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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 3. 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>
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| + | <p style="font-size:16px;text-align:justify">Based on knowledge regarding the structure of Trim21 and the working model of Trim21-mediated degradation, 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">The Mechanism of Trim21-mediated degradation</p>
| + | <p style="font-size:20px">Our Hypothesis:</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>
| + | <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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− | <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>
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− | <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> | + | |
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| </div> | | </div> |
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| <p style="font-size:16px;text-align:justify">● The constitutive dimerization of DocS and Coh2 would function as the interface to bring the truncated Trim21 into proximity with GFP protein and trigger the degradation of GFP (Fig 4A).</p> | | <p style="font-size:16px;text-align:justify">● The constitutive dimerization of DocS and Coh2 would function as the interface to bring the truncated Trim21 into proximity with GFP protein and trigger the degradation of GFP (Fig 4A).</p> |
| <p style="font-size:16px;text-align:justify">● ~55% decrease of green fluorescence was observed in pEGFP plasmid and GFP PrePro plasmid co-transfected groups 48 hours after transfection (Fig 4B).</p> | | <p style="font-size:16px;text-align:justify">● ~55% decrease of green fluorescence was observed in pEGFP plasmid and GFP PrePro plasmid co-transfected groups 48 hours after transfection (Fig 4B).</p> |
− | <p style="font-size:16px;text-align:justify">● Western blotting analysis showed that GFP was significantly degraded to ~40% of the control group (Fig 4C), which confirmed that <span style="color:#6aa8a2;font-size:16px;font-weight: bold">PREDATOR Pro could be used to degrade target protein with high efficiency</span>.</p> | + | <p style="font-size:16px;text-align:justify">● Western blotting analysis showed that GFP was significantly degraded to ~40% of the control group (Fig 4C), which confirmed that <span style="color:#0069A6;font-size:16px;font-weight: bold">PREDATOR Pro could be used to degrade target protein with high efficiency</span>.</p> |
− | <p style="font-size:16px;text-align:justify">● Significant lower GFP fluorescence could be observed in GFP PrePro expressing group in every time point from 12 hours to 144 hours post transfection (Fig 1D and 1E). Of note, such trend was also well aligned to the prediction of our mathematical model (Fig 4D and 4E).</p><br> | + | <p style="font-size:16px;text-align:justify">● Significant lower GFP fluorescence could be observed in GFP PrePro expressing group in every time point from 12 hours to 144 hours post transfection. Of note, such trend was also well aligned to the prediction of our mathematical model (Fig 4D and 4E).</p><br> |
| <p style="font-size:20px;text-align:justify"> Conclusion:</p> <p style="font-size:16px;text-align:justify"> <span style="color:#e15f9f;font-size:16px;font-weight: bold">the original PRYSPRY/IgG-Fc interface in the PREDATOR system could be replaced with other protein dimerization pairs while maintaining the protein targeting and degradation activity.</span></p> | | <p style="font-size:20px;text-align:justify"> Conclusion:</p> <p style="font-size:16px;text-align:justify"> <span style="color:#e15f9f;font-size:16px;font-weight: bold">the original PRYSPRY/IgG-Fc interface in the PREDATOR system could be replaced with other protein dimerization pairs while maintaining the protein targeting and degradation activity.</span></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 5A), 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 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> | + | <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).</p> |
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| + | <p style="font-size:20px;text-align:justify"> Conclusion:</p> <p style="font-size:16px;text-align:justify"> <span style="color:#e15f9f;font-size:16px;font-weight: bold">GFP PrePro has impressive efficiency in degrading the target protein</p></div> |
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| <!--Write the text explaining this section --> | | <!--Write the text explaining this section --> |
− | <div class="text"> <p style="font-size:16px">With the implications obtained from the model, we changed the GFPnano-FKBP unit into GFPnano-FKBP-FKBP.</p> | + | <div class="text"> <p style="font-size:16px">With the implications obtained from the model, we changed the GFPnano-FKBP unit into GFPnano-FKBP-FKBP(Fig 8A).</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> |
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| <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">● 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 8E). </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> | + | <br> |
| + | <p style="font-size:20px;text-align:justify"> Conclusion:</p> <p style="font-size:16px;text-align:justify"> <span style="color:#e15f9f;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> |
| </div> | | </div> |
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| <div class="info"> | | <div class="info"> |
| <!--Write the title of the section --> | | <!--Write the title of the section --> |
− | <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"></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"> </p></div> | | <div class="text"><p style="font-size:16px;text-align:justify"> </p></div> |
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| <div class="title"><p style="font-size:30px">Partnership</p></div> | | <div class="title"><p style="font-size:30px">Partnership</p></div> |
| <!--Write the text explaining this section --> | | <!--Write the text explaining this section --> |
− | <div class="text"> <p style="font-size:18px"> <span style="color:#e15f9f;font-size:18px;font-weight: bold">Long-term cooperation mechanism with CSU_CHINA</span> </p>
| + | <div class="text"> <p style="font-size:18px"> <span style="color:#e15f9f;font-size:18px;font-weight: bold">Long-term cooperation mechanism with CSU_CHINA</span> </p> |
− | <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/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 20. 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 20. The online and offline meeting with CSU_CHINA</b></p> |
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− | <p style="font-size:16px;text-align:justify">● Held multiple online and offline meetings for project and idea sharing. </p> | + | <p style="font-size:16px;text-align:justify">● Held <span style="color:#e15f9f;font-size:16px;font-weight: bold">multiple online and offline meetings</span> for project and idea sharing. </p> |
− | <p style="font-size:16px;text-align:justify">● CSU_CHINA helped with the visualization of our fluorescent images analyzing. </p> | + | <p style="font-size:16px;text-align:justify">● CSU_CHINA helped us with the <span style="color:#e15f9f;font-size:16px;font-weight: bold">visualization of our massive amount of fluorescent images</span>. </p> |
− | <p style="font-size:16px;text-align:justify">● We helped CSU_CHINA to measure the cadmium-uptaking levels of Synechocystis in different growth states.</p> | + | <p style="font-size:16px;text-align:justify">● We helped CSU_CHINA to <span style="color:#e15f9f;font-size:16px;font-weight: bold">measure the cadmium-uptaking levels </span> of Synechocystis in different growth states.</p> |
− | <p style="font-size:16px;text-align:justify">● We designed our joint brochure for educational propaganda of synthetic biology.</p> | + | <p style="font-size:16px;text-align:justify">● We designed our <span style="color:#e15f9f;font-size:16px;font-weight: bold">joint brochure for education purposes</span>.</p> |
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| <span style="font-weight: bold;color:#0069A6;font-size:30px;font-family: Freeroad_Regular, Arial;line-height:35px">Attribution</span> | | <span style="font-weight: bold;color:#0069A6;font-size:30px;font-family: Freeroad_Regular, Arial;line-height:35px">Attribution</span> |
| + | <p style="font-size:16px"><b style="font-size:16px">Experiment:</b> Tianyi Zhang, Hanxiao Feng, Junzi Gu, Yuxin Liu, Changtai Xiao, Yingqian Ye, Zhenyu Zhou, Yuxuan Wang, Huiying Liu, Weiqian Zhou. </p> |
| <p style="font-size:16px"><b style="font-size:16px">Model:</b> Jie Cai, Yanchen Gou, Yongjiang Li, Zhiliang Pan, Haoyu Zhang. Our PI Xinyuan Qiu and Instructor Chuanyang Liu helped us with modeling ideas and methods. </p> | | <p style="font-size:16px"><b style="font-size:16px">Model:</b> Jie Cai, Yanchen Gou, Yongjiang Li, Zhiliang Pan, Haoyu Zhang. Our PI Xinyuan Qiu and Instructor Chuanyang Liu helped us with modeling ideas and methods. </p> |
| <p style="font-size:16px"><b style="font-size:16px">Human Practice:</b> Lunhao Ju, Zuyu Dai, Qingyi Liu, Xinlin Liu, Yuxin Liu, Linjie Li, Yulan Chen</p> | | <p style="font-size:16px"><b style="font-size:16px">Human Practice:</b> Lunhao Ju, Zuyu Dai, Qingyi Liu, Xinlin Liu, Yuxin Liu, Linjie Li, Yulan Chen</p> |