Difference between revisions of "Part:BBa K5036030"
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span | <p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span | ||
lang=EN style='padding-bottom:30px;font-size:11.0pt;line-height:115%'>The figure displays the interaction between two receptor chains and VEGFA, The (VEGFR2-Cdcas9) chain appears in the red colour, and the (VEGFR2-Ndcas9) chain appears in the blue colour. The VEGFA is in green colour. The calculated binding stability (ΔG) of the combination is -12.5 kcal mol-1 | lang=EN style='padding-bottom:30px;font-size:11.0pt;line-height:115%'>The figure displays the interaction between two receptor chains and VEGFA, The (VEGFR2-Cdcas9) chain appears in the red colour, and the (VEGFR2-Ndcas9) chain appears in the blue colour. The VEGFA is in green colour. The calculated binding stability (ΔG) of the combination is -12.5 kcal mol-1 | ||
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+ | The final form of our receptor is composed of two chains, each chain is built of internal and external domains, so we validated these interactions by calculating the binding affinity between the two chains and VEGFA, which simulate the final design of our receptor. | ||
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+ | (VEGFR1-Cdcas9 – VEGFR2-Ndcas9) with VEGFA | ||
+ | <html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style=" max-width:850px; | ||
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+ | "src="https://static.igem.wiki/teams/5036/part-software/r1c-r2n-vegfaannotated-ezgif-com-video-to-gif-converter.gif | ||
+ | "> | ||
+ | <p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span | ||
+ | lang=EN style='padding-bottom:30px;font-size:11.0pt;line-height:115%'>The figure displays the interaction between two receptor chains and VEGFA, The (VEGFR1-Cdcas9) chain appears in the red colour, and the (VEGFR2-Ndcas9) chain appears in the blue colour. The VEGFA is in green colour. The calculated binding stability (ΔG) of the combination is -13.7 kcal mol-1 | ||
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<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here |
Revision as of 08:50, 25 September 2024
dCas9(N)_NES-Syn-VEGFR-2 (VEGF-R2, N-TEV, NES, TCS (Q, G), HA, dCas9(N),mCherry)
Part Description
In our second receptor chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R2, is attached to an internal domain composed of N terminal domain of TEV protease, a nuclear export signal (NES), a TEV cleavage site(TCS(Q,G)), and dCas9(N).
Usage
this is our receptor's second chain. our receptor is activated after binding of VEGF to the external domain which is designed to attach specifically to it. after activation the two domains of TEV dimerizes forming catalytically active TEV protease which will cleave the two chains at TCS. upon cleavage of the two chains the two domains of dCas9 dimerize and is released attached to transcription activator to be guided to its direction.
this figure illustrates variant of our receptor's second chain where TCS (Q, G) is attached to it. .
Software Characterization
we had the chance to match the external domains with different internal domain components to select single suitable receptor chain. The whole chain affinity is affected by the internal domain, thus we had to try VEGFR2Ndcas with VEGFA:
VEGFR2Ndcas-VEGFA
The interaction between the chain composed of VEGFR2 as an external domain and Ndcas9 as an internal domain with VEGFA yields ΔG of -10.2 kcal mol-1 .
Then we have made a comparison between the four receptor chain variants’ binding stability with VEGFA.
This figure shows that VEGFR2Cdcas-VEGFA complex has the highest stability among other variants and VEGFR1NdCas9-VEGFA complex has the lowest stability among other variants .
The final form of our receptor is composed of two chains, each chain is built of internal and external domains, so we validated these interactions by calculating the binding affinity between the two chains and VEGFA, which simulate the final design of our receptor.
(VEGFR2-Cdcas9 – VEGFR2-Ndcas9) with VEGFA
The figure displays the interaction between two receptor chains and VEGFA, The (VEGFR2-Cdcas9) chain appears in the red colour, and the (VEGFR2-Ndcas9) chain appears in the blue colour. The VEGFA is in green colour. The calculated binding stability (ΔG) of the combination is -12.5 kcal mol-1 .
The final form of our receptor is composed of two chains, each chain is built of internal and external domains, so we validated these interactions by calculating the binding affinity between the two chains and VEGFA, which simulate the final design of our receptor.
(VEGFR1-Cdcas9 – VEGFR2-Ndcas9) with VEGFA
The figure displays the interaction between two receptor chains and VEGFA, The (VEGFR1-Cdcas9) chain appears in the red colour, and the (VEGFR2-Ndcas9) chain appears in the blue colour. The VEGFA is in green colour. The calculated binding stability (ΔG) of the combination is -13.7 kcal mol-1 .
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 982
Illegal BglII site found at 2341
Illegal BglII site found at 3647 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4474
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1854
Illegal BsaI.rc site found at 664
Illegal BsaI.rc site found at 1442
Illegal BsaI.rc site found at 2731
Illegal SapI.rc site found at 3289