><img style="width:100%;" src="https://static.igem.org/mediawiki/2020/2/22/T--NUDT_CHINA--Poster_phase2.png"></div> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 1. Dual luciferase assay showing the GFP degradation mediated by GFP PrePro system.</b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 5. Dual luciferase assay showing the GFP degradation mediated by GFP PrePro system.</b></p> |
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− | <p style="font-size:16px;text-align:justify">● A dual luciferase reporter (pEFR) was used to obtain more accurate GFP abundance changes (Figure 1A), in which firefly luciferase (Fluc) was fused with GFP. </p> | + | <p style="font-size:16px;text-align:justify">● A dual luciferase reporter (pEFR) was used to obtain more accurate GFP abundance changes (Figure 5A), in which firefly luciferase (Fluc) was fused with GFP. </p> |
− | <p style="font-size:16px;text-align:justify">● Results showed <span style="color:#0069A6;font-size:16px;font-weight: bold;">significant lower</span> (~50% lower) Fluc/Rluc ratio in GFP PrePro group comparing to the Ctr group (Figure 1B), <span style="color:#0069A6;font-size:16px;font-weight: bold;">indicating that GFP PrePro has impressive efficiency in degrading the target protein. </p></div> | + | <p style="font-size:16px;text-align:justify">● Results showed <span style="color:#0069A6;font-size:16px;font-weight: bold;">significant lower</span> (~50% lower) Fluc/Rluc ratio in GFP PrePro group comparing to the Ctr group (Figure 5B), <span style="color:#0069A6;font-size:16px;font-weight: bold;">indicating that GFP PrePro has impressive efficiency in degrading the target protein. </p></div> |
| </div> | | </div> |
| </div> | | </div> |
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| <div style="text-align:center;"><img style="width:100%;" src="https://static.igem.org/mediawiki/2020/2/24/T--NUDT_CHINA--Poster_phase3-1.png"> </div> | | <div style="text-align:center;"><img style="width:100%;" src="https://static.igem.org/mediawiki/2020/2/24/T--NUDT_CHINA--Poster_phase3-1.png"> </div> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 1. Rapamycin-induced PREDATOR Pro system based on the FRB-FKBP interaction module.</b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 6. Rapamycin-induced PREDATOR Pro system based on the FRB-FKBP interaction module.</b></p> |
| <br> | | <br> |
− | <p style="font-size:16px;text-align:justify">● We further engineered a Rapamycin-induced PREDATOR Pro system (RiPrePro-1) by replacing DocS-Coh2 with FRB-FKBP(Fig 1A). </p> | + | <p style="font-size:16px;text-align:justify">● We further engineered a Rapamycin-induced PREDATOR Pro system (RiPrePro-1) by replacing DocS-Coh2 with FRB-FKBP(Fig 6A). </p> |
− | <p style="font-size:16px;text-align:justify">● Fluorescent imaging showed <span style="color:#0069A6;font-size:16px;font-weight: bold;">slight decrease (~20%) of GFP fluorescence in RiPrePro1.0 expressing group</span>(Fig 1B and 1C) under 1 ng/μL rapamycin induction.</p> | + | <p style="font-size:16px;text-align:justify">● Fluorescent imaging showed <span style="color:#0069A6;font-size:16px;font-weight: bold;">slight decrease (~20%) of GFP fluorescence in RiPrePro1.0 expressing group</span>(Fig 6B and 1C) under 1 ng/μL rapamycin induction.</p> |
| <p style="font-size:16px;text-align:justify">● The model group performed Sensitivity Analysis, finding <span style="color:#0069A6;font-size:16px;font-weight: bold;">the parameter reflecting the interaction strength of the protein dimerization pair</span>: </p> | | <p style="font-size:16px;text-align:justify">● The model group performed Sensitivity Analysis, finding <span style="color:#0069A6;font-size:16px;font-weight: bold;">the parameter reflecting the interaction strength of the protein dimerization pair</span>: </p> |
| <p style="font-size:16px" > > increasing the concentration of rapamycin </p> | | <p style="font-size:16px" > > increasing the concentration of rapamycin </p> |
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| <br><img src="https://static.igem.org/mediawiki/2020/d/d7/T--NUDT_CHINA--Poster_phase3.png"> | | <br><img src="https://static.igem.org/mediawiki/2020/d/d7/T--NUDT_CHINA--Poster_phase3.png"> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 2. Heatmap of fluorescence data detecting GFP and GFP-coupled firefly luciferase showing the degradation of GFP in RiPrePro-1 transfected groups with different concentrations of Rapamycin.</b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 7. Heatmap of fluorescence data detecting GFP and GFP-coupled firefly luciferase showing the degradation of GFP in RiPrePro-1 transfected groups with different concentrations of Rapamycin.</b></p> |
| <br> | | <br> |
| <p style="font-size:16px;text-align:justify">● Fluorescence image detecting GFP and GFP-coupled firefly luciferase showing the degradation of GFP in RiPrePro-1 transfected groups with different concentrations of Rapamycin. </p> | | <p style="font-size:16px;text-align:justify">● Fluorescence image detecting GFP and GFP-coupled firefly luciferase showing the degradation of GFP in RiPrePro-1 transfected groups with different concentrations of Rapamycin. </p> |
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| <div style="text-align:center;"><img style="width:100%;" src="https://static.igem.org/mediawiki/2020/a/aa/T--NUDT_CHINA--Poster_phase4.png"></div> | | <div style="text-align:center;"><img style="width:100%;" src="https://static.igem.org/mediawiki/2020/a/aa/T--NUDT_CHINA--Poster_phase4.png"></div> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 1. Rapamycin-induced PREDATOR Pro system based on the FRB-FKBP-FKBP interaction module. </b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 8. Rapamycin-induced PREDATOR Pro system based on the FRB-FKBP-FKBP interaction module. </b></p> |
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| <br> | | <br> |
− | <p style="font-size:16px;text-align:justify">● Dual luciferase assay showed that the normalized GFP abundance in RiPrePro2.0 group was significantly lower than the RiPrePro1.0 group in most time points (Fig 1B), <span style="color:#0069A6;font-size:16px;font-weight: bold;">indicating an improved degradation efficiency under increased FKBP copies</span>. </p> | + | <p style="font-size:16px;text-align:justify">● Dual luciferase assay showed that the normalized GFP abundance in RiPrePro2.0 group was significantly lower than the RiPrePro1.0 group in most time points (Fig 8B), <span style="color:#0069A6;font-size:16px;font-weight: bold;">indicating an improved degradation efficiency under increased FKBP copies</span>. </p> |
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− | <p style="font-size:16px;text-align:justify">● Further analysis on RiPrePro2.0 further revealed that the GFP degradation activity of RiPrePro2.0 was <span style="color:#0069A6;font-size:16px;font-weight: bold;">dose dependent</span> to the rapamycin concentration (Fig 1C). </p> | + | <p style="font-size:16px;text-align:justify">● Further analysis on RiPrePro2.0 further revealed that the GFP degradation activity of RiPrePro2.0 was <span style="color:#0069A6;font-size:16px;font-weight: bold;">dose dependent</span> to the rapamycin concentration (Fig 8C). </p> |
− | <p style="font-size:16px;text-align:justify">● Western blotting analysis(Fig 1D) is <span style="color:#0069A6;font-size:16px;font-weight: bold;">in alignment with the dual luciferase assay results</span>. </p> | + | <p style="font-size:16px;text-align:justify">● Western blotting analysis(Fig 8D) is <span style="color:#0069A6;font-size:16px;font-weight: bold;">in alignment with the dual luciferase assay results</span>. </p> |
− | <p style="font-size:16px;text-align:justify">● Reduction on Fluc/Rluc ratio in the RiPrePro2.0 group could be observed in different host cell lines , suggesting <span style="color:#0069A6;font-size:16px;font-weight: bold;">good robustness of such RiPrePro system</span> (Fig 1E). </p> | + | <p style="font-size:16px;text-align:justify">● Reduction on Fluc/Rluc ratio in the RiPrePro2.0 group could be observed in different host cell lines , suggesting <span style="color:#0069A6;font-size:16px;font-weight: bold;">good robustness of such RiPrePro system</span> (Fig 8E). </p> |
| <p style="font-size:16px;text-align:justify">In general, these results showed that our <span style="color:#0069A6;font-size:16px;font-weight: bold;">PREDATOR Pro system can be successfully engineered to degrade the target protein under the control of exogenous signals with decent performance and satisfying robustness</span>.</p> | | <p style="font-size:16px;text-align:justify">In general, these results showed that our <span style="color:#0069A6;font-size:16px;font-weight: bold;">PREDATOR Pro system can be successfully engineered to degrade the target protein under the control of exogenous signals with decent performance and satisfying robustness</span>.</p> |
| </div> | | </div> |
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| <div class="text"> | | <div class="text"> |
| <div style="text-align:center;"> <img style="width:60%" align="middle" src="https://static.igem.org/mediawiki/2020/4/44/T--NUDT_CHINA--Poster_p1.png"></div> | | <div style="text-align:center;"> <img style="width:60%" align="middle" src="https://static.igem.org/mediawiki/2020/4/44/T--NUDT_CHINA--Poster_p1.png"></div> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 1. the time-dependent characteristic of GFP PrePro system <i style="font-size:14px">in silico</i> and <i style="font-size:14px">in vitro</i></b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 9. the time-dependent characteristic of GFP PrePro system <i style="font-size:14px">in silico</i> and <i style="font-size:14px">in vitro</i></b></p> |
| <p style="font-size:16px;text-align:justify">● The module group simulated the function according to the experimental data and the figure showed that<span style="color:#0069A6;font-size:16px;font-weight: bold;"> the degradation rate of GFP is basically stable within 48 hours</span>.</p> | | <p style="font-size:16px;text-align:justify">● The module group simulated the function according to the experimental data and the figure showed that<span style="color:#0069A6;font-size:16px;font-weight: bold;"> the degradation rate of GFP is basically stable within 48 hours</span>.</p> |
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| <div class="text"> | | <div class="text"> |
| <br><img src="https://static.igem.org/mediawiki/2020/0/08/T--NUDT_CHINA--Poster_p21.png"><br> | | <br><img src="https://static.igem.org/mediawiki/2020/0/08/T--NUDT_CHINA--Poster_p21.png"><br> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 1. Interview Prof.Xia.</b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 10. Interview Prof.Xia.</b></p> |
| <p style="font-size:16px;text-align:justify">● Interview Professor Zanxian Xia with our preliminary trial experiment data, We were suggested to:</p> | | <p style="font-size:16px;text-align:justify">● Interview Professor Zanxian Xia with our preliminary trial experiment data, We were suggested to:</p> |
| <p style="font-size:16px;text-align:justify"> > Change the GFP reporter into a Dual Luciferase Reporter system to normalize irrelevant factors affecting GFP abundance. </p> | | <p style="font-size:16px;text-align:justify"> > Change the GFP reporter into a Dual Luciferase Reporter system to normalize irrelevant factors affecting GFP abundance. </p> |
| <br><img src="https://static.igem.org/mediawiki/2020/5/5e/T--NUDT_CHINA--Poster_p22.png"><br> | | <br><img src="https://static.igem.org/mediawiki/2020/5/5e/T--NUDT_CHINA--Poster_p22.png"><br> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 2. CCiC meet-up.</b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 11. CCiC meet-up.</b></p> |
| <p style="font-size:16px;text-align:justify">With this new reporter, the abovementioned experiments were repeated and the results clearly showed that<span style="color:#0069A6;font-size:16px;font-weight: bold;"> GFP PrePro could degrade the target protein significantly</span>.</p> | | <p style="font-size:16px;text-align:justify">With this new reporter, the abovementioned experiments were repeated and the results clearly showed that<span style="color:#0069A6;font-size:16px;font-weight: bold;"> GFP PrePro could degrade the target protein significantly</span>.</p> |
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| <br><div style="text-align:center;"><img style="width:80%" src="https://static.igem.org/mediawiki/2020/c/c2/T--NUDT_CHINA--Poster_p31.png"></div><br> | | <br><div style="text-align:center;"><img style="width:80%" src="https://static.igem.org/mediawiki/2020/c/c2/T--NUDT_CHINA--Poster_p31.png"></div><br> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 1. the sensitivity analysis of parameters in the model.</b></p><br> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 12. the sensitivity analysis of parameters in the model.</b></p><br> |
− | <p style="font-size:16px;text-align:justify">● We approached our modele group to figure out the most important factors affecting the PREDATOR Pro system. By performing Sensitivity Analysis (Figure A), we noticed that <span style="color:#0069A6;font-size:16px;font-weight: bold;">the degradation effect can be improved by increasing the concentration of rapamycin or increasing the binding strength of the interface part</span>.</p> | + | <p style="font-size:16px;text-align:justify">● We approached our modele group to figure out the most important factors affecting the PREDATOR Pro system. By performing Sensitivity Analysis (Figure 12A), we noticed that <span style="color:#0069A6;font-size:16px;font-weight: bold;">the degradation effect can be improved by increasing the concentration of rapamycin or increasing the binding strength of the interface part</span>.</p> |
| <p style="font-size:16px;text-align:justify">With the increment of rapamycin concentration, the degradation efficiency of RiPrePro did increase impressively.</p> | | <p style="font-size:16px;text-align:justify">With the increment of rapamycin concentration, the degradation efficiency of RiPrePro did increase impressively.</p> |
| </div> | | </div> |
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| <div class="title"><p style="font-size:30px;text-align:justify">Literature research to determine protein dimerization pairs regulated by exogenous signals</p></div> | | <div class="title"><p style="font-size:30px;text-align:justify">Literature research to determine protein dimerization pairs regulated by exogenous signals</p></div> |
| <!--Write the text explaining this section --> | | <!--Write the text explaining this section --> |
− | <div class="text"><p style="font-size:16px;text-align:justify">● We performed literature research to find a set of widely used and fully characterized heterodimerizing components to achieve sifnal responsiveness. We noticed that the dimerization of FK506 binding protein (FKBP) domain and the T2089L mutant of FKBP-rapamycin binding domain (FRB) could be initiated by external rapamycin signals. Therefore, we substituted the interface with FRB-FKBP protein interaction pairs. </p></div> | + | <div class="text"><p style="font-size:16px;text-align:justify">● We performed literature research to find a set of widely used and fully characterized heterodimerizing components to achieve sifnal responsiveness. We noticed that the dimerization of FK506 binding protein (FKBP) domain and the T2089L mutant of FKBP-rapamycin bindin433g domain (FRB) could be initiated by external rapamycin signals. Therefore, we substituted the interface with FRB-FKBP protein interaction pairs. </p></div> |
| </div> | | </div> |
| </div> | | </div> |
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| <br><img src="https://static.igem.org/mediawiki/2020/1/17/T--NUDT_CHINA--Poster_p51.png"><br> | | <br><img src="https://static.igem.org/mediawiki/2020/1/17/T--NUDT_CHINA--Poster_p51.png"><br> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 1. Representative fluorescence images of HEK-293T cells carrying pEFR and RiPrePro-2 plasmids in comparison to the Ctr group 48 h post 2 ng/μL rapamycin induction.</b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 13. Representative fluorescence images of HEK-293T cells carrying pEFR and RiPrePro-2 plasmids in comparison to the Ctr group 48 h post 2 ng/μL rapamycin induction.</b></p> |
| <p style="font-size:16px;text-align:justify">● The model group discovered that a parameter regarding the protein interaction intensity of FKBP and FRB was pivotal to the degradation efficiency after performing Sensitivity Analysis. Therefore, we designed RiPrePro2.0 composing GFPnano-FKBP*2 and HA-Trim21-FRB. </p> | | <p style="font-size:16px;text-align:justify">● The model group discovered that a parameter regarding the protein interaction intensity of FKBP and FRB was pivotal to the degradation efficiency after performing Sensitivity Analysis. Therefore, we designed RiPrePro2.0 composing GFPnano-FKBP*2 and HA-Trim21-FRB. </p> |
| <p style="font-size:16px;text-align:justify">● Under 2 ng/μL rapamycin induction, <span style="color:#0069A6;font-size:16px;font-weight: bold;">the fluorescence intensity of HEK-293T cells co-transfected with pEGFP and RiPrePro2.0 plasmid was significantly lower than the control group</span>.</p></div> | | <p style="font-size:16px;text-align:justify">● Under 2 ng/μL rapamycin induction, <span style="color:#0069A6;font-size:16px;font-weight: bold;">the fluorescence intensity of HEK-293T cells co-transfected with pEGFP and RiPrePro2.0 plasmid was significantly lower than the control group</span>.</p></div> |
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− | <img src="https://static.igem.org/mediawiki/2020/f/fc/T--NUDT_CHINA--Poster_model1-2.svg"><p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 1. Framework of the mathematical model</b></p> | + | <img src="https://static.igem.org/mediawiki/2020/f/fc/T--NUDT_CHINA--Poster_model1-2.svg"><p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 14. Framework of the mathematical model</b></p> |
| <p style="font-size:16px;text-align:justify">To understand the performance of our design comprehensively and offer guidance on wet-lab work, a mathematical model composed of <span style="color:#0069A6;font-size:16px;font-weight: bold">two modules (protein interaction module and a protein degradation module)</span> was constructed based on fundamental biochemical principles.</p> | | <p style="font-size:16px;text-align:justify">To understand the performance of our design comprehensively and offer guidance on wet-lab work, a mathematical model composed of <span style="color:#0069A6;font-size:16px;font-weight: bold">two modules (protein interaction module and a protein degradation module)</span> was constructed based on fundamental biochemical principles.</p> |
| <br><div style="text-align:center;"><img style="width:70%" src="https://static.igem.org/mediawiki/2020/0/07/T--NUDT_CHINA--Poster_modefig2.png"></div> | | <br><div style="text-align:center;"><img style="width:70%" src="https://static.igem.org/mediawiki/2020/0/07/T--NUDT_CHINA--Poster_modefig2.png"></div> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 2. the side-by-side comparison of model prediction and experimental data</b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 15. the side-by-side comparison of model prediction and experimental data</b></p> |
| <p style="font-size:16px;text-align:justify"> ● The simulation showed that the model predictions <span style="color:#0069A6;font-size:16px;font-weight: bold">matched well</span> with the corresponding wet-lab data.</p> | | <p style="font-size:16px;text-align:justify"> ● The simulation showed that the model predictions <span style="color:#0069A6;font-size:16px;font-weight: bold">matched well</span> with the corresponding wet-lab data.</p> |
− | <br><img src="https://static.igem.org/mediawiki/2020/7/70/T--NUDT_CHINA--Poster_m3.png"><p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 3. the influence of plasmid dosage and ratio on GFP degradation rate</b></p> | + | <br><img src="https://static.igem.org/mediawiki/2020/7/70/T--NUDT_CHINA--Poster_m3.png"><p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 16. the influence of plasmid dosage and ratio on GFP degradation rate</b></p> |
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| <p style="font-size:16px;text-align:justify">● Model results showed that<span style="color:#0069A6;font-size:16px;font-weight: bold"> 1:1 </span>is the optimal plasmid ratio and 0.25 mg dosage for both plasmids should be prioritized.</p> | | <p style="font-size:16px;text-align:justify">● Model results showed that<span style="color:#0069A6;font-size:16px;font-weight: bold"> 1:1 </span>is the optimal plasmid ratio and 0.25 mg dosage for both plasmids should be prioritized.</p> |
− | <br><div style="text-align:center;"><img style="width:80%" src="https://static.igem.org/mediawiki/2020/c/ce/T--NUDT_CHINA--Poster_m4.png"></div><p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 4. the sensitivity analysis of parameters in the model</b></p> | + | <br><div style="text-align:center;"><img style="width:80%" src="https://static.igem.org/mediawiki/2020/c/ce/T--NUDT_CHINA--Poster_m4.png"></div><p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 17. the sensitivity analysis of parameters in the model</b></p> |
| <p style="font-size:16px;text-align:justify">● The <span style="color:#e15f9f;font-size:16px;font-weight: bold">Sensitivity Analysis</span> of all parameters in the model calculate the time-dependent sensitivities (derivatives) of GFP with respect to each parameter.</p> | | <p style="font-size:16px;text-align:justify">● The <span style="color:#e15f9f;font-size:16px;font-weight: bold">Sensitivity Analysis</span> of all parameters in the model calculate the time-dependent sensitivities (derivatives) of GFP with respect to each parameter.</p> |
− | <p style="font-size:16px;text-align:justify"><span style="color:#f38c68;font-size:16px;font-weight: bold">k3f</span>, the rate parameter characterizing the dimerization of FRB and FKBP, <span style="color:#f38c68;font-size:16px;font-weight: bold">manifested the highest sensitivity across the board(Figure 3)</span>.</p> | + | <p style="font-size:16px;text-align:justify"><span style="color:#f38c68;font-size:16px;font-weight: bold">k3f</span>, the rate parameter characterizing the dimerization of FRB and FKBP, <span style="color:#f38c68;font-size:16px;font-weight: bold">manifested the highest sensitivity across the board(Figure 17)</span>.</p> |
| <p style="font-size:16px;text-align:justify"> Results indicated that we could optimize the RiPrePro by increasing the value of k3f, or in other words, <span style="color:#e15f9f;font-size:16px;font-weight: bold">increasing the dosage of rapamycin</span> or <span style="color:#e15f9f;font-size:16px;font-weight: bold">the number of FKBP domains</span>. Considering the toxicity of rapamycin to cells, the future experiment focused on adding up the number of FKBP domains.</p> | | <p style="font-size:16px;text-align:justify"> Results indicated that we could optimize the RiPrePro by increasing the value of k3f, or in other words, <span style="color:#e15f9f;font-size:16px;font-weight: bold">increasing the dosage of rapamycin</span> or <span style="color:#e15f9f;font-size:16px;font-weight: bold">the number of FKBP domains</span>. Considering the toxicity of rapamycin to cells, the future experiment focused on adding up the number of FKBP domains.</p> |
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| <br><img src="https://static.igem.org/mediawiki/2020/a/a3/T--NUDT_CHINA--Poster_interxie.jpg"><br> | | <br><img src="https://static.igem.org/mediawiki/2020/a/a3/T--NUDT_CHINA--Poster_interxie.jpg"><br> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 1. Interview Prof.Xie</b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 18. Interview Prof.Xie</b></p> |
| <p style="font-size:16px;text-align:justify">● We approached Dr. Mingqi Xie after we obtained most of our data. Through the interview, we first showed Dr.Xie the design of Predator Pro system, as well as the data we've already obtained. </p> | | <p style="font-size:16px;text-align:justify">● We approached Dr. Mingqi Xie after we obtained most of our data. Through the interview, we first showed Dr.Xie the design of Predator Pro system, as well as the data we've already obtained. </p> |
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| <p style="font-size:16px"> > A long-term cooperation mechanism with CSU_CHINA</p> | | <p style="font-size:16px"> > A long-term cooperation mechanism with CSU_CHINA</p> |
| <br><img style="width:45%" src="https://static.igem.org/mediawiki/2020/e/e1/T--NUDT_CHINA--Poster_par1.jpg"> | | <br><img style="width:45%" src="https://static.igem.org/mediawiki/2020/e/e1/T--NUDT_CHINA--Poster_par1.jpg"> |
− | <img style="width:45%" src="https://static.igem.org/mediawiki/2020/6/6c/T--NUDT_CHINA--Poster_par2.jpg"><p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 1. The online and offline meeting with CSU_CHINA</b></p> | + | <img style="width:45%" src="https://static.igem.org/mediawiki/2020/6/6c/T--NUDT_CHINA--Poster_par2.jpg"><p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 19. The online and offline meeting with CSU_CHINA</b></p> |
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| <p style="font-size:16px;text-align:justify"> > Offline meeting to deepen the communication between our teams. Both teams agreed on further and more concrete collaboration.</p> | | <p style="font-size:16px;text-align:justify"> > Offline meeting to deepen the communication between our teams. Both teams agreed on further and more concrete collaboration.</p> |
− | <br><img src="https://static.igem.org/mediawiki/2020/e/ed/T--NUDT_CHINA--Poster_par3.jpg"><p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 2. The partnership with CSU_CHINA</b></p><br> | + | <br><img src="https://static.igem.org/mediawiki/2020/e/ed/T--NUDT_CHINA--Poster_par3.jpg"><p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 20. The partnership with CSU_CHINA</b></p><br> |
− | <p style="font-size:16px;text-align:justify"><span style="color:#e15f9f;font-size:16px;font-weight: bold">Partnership:</span> CSU_CHINA helped us with our massive fluorescent images analyzing. With their brilliant R language skill, we gained <span style="color:#e15f9f;font-size:16px;font-weight: bold">a beautiful visualization</span> of our fluorescent images (Fig 2A). As CSU_CHINA has limited access to the cadmium ion detection kits, we lent atomic absorption spectrometer and <span style="color:#e15f9f;font-size:16px;font-weight: bold">helped them measure the cadmium-uptaking levels of Synechocystis</span> while in different growth states(Fig 2B).</p> | + | <p style="font-size:16px;text-align:justify"><span style="color:#e15f9f;font-size:16px;font-weight: bold">Partnership:</span> CSU_CHINA helped us with our massive fluorescent images analyzing. With their brilliant R language skill, we gained <span style="color:#e15f9f;font-size:16px;font-weight: bold">a beautiful visualization</span> of our fluorescent images (Fig 20A). As CSU_CHINA has limited access to the cadmium ion detection kits, we lent atomic absorption spectrometer and <span style="color:#e15f9f;font-size:16px;font-weight: bold">helped them measure the cadmium-uptaking levels of Synechocystis</span> while in different growth states(Fig 20B).</p> |
− | <br><img src="https://static.igem.org/mediawiki/2020/6/68/T--NUDT_CHINA--Poster_par4.jpg"><p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 3. The joint brochure for education</b></p> | + | <br><img src="https://static.igem.org/mediawiki/2020/6/68/T--NUDT_CHINA--Poster_par4.jpg"><p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 21. The joint brochure for education</b></p> |
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− | <p style="font-size:16px;text-align:justify">Furthermore, we designed our joint brochure for educational propaganda of synthetic biology (Fig 2).</p> | + | <p style="font-size:16px;text-align:justify">Furthermore, we designed our joint brochure for educational propaganda of synthetic biology (Fig 21).</p> |
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| <img style="width:45%"src="https://static.igem.org/mediawiki/2020/2/20/T--NUDT_CHINA--Poster_sci1.jpg"> | | <img style="width:45%"src="https://static.igem.org/mediawiki/2020/2/20/T--NUDT_CHINA--Poster_sci1.jpg"> |
| <img style="width:45%" src="https://static.igem.org/mediawiki/2020/1/1b/T--NUDT_CHINA--Poster_sci2.jpg"> | | <img style="width:45%" src="https://static.igem.org/mediawiki/2020/1/1b/T--NUDT_CHINA--Poster_sci2.jpg"> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 1. Spreading the SynBio</b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 22. Spreading the SynBio</b></p> |
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| <p style="font-size:16px;text-align:justify">● Prepare the brochure about synthetic biology and our projects for students in lower age </p> | | <p style="font-size:16px;text-align:justify">● Prepare the brochure about synthetic biology and our projects for students in lower age </p> |
| <img src="https://static.igem.org/mediawiki/2020/6/68/T--NUDT_CHINA--Poster_par4.jpg"> | | <img src="https://static.igem.org/mediawiki/2020/6/68/T--NUDT_CHINA--Poster_par4.jpg"> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 2. The joint brochure for education</b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 23. The joint brochure for education</b></p> |
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| <p style="font-size:16px;text-align:justify">● After we finished our project promotion video, we prepared a Chinese version and uploaded it online allowing others to know our team and our project</p> | | <p style="font-size:16px;text-align:justify">● After we finished our project promotion video, we prepared a Chinese version and uploaded it online allowing others to know our team and our project</p> |
| <div style="text-align:center;"> <img style="width:50%;" src="https://static.igem.org/mediawiki/2020/1/1c/T--NUDT_CHINA--Poster_sci-1.png"></div> | | <div style="text-align:center;"> <img style="width:50%;" src="https://static.igem.org/mediawiki/2020/1/1c/T--NUDT_CHINA--Poster_sci-1.png"></div> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 3. The localized propaganda of our SynBio project</b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 24. The localized propaganda of our SynBio project</b></p> |
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| <div style="text-align:center;"><img style="width:40%;" src="https://static.igem.org/mediawiki/2020/a/a2/T--NUDT_CHINA--Poster_tr21.png"></div> | | <div style="text-align:center;"><img style="width:40%;" src="https://static.igem.org/mediawiki/2020/a/a2/T--NUDT_CHINA--Poster_tr21.png"></div> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 1. The truncated Trim21</b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 25. The truncated Trim21</b></p> |
| <p style="font-size:16px;text-align:justify">Our favorite Basic parts are Truncated Trim21 and CMV-Replaceable-1-Fluc-P2A-Rluc. Truncated Trim21 is the core of the newly registered Predator Pro system. This truncate protein maintained the E3 ubiquitin ligase activity, and provided an open interface for other protein dimerization pairs to be added.</p> | | <p style="font-size:16px;text-align:justify">Our favorite Basic parts are Truncated Trim21 and CMV-Replaceable-1-Fluc-P2A-Rluc. Truncated Trim21 is the core of the newly registered Predator Pro system. This truncate protein maintained the E3 ubiquitin ligase activity, and provided an open interface for other protein dimerization pairs to be added.</p> |
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| <div style="text-align:center;"><img style="width:50%;" src="https://static.igem.org/mediawiki/2020/5/5c/T--NUDT_CHINA--Poster_parts2.jpg"></div> | | <div style="text-align:center;"><img style="width:50%;" src="https://static.igem.org/mediawiki/2020/5/5c/T--NUDT_CHINA--Poster_parts2.jpg"></div> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 2. a new reporter system</b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 26. a new reporter system</b></p> |
| <p style="font-size:16px;text-align:justify">Another part is a reporter system to quantify the abundance of specific target protein.</p> | | <p style="font-size:16px;text-align:justify">Another part is a reporter system to quantify the abundance of specific target protein.</p> |
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| <div style="text-align:center;" ><img style="width:70%;" src="https://static.igem.org/mediawiki/2020/e/e3/T--NUDT_CHINA--Poster_parts3.jpg"></div> | | <div style="text-align:center;" ><img style="width:70%;" src="https://static.igem.org/mediawiki/2020/e/e3/T--NUDT_CHINA--Poster_parts3.jpg"></div> |
− | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 3. a composite part for controllable protein degradation</b></p> | + | <p style="font-size:14px;text-align:center"><b style="font-size:14px">Figure 27. a composite part for controllable protein degradation</b></p> |
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| <p style="font-size:16px;text-align:justify">Our favorite composite part is Replaceable-1-Replaceable-2-P2A-TRIM21-Replaceable-3. It is a plasmid platform on which different targeting proteins and protein dimerization pairs can be easily installed to achieve the controllable degradation of specific target protein.</p> | | <p style="font-size:16px;text-align:justify">Our favorite composite part is Replaceable-1-Replaceable-2-P2A-TRIM21-Replaceable-3. It is a plasmid platform on which different targeting proteins and protein dimerization pairs can be easily installed to achieve the controllable degradation of specific target protein.</p> |