Part:BBa_K5036028
dCas9(N)_NES-Syn-VEGFR-2 (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.As the whole chain affinity is affected by the internal domain, thus we had to try VEGFR2Ndcas with VEGFA:
VEGFR2Ndcas-VEGFA binding stability
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 binding stability
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 binding stability
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 .
Characterization by Mathematical Modeling
The model provides the interaction kinetics of VEGFR2 external domain upon binding of VEGF to it , the result shows satisfactory binding state and stability based on parametric values from literature
Graph (1). illustrates the decreasing of VEGFR2 (Red line) upon binding of VEGF (A) to form VEGF-VEGFR2 complex (R2A) (Yellow line) which increases till its binding to VEGFR1 (R1), so (R1) also decreases after will ( Black line). To finally form a binding state for fitted ligand receptor complex (R2AR1) (Blue line) .
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 our receptor chains to take place. The result shows sufficient TEV protease activation based on parametric values from literature
Graph (2). Illustrates the dimerization level (Blue line) that reaches steady state upon binding of VEGF to its receptor to activate TEV protease (Red line), The activation level of TEV protease reaches (14) 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 (240), upon activation of TEV protease .
Experimental Characterization
N-TEV and C-TEV segments Was connected to NES-dCas9(N) and NLS-dCas9(C)VP64, respectively, using a flexible link . To find the best dCas9-synVEGFR structure, TEV(N)-NES-dCas9(N) and TEV(C)-NLS-dCas9(C)VP64 was joined to the VEGFR1(FLT1) and VEGFR2(KDR) ectodomains . These combinations were tested in HEK293T cells and the activity of each homo- and hetero-dimer was measured with and without transgenically expressed VEGFA121
(Baeumler, T.A., Ahmed, A.A. and Fulga, T.A., 2017. Engineering synthetic signaling pathways with programmable dCas9-based chimeric receptors. Cell reports, 20(11), pp.2639-2653.)
The graph demonstrates that the EYFP reporter, a marker of activation score, was significantly upregulated when a C-terminal dCas9 was fused to VEGFR-1 and an N-terminal dCas9 was fused to VEGFR-2 were employed in combination. This effect was particularly pronounced in the presence of VEGFA121 outperforming other dCas9-VEGFR constructs. In addition to this, the combination of VEGFR2: TEV(N)-NES-dCas9(N)/VEGFR1: TEV(C)-NLS-dCas9(C)VP64 heterodimer shows very low activation score in absence of VEGFA121 .
It was hypothesized that by fine-tuning the TCS modules, the assembly of dCas9-VP64 could be controlled so that it only occurs upon simultaneous activation of VEGFR1 and VEGFR2. To investigate this, a set of dCas9(N)-synVEGFR2 and dCas9(C)-synVEGFR1 variants with TCS sequences that had been engineered to weaken their interaction with TEV were designed.Then These combinations were tested in HEK293T cells and the activity of different dCas9-VEGFR variants with TCS sequences was measured with and without transgenically expressed VEGFA121
(Baeumler, T.A., Ahmed, A.A. and Fulga, T.A., 2017. Engineering synthetic signaling pathways with programmable dCas9-based chimeric receptors. Cell reports, 20(11), pp.2639-2653.)
Among the tested dCas9-VEGFR constructs, a combination of dCas9-VEGFR2 with a low-affinity TCS(QL) and dCas9-VEGFR1 with a high-affinity TCS(QG) exhibited relatively low basal activity in absence of VEGFA 121 while preserving high activation score in comparison to other constructs in presence of VEGFA 121. In other words, the previous combination shows the most specific activation of the EYFP reporter in response to VEGFA 121 .
A HEK293 cell line was engineered to express the VEGFR2: TEV(N)-NES-dCas9(N)/VEGFR1: TEV(C)-NLS-dCas9(C)VP64 heterodimer. To assess the effectiveness of the system, flow cytometry was used to compare EYFP reporter expression in cells transfected with either a control sgRNA or one targeting EYFP.
(Baeumler, T.A., Ahmed, A.A. and Fulga, T.A., 2017. Engineering synthetic signaling pathways with programmable dCas9-based chimeric receptors. Cell reports, 20(11), pp.2639-2653.)
Flow cytometry analysis of cells expressing dCas9-VEGFR constructs with either a control sgRNA (sgSCR) or a targeting sgRNA (sgEYFP) revealed that the receptor configuration treated with the targeting sgRNA was unresponsive to activation without VEGFA121 but exhibited a strong response to VEGFA121, with up to a 1,000-fold increase in EYFP activation .
We have done DNA gel electrophoresis to validate the cloning of our mCherry into dCas9(N)_NES-Syn-VEGFR-2
This figure illustrates the amplified fragments of our insert mCherry within P2 .
The final ligated form of plasmid dCas9(N)_NES-Syn-VEGFR-2 was transformed into competent BL 21
This figure illustrates the transformed dCas9(C)_NLS-Syn-VEGFR-1 and dCas9(N)_NES-Syn-VEGFR-2 plasmid to BL21 competent cells .
Reference
White C, Rottschäfer V, Bridge LJ. Insights into the dynamics of ligand-induced dimerisation via mathematical modelling and analysis. J Theor Biol. 2022 Apr 7;538:110996. doi: 10.1016/j.jtbi.2021.110996. Epub 2022 Jan 24. PMID: 35085533.
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.
Paththamperuma C, Page RC. Fluorescence dequenching assay for the activity of TEV protease. Anal Biochem. 2022 Dec 15;659:114954. doi: 10.1016/j.ab.2022.114954. Epub 2022 Oct 18. PMID: 36265691; PMCID: PMC9662696.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 982
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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
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Illegal SapI.rc site found at 3289
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