Difference between revisions of "Part:BBa K5036030"

Revision as of 07:34, 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 .