Composite

Part:BBa_K5036031

Designed by: Emad hamdy Matter   Group: iGEM24_AFCM-Egypt   (2024-09-21)


VEGF-R1, C-TEV, NLS, TCS(Q,G),HA,dCas9(N),VP64,GFP

Part Description

In our first receptor chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R1, is attached to an internal domain composed of C terminal domain of TEV protease, a nuclear localization signal (NLS), a TEV cleavage site(TCS(Q,G)), and dCas9(N) which is linked to transcription activator VP64.

Usage

this is our receptor's first 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 first chain where dCas9(N) is to it.

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 VEGFR1Ndcas with VEGFA:

VEGFR1Ndcas-VEGFA

The interaction between the chain composed of VEGFR1 as an external domain and Ndcas9 as an internal domain with VEGFA yields ΔG of -8.3 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.

(VEGFR1-Cdcas9 – VEGFR1-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 (VEGFR1-Ndcas9) chain appears in the blue colour. The VEGFA is in green colour. The calculated binding stability (ΔG) of the combination is -12.9 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.

(VEGFR2-Cdcas9 – VEGFR1-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 (VEGFR1-Ndcas9) chain appears in the blue colour. The VEGFA is in green colour. The calculated binding stability (ΔG) of the combination is -13.0 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 .

Characterization by Mathematical Modeling

The model provides the interaction kinetics of VEGFR1 receptor upon binding of VEGF to it , the result shows satisfactory binding affinity and stability based on parametric values from literature

Graph (1). Illustrates the decreasing of free VEGFR (Black line) upon binding of VEGF to one of the receptor chains to form (RA) complex (Yellow line), that bind directly to the other chain of the receptor to final form the fitted ligand receptor complex (RAR) to reach 0.2 .

The continuation of the first model provides the activation kinetics of the TEV protease which occurs subsequent to the binding of VEGF to our receptor allowing the dimerization process for the receptor chains to take place. The result shows TEV protease activation based on parametric values from literature

Graph (2). Illustrates the dimerization level (Blue line) that reaches (7.8) upon binding of VEGF to its receptor to activate TEV protease (Red line), The activation level reaches (6.5) to release d-Cas9 system .


The continuation of the second model provides the activation kinetics of the d-Cas9 system which occurs subsequent to cleavage activity of TEV protease after its activation. The result shows increase in d-Cas9 activity which implies successful cleavage of the TEV protease for releasing the N and C terminal of the d-Cas9 system and its assembly based on parametric values from literature

Graph (3). Illustrates the released d-Cas9 system that activation reaches (110), upon activation of TEV protease. .

Reference

Mac Gabhann F, Popel AS. Dimerization of VEGF receptors and implications for signal transduction: a computational study. Biophys Chem. 2007 Jul;128(2-3):125-39. doi: 10.1016/j.bpc.2007.03.010. Epub 2007 Mar 24. PMID: 17442480; PMCID: PMC2711879.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2173
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3644
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 4471
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 5807
    Illegal SapI.rc site found at 3268


[edit]
Categories
Parameters
None