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| <!--Write the text explaining this section --> | | <!--Write the text explaining this section --> |
| <p style="font-size:16px">To sum up, PREDATOR Pro is tunable, reversible and highly modularized protein degradation tool with promising applications.</p> | | <p style="font-size:16px">To sum up, PREDATOR Pro is tunable, reversible and highly modularized protein degradation tool with promising applications.</p> |
− | <p style="font-size:16px;text-align:justify">A: PREDATOR Pro would be helpful for SynBio researchers to <span style="color:#679b9b;font-size:16px;font-weight: bold;text-decoration:underline">manipulate untagged endogenous protein abundance in a freely selectable temporal and dose-dependent combinations.</span></p> | + | <p style="font-size:16px;text-align:justify">A: PREDATOR Pro would be helpful for SynBio researchers to <span style="color:#679b9b;font-size:16px;font-weight: bold;text-decoration:underline">manipulate untagged endogenous protein abundance</span> in a freely selectable temporal and dose-dependent combinations.</p> |
− | <p style="font-size:16px;text-align:justify">B: For scientists working on gene function researches, PREDATOR Pro can also provide solution on developing cellular or animal model to <span style="color:#679b9b;font-size:16px;font-weight: bold;text-decoration:underline">knockdown the expression of specific gene in a tissue specific manner</span> under given stimulus.</p> | + | <p style="font-size:16px;text-align:justify">B: For scientists working on gene function researches, PREDATOR Pro can also provide solution on developing cellular or animal model to <span style="color:#679b9b;font-size:16px;font-weight: bold;text-decoration:underline">knockdown the expression of specific gene in a tissue specific manner under given stimulus.</span></p> |
− | <p style="font-size:16px;text-align:justify">C: It's possible that our project might be used for<span style="color:#679b9b;font-size:16px;font-weight: bold;text-decoration:underline"> therapeutic purposes</span>. PREDATOR Pro can be used to control the target protein abundance within physical level, thus maintaining the original function of such protein. </p> | + | <p style="font-size:16px;text-align:justify">C: It's possible that our project might be used for <span style="color:#679b9b;font-size:16px;font-weight: bold;text-decoration:underline">therapeutic purposes</span>. PREDATOR Pro can be used to control the target protein abundance within physical level, thus maintaining the original function of such protein. </p> |
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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> |
− | <br>
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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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− | <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 1A).</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 1B).</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 1C), which confirmed that PREDATOR Pro could be used to degrade target protein with high efficiency.</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 1D).</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. 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">● These results suggested that<span style="color:#0069A6;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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− | <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> |
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− | <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:80%" align="middle" src="https://static.igem.org/mediawiki/2020/9/98/T--NUDT_CHINA--Poster_jiaohu1.png"></div> |
− | <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:14px;text-align:center"><b style="font-size:14px">Figure 9. Schematic representation showing the experimental workflow (left panel) and quantified GFP fluorescence heatmap 48 h post transfection of corresponding amount of plasmid (right panel). </b></p> |
| <p style="font-size:16px;text-align:justify">● To find the optimal GFP PrePro transfection dosage, fluorescent intensity was transformed into heatmap for better visualization. Through the difference of color shade, a GFP PrePro dose dependent GFP degradation could be clearly observed.</p> | | <p style="font-size:16px;text-align:justify">● To find the optimal GFP PrePro transfection dosage, fluorescent intensity was transformed into heatmap for better visualization. Through the difference of color shade, a GFP PrePro dose dependent GFP degradation could be clearly observed.</p> |
| <p style="font-size:16px;text-align:justify">● Furthermore, we provided the data to Model hoping that they could obtain the optimal concentration ratio of GFP and GFP PrePro plasmids. With their advice, <span style="color:#0069A6;font-size:16px;font-weight: bold;">the plasmid ratio was set as 1:1 in further experiment</span>.</p> | | <p style="font-size:16px;text-align:justify">● Furthermore, we provided the data to Model hoping that they could obtain the optimal concentration ratio of GFP and GFP PrePro plasmids. With their advice, <span style="color:#0069A6;font-size:16px;font-weight: bold;">the plasmid ratio was set as 1:1 in further experiment</span>.</p> |
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| <!--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">● 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 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;text-align:justify">Focusing on various ways for collaborations to occur, both online and offline</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> |
− | <p style="font-size:16px"> > A long-term cooperation mechanism with CSU_CHINA</p>
| + | <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 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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| + | <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 21. Project collaboration with CSU_CHINA</b></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> |
− | <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 21. The partnership with CSU_CHINA</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 22. The joint brochure for education</b></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 21A). 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 21B).</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 22. The joint brochure for education</b></p> | + | |
| <br> | | <br> |
− | <p style="font-size:16px;text-align:justify">Furthermore, we designed our joint brochure for educational propaganda of synthetic biology (Fig 22).</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 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 <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 <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> |
| <p style="font-size:16px"><b style="font-size:16px">Art Design:</b> Guangyi Lin, Ruoxi Wang, Tianyi Zhang.</p> | | <p style="font-size:16px"><b style="font-size:16px">Art Design:</b> Guangyi Lin, Ruoxi Wang, Tianyi Zhang.</p> |
| <p style="font-size:16px"><b style="font-size:16px">Wiki Design:</b> Qinrui Jiang, Guangyi Lin, Ruoxi Wang.</p> | | <p style="font-size:16px"><b style="font-size:16px">Wiki Design:</b> Qinrui Jiang, Guangyi Lin, Ruoxi Wang.</p> |
− | <p style="font-size:16px"><b style="font-size:16px">General Support: </b>The project was supported by the Department of Biology and Chemistry of College of liberal art and Science, National University of Defense Technology.</p> | + | <br> |
− | <p style="font-size:16px"><b style="font-size:16px">Project Support:</b> PI Lingyun Zhu, Xinyuan Qiu; Instructor Chuanyang Liu, Lu Min, Lvyun Zhu, Xiaomin Wu, Jingyu Kuang, Wenying Li; Advisor Jiaxin Ma.</p> | + | <span style="font-weight: bold;color:#0069A6;font-size:30px;font-family: Freeroad_Regular, Arial;line-height:35px">Support</span> |
− | <p style="font-size:16px"><b style="font-size:16px">Fundraising help and advice: </b>PI Lingyun Zhu, advisor Lu Min was in charge of expenses management. </p> | + | <p style="font-size:16px"><b style="font-size:16px">General Support: </b>The project was supported by the Department of Biology and Chemistry of College of liberal art and Science, National University of Defense Technology.</p> |
− | <p style="font-size:16px"><b style="font-size:16px">Lab support:</b> Department of Biology and Chemistry of College of Liberal arts and Sciences. Difficult technique support: Sisi Xie, Wenying Li, Xinyuan Qiu.</p> | + | <p style="font-size:16px"><b style="font-size:16px">Project Support:</b> PI Lingyun Zhu, Xinyuan Qiu; Instructor Chuanyang Liu, Lu Min, Lvyun Zhu, Xiaomin Wu, Jingyu Kuang, Wenying Li; Advisor Jiaxin Ma.</p> |
− | <p style="font-size:16px"><b style="font-size:16px">Human Practice & Wiki support:</b> Our advisor Xinyuan Qiu, Chuanyang Liu provided us with solutions when we face difficulties in the process of wiki production.</p> | + | <p style="font-size:16px"><b style="font-size:16px">Fundraising help and advice: </b>PI Lingyun Zhu, advisor Lu Min was in charge of expenses management. </p> |
| + | <p style="font-size:16px"><b style="font-size:16px">Lab support:</b> Department of Biology and Chemistry of College of Liberal arts and Sciences. Difficult technique support: Sisi Xie, Wenying Li, Xinyuan Qiu.</p> |
| + | <p style="font-size:16px"><b style="font-size:16px">Human Practice & Wiki support:</b> Our advisor Xinyuan Qiu, Chuanyang Liu provided us with solutions when we face difficulties in the process of wiki production.</p> |
| <br> | | <br> |
| <span style="font-weight: bold;color:#0069A6;font-size:30px;font-family: Freeroad_Regular, Arial;line-height:35px">Acknowledgement</span> | | <span style="font-weight: bold;color:#0069A6;font-size:30px;font-family: Freeroad_Regular, Arial;line-height:35px">Acknowledgement</span> |