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− | <p style="font-size:16px">● Fig A. Schematic representation showing the design and function of GFP PrePro </p> | + | <p style="font-size:16px">● Schematic representation showing the design and function of GFP PrePro(Fig 1A). </p> |
− | <p style="font-size:16px">● Fig B. Schematic representation showing the experimental workflow (left panel) and quantified GFP fluorescence heatmap 48 h post transfection of corresponding amount of plasmid (right panel). </p> | + | <p style="font-size:16px">● 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">● Fig C. Fluorescence images and quantified fluorescent intensity of HEK-293T cells 48 h post co-transfection with pEGFP plasmid and GFP PrePro/control plasmid. </p> | + | <p style="font-size:16px">● 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">● Fig D. Representative Western blotting determining the GFP plasmid protein abundance in HEK-293T cells 48 h post co-transfected with pEGFP plasmid and GFP PrePro/control plasmid (left panel), and quantified GFP protein level in three biological replicates (right panel). Relative protein level was calculated by normalizing the gray scale data of each group to the control group. </p> | + | <p style="font-size:16px">● 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">● Fig E. Representative fluorescence images in HEK-293T cell imaged 12-144 h post co-transfection with pEGFP plasmid and GFP PrePro /control plasmid.</p> | + | <p style="font-size:16px">● 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> |
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| <p style="font-size:16px">These results suggested that | | <p style="font-size:16px">These results suggested that |
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− | <p style="font-size:16px">We used a dual luciferase reporter (pEFR), in which firefly luciferase (Fluc) was fused with GFP, to obtain more accurate GFP abundance changes (Figure 1A). </p> | + | |
− | <p style="font-size:16px">Fluc activity was used as an indicator of GFP abundance, and the activity of a constitutively expressed Renilla luciferase (Rluc) reporter was used to normalize the noise caused by irrelevant factors such as transfection efficiency, cellular protein synthesis and cell growth. </p>
| + | <p style="font-size:16px">● 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">Results also 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">Results also 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> |
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| <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 1. Rapamycin-induced PREDATOR Pro system based on the FRB-FKBP interaction module.</b></p> |
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− | <p style="font-size:16px">To push our new PREDATOR Pro system towards signal responsiveness, 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">● To push our new PREDATOR Pro system towards signal responsiveness, 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">To test whether the GFP levels can be tuned and continuously regulated by rapamycin, HEK-293T cells were co-transfected with GFP expressing plasmid and RiPrePro1.0 plasmid. 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">● 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">To improve the degradation effect of RiPrePro system, modeling group noticed that the degradation efficiency was highly sensitive to the parameter reflecting the interaction strength of the protein dimerization pairby by performing Sensitivity Analysis among all parameters in our model. In the case of RiPrePro system, such result can be improved by increasing the concentration of rapamycin or increasing the binding strength of the interface part of the protein. | + | <p style="font-size:16px">To improve the degradation effect of RiPrePro system, modeling group noticed that <span style="color:#0069A6;font-size:16px;font-weight: bold;">the degradation efficiency was highly sensitive to the parameter reflecting the interaction strength of the protein dimerization pair</span> by performing Sensitivity Analysis among all parameters in our model. In the case of RiPrePro system, such result can be improved by increasing the concentration of rapamycin or increasing the binding strength of the interface part of the protein. |
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| <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 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> |
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− | <p style="font-size:16px">Therefore observed 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">● 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">As expected, <span style="color:#0069A6;font-size:16px;font-weight: bold;">the degradation effect could be significantly improved</span> with the increasing concentration of rapamycin. However, huge amount of rapamycin would significantly reduce the cellular protein synthesis. Therefore, we come to another solution to multiplying FKBP copies for a higher intensity.</p></div> | | <p style="font-size:16px">As expected, <span style="color:#0069A6;font-size:16px;font-weight: bold;">the degradation effect could be significantly improved</span> with the increasing concentration of rapamycin. However, huge amount of rapamycin would significantly reduce the cellular protein synthesis. Therefore, we come to another solution to multiplying FKBP copies for a higher intensity.</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 in the RiPrePro1.0 into GFPnano-FKBP-FKBP unit to further explored whether our Rapamycin-induced PREDATOR Pro system with two FKBP fragment (RiPrePro-2 or RiPrePro2.0) could show better performance.</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 to further explore whether our Rapamycin-induced PREDATOR Pro system with two FKBP fragment (RiPrePro-2 or RiPrePro2.0) could show better performance.</p> |
| <br><img src="https://static.igem.org/mediawiki/2020/a/aa/T--NUDT_CHINA--Poster_phase4.png"> | | <br><img src="https://static.igem.org/mediawiki/2020/a/aa/T--NUDT_CHINA--Poster_phase4.png"> |
| <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 1. Rapamycin-induced PREDATOR Pro system based on the FRB-FKBP-FKBP interaction module. </b></p> |
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− | <p style="font-size:16px">RiPrePro2.0 group is consists of HEK-293T cells, pEFR and plasmid expressing RiPrePro2.0.In Ctr group,there are HEK-293T cells, pEFR and empty vector. </p> | + | <p style="font-size:16px">● 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> |
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− | <p style="font-size:16px">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>. 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">● 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">Similarly, western blotting analysis showed ~40% lower GFP protein level in RiPrePro2.0 group (Fig 1D), which 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">● Western blotting analysis showed ~40% lower GFP protein level in RiPrePro2.0 group (Fig 1D), which 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">Moreover, 30%-60% reduction on Fluc/Rluc ratio in the RiPrePro2.0 group could be observed in different host cell lines we tested 48 h post 2 ng/μL rapamycin induction, suggesting good robustness of such RiPrePro system (Fig 1E). </p> | + | <p style="font-size:16px">● 30%-60% 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">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">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"> |
| <br><img style="width:70%" align="middle" src="https://static.igem.org/mediawiki/2020/4/44/T--NUDT_CHINA--Poster_p1.png"><br> | | <br><img style="width:70%" align="middle" src="https://static.igem.org/mediawiki/2020/4/44/T--NUDT_CHINA--Poster_p1.png"><br> |
− | <p style="font-size:20px">In order to determine the experimental time of degradation of GFP PrePro and GFP, we simulated the function according to the experimental data, and drew the corresponding function curve. According to the above figure, we can see that<span style="color:#0069A6;font-size:20px;font-weight: bold;"> the degradation rate of GFP is basically stable within 48 hours</span>. In other words, the reaction between p18 and GFP was basically completed. Therefore, we think that <span style="color:#0069A6;font-size:20px;font-weight: bold;">the experimental group can take 48 hours as the node of experimental data collection</span>, so as to obtain stable data and follow-up experimental observation results.</p> | + | <p style="font-size:16px">● The module group simulated the function according to the experimental data and the figure showed that<span style="color:#0069A6;font-size:20px;font-weight: bold;"> the degradation rate of GFP is basically stable within 48 hours</span>. In other words, the reaction between p18 and GFP was basically completed. </p> |
| + | <p style="font-size:16px">Therefore, we think that <span style="color:#0069A6;font-size:20px;font-weight: bold;">the experimental group can take 48 hours as the node of experimental data collection</span>, so as to obtain stable data and follow-up experimental observation results.</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:20px">After the preliminary trial experiment of replacing PRYSPRY/Fc domain with DocS-Coh2 protein pairs, we consulted Professor Zanxian Xia and presented our data in CCiC meetup. We were suggested to change the GFP reporter into a Dual Luciferase Reporter system to normalize irrelevant factors affecting GFP abundance. </p> | + | <p style="font-size:16px">● We consulted Professor Zanxian Xia and presented our preliminary trial experiment data in CCiC meetup. We were suggested to 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:20px">With this new reporter, the abovementioned experiments were repeated and the results clearly showed that<span style="color:#0069A6;font-size:20px;font-weight: bold;"> GFP PrePro could degrade the target protein significantly</span>.</p> | + | <p style="font-size:20px">● With this new reporter, the abovementioned experiments were repeated and the results clearly showed that<span style="color:#0069A6;font-size:20px;font-weight: bold;"> GFP PrePro could degrade the target protein significantly</span>.</p> |
| <br><img style="width:40%" align="middle" src="https://static.igem.org/mediawiki/2020/2/20/T--NUDT_CHINA--Poster_p23.png"><br> | | <br><img style="width:40%" align="middle" src="https://static.igem.org/mediawiki/2020/2/20/T--NUDT_CHINA--Poster_p23.png"><br> |
| </div> | | </div> |
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| <div class="text"> | | <div class="text"> |
| <br><img src="https://static.igem.org/mediawiki/2020/c/c2/T--NUDT_CHINA--Poster_p31.png"><br> | | <br><img src="https://static.igem.org/mediawiki/2020/c/c2/T--NUDT_CHINA--Poster_p31.png"><br> |
− | <p style="font-size:20px">To improve the degradation effect of our RiPrePro system, we approached our modeling group to figure out the most important factors affecting the PREDATOR Pro system. By performing Sensitivity Analysis (Figure A) among all parameters in our model, we noticed that the degradation efficiency was highly sensitive to the parameter reflecting the interaction strength of the protein dimerization pair(Figure B and C). In the case of RiPrePro system, such result implied that <span style="color:#0069A6;font-size:20px;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">To improve the degradation effect of our RiPrePro system, we approached our modeling group to figure out the most important factors affecting the PREDATOR Pro system. By performing Sensitivity Analysis (Figure A) among all parameters in our model, we noticed that the degradation efficiency was highly sensitive to the parameter reflecting the interaction strength of the protein dimerization pair(Figure B and C). In the case of RiPrePro system, such result implied that <span style="color:#0069A6;font-size:20px;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> |
| <br><img src="https://static.igem.org/mediawiki/2020/d/d7/T--NUDT_CHINA--Poster_phase3.png"><br> | | <br><img src="https://static.igem.org/mediawiki/2020/d/d7/T--NUDT_CHINA--Poster_phase3.png"><br> |
− | <p style="font-size:20px">With the increment of rapamycin concentration, the degradation efficiency of RiPrePro did increase impressively. However, it has been well established that exorbitant amount of rapamycin would significantly reduce the cellular protein synthesis. Therefore, we come to another solution to multiply FKBP copies for higher binding strength.</p> | + | <p style="font-size:16px">With the increment of rapamycin concentration, the degradation efficiency of RiPrePro did increase impressively. However, it has been well established that exorbitant amount of rapamycin would significantly reduce the cellular protein synthesis. Therefore, we come to another solution to multiply FKBP copies for higher binding strength.</p> |
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| <div class="text"> | | <div class="text"> |
| <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:20px">To promote the degradation efficiency, we turned to the model group for help. After performing Sensitivity Analysis of our system, they discovered that a parameter regarding the protein interaction intensity of FKBP and FRB was pivotal to the degradation efficiency. Therefore, we designed a Rapamycin-inducible PREDATOR Pro2.0 plasmid (RiPrePro2.0) composing GFPnano-FKBP*2 and HA-Trim21-FRB. It was observed that under 2 ng/μL rapamycin induction, the fluorescence intensity of HEK-293T cells co-transfected with pEGFP and RiPrePro2.0 plasmid was significantly lower than the control group transfected with pEGFP and empty vector.</p></div> | + | <p style="font-size:16px">To promote the degradation efficiency, we turned to the model group for help. After performing Sensitivity Analysis of our system, they discovered that a parameter regarding the protein interaction intensity of FKBP and FRB was pivotal to the degradation efficiency. Therefore, we designed a Rapamycin-inducible PREDATOR Pro2.0 plasmid (RiPrePro2.0) composing GFPnano-FKBP*2 and HA-Trim21-FRB. It was observed that under 2 ng/μL rapamycin induction, the fluorescence intensity of HEK-293T cells co-transfected with pEGFP and RiPrePro2.0 plasmid was significantly lower than the control group transfected with pEGFP and empty vector.</p></div> |
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