Composite

Part:BBa_K5036028

Designed by: Emad hamdy Matter   Group: iGEM24_AFCM-Egypt   (2024-09-21)
Revision as of 16:59, 25 September 2024 by Alisalah (Talk | contribs) (→‎Software Characterization)


VEGF-R2, N-TEV, NES, TCS (Q, L), 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,L)), 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 our receptor's second chain structure.

Dry lab 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 .


The receptor chains’ affinity could be affected by the internal domains interactions with the external domains, so we compared between the receptors’ variants to choose the best receptor design in our project.

The figure shows that the combination of VEGFR1-Cdcas9 and VEGFR2-Ndcas9 took the upper hand among other variants .

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 982
    Illegal BglII site found at 2341
    Illegal BglII site found at 3647
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 4474
  • 1000
    INCOMPATIBLE 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


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