Along the way of developing and engineering our approach, we ensured validation of every aspect and milestone of the project by relying on the available different methods including experts and literature validation besides dry lab and wet lab experimental validation.
We have designed our dry lab based on the steps of our system’s activation. On account of our design complexity, we went through multiple dry lab validations to increase our design reliability. Firstly, we divided our projects into two different main systems: each one was subdivided into multiple steps.
The first system is the dCas-Syn-RTK receptor which is composed of a signal sensing domain (external domain), transmembrane domain, and internal domain. The receptor activation starts by sensing the VEGFA by the signal sensing domain. Thus, we started our receptor’s dry lab validation by testing the external domain structure, function, and stability using different homology modeling tools, docking simulations, and molecular dynamics,respectively.
We also performed directed evolution to scan our receptor’s mutational landscape and define the mutation associated with the highest epistatic fitness and independent score. In response to the receptor activation, the internal domain went through multiple changes to transport the signals intracellularly. These changes started by receptor dimerization which induces Tobacco Etch Virus (TEV) protease assembly to cut the internal domain at TEV Cleavage Site (TCS). We tested the TEV protease assembly by docking simulation between its two domains. Furthermore, we tested its function by measuring its affinity with the TCS. Additionally, we induced single point mutation in each chain’s TCS: Q.L and Q.G ,making our receptor modular and usable for other applications. Dependently, the TEV activation results in dCas9 domains release and assembly. The binding between dCas9 domains were tested by docking simulations with and without its gRNA to ensure that the gRNA wouldn’t affect their assembly. Furthermore, we tested single receptor chain variants, and performed multi-docking simulations for the 2 chains with VEGFA to determine the best combination for our receptors’ chains.
Our second system is Translational Initiation Device (TID) which is an mRNA based translational switch. Despite the importance of our project’s safety, we put our mRNA stability a priority during the design milestones. Indeed, we measured our mRNA stability with and without the poly A tail which is responsible for a minimal basal activity of our mRNA switch. After ensuring our system’s stability, we conduct by testing our nanobodies affinity to their ligand (MMP-9) based on docking simulations and the complex's stability using molecular dynamics. Based on the previous step, the highest two nanobodies' affinity to the MMP-9 were tested on MCP and NSP3A respectively to measure their effects on the binding affinity to the two poles of mRNA to mediate the closed-loop model sites we put each one to its appropriate site.
Plasmids
| Name | Antibiotic selection | Reporter gene | Tags |
|---|---|---|---|
| pVEGFR1_TEV(C)-TCS(Q G)_NLS-HA-dCas9(C)-VP64 | Ampicillin, 100 μg/mL | GFP | Hemagglutinin(HA) |
| pVEGFR2_TEV(N)-TCS(Q G)_NLS-HA-dCas9(C)-VP64 | Ampicillin, 100 μg/mL Puromycin | mcherry fluorescence | Hemagglutinin(HA) |
| L1 PGK-BFP positive control plasmid | Kanamycin | BFP | His tag ,flag |
| pEYFP-YAP_MS2(N)-(HHR) | Chloramphenicol | EYFP | Flag tag |
The structural validation of our receptor’s two chains is done through tagging both of them with HA tag as there is a significant difference in length that would be reflected in western plot.
Moreover, we validate our switch’s structure by tagging NSP3-NB3 and MCP-NB1 with HIS and FLAG tags, respectively. Since both tags are approximately of the same length, they can be validated by detecting their corresponding antibodies with flow cytometry.
Receptor functional validation
The expression of the dCas9-TF Syn-RTK chains is mediated through CMV promoter activity; on the other hand, the gRNA expression is mediated through U6 promoter. The system performance require measuring the target EYFP activation score in two populations:
The EYFP activation score has to be measured in the absence and presence of VEGF, within the media, to assess the basal activity of the system.
Switch functional Validation
TID activity will be assessed by simulating the cells in a microenvironment similar to that of an injured wound. Therefore, an MMP-9 expressing vector was needed for the injured cells model. The assessment of TID performance will be done through measuring the reporter gene activation score in multiple populations:
YAP-1 Functional Validation
After the functional validation of each system independently, we would like to increase the endogenous YAP-1 production through directing our dCas9 system toward its gene using gRNA. Accordingly, the YAP-1 function would be assessed by analyzing the proliferative pattern of these cells through direct cell counting or proliferative assays such as MTT assay.
Validation plan of our constructed plasmids
We prepared our IDT gBlocks with restriction enzymes based cloning, then, we redigested the plasmid constructs and ran the digest into gel electrophoresis to characterize the plasmid and the inserted parts. Additionally, we used primers, flanking on the plasmid and the genetic part, and performed PCR to confirm the insert's presence within the plasmid and its correct orientation.
Validation of our system transcript structurally and functionally
We have chosen the HEK293T cell line, instead, due to their high transfection efficiency and rapid growth rate that would allow us to gain our validation result as early as possible.
We planned to study, test, and analyze the results of our validation plan into four different groups of HEK293K culture including two control groups reducing the bias in the results
Cell viability assay (MTT) will be used to assess the HEK293k cell integrity after the poly transfection technique, considering the cell toxicity that could result from the transfection.
1-Real Time qPCR to measure the expression levels of our genetic circuit. Western blotting and immunofluorescence confocal microscopy images will be processed by imageJ package to quantify our tagged parts and measure the expression of our system.
We have linked the fluorescence reporter gene to our final effector protein to expression level through detecting the potential of our system to induce the expression of (YAP-1)
Reporter gene expression quantification will be done through flow cytometry to measure the reporter gene activation score; moreover Forward scatter and side scatter will be used to identify the cell population and subsequently live single cells and data will be analyzed and compensated using the FlowJo package (FLOWJO LLC).
Real-time qPCR to measure the amount of (YAP) mRNA transcript to assess the functionality of our system.
Mix HMW-HA and LMW-HA in distilled water to create a homogeneous solution in a specific proportion depending on hydrogel’s final characteristics, And different ratios will give Hydrogel (A, B, C, D, and E). And then make a BDDE (1, 4-Butanediol diglycidyl ether) in a buffer solution the optimal concentration of BDDE should be determined according to the intended level of crosslinking. Combine the BDDE solution with the HA solution in the presence of a strong base in order to regulate the pH within the alkaline scale (pH 10-12). This makes it easier for the epoxy groups of BDDE to react with the hydroxyl groups of HA. Let the reaction continue for a few hours at room temperature (according to the intended final characteristic). After the reaction is complete, use an acid to neutralize the base and return the pH to neutral. Eliminate any leftover BDDE which is cytotoxic.
[1] Xue Y, Chen H, Xu C, Yu D, Xu H, Hu Y. Synthesis of hyaluronic acid hydrogels by crosslinking the mixture of high-molecular-weight hyaluronic acid and low-molecular-weight hyaluronic acid with 1,4-butanediol diglycidyl ether. RSC Adv. 2020 Feb 18;10(12):7206-7213. doi: 10.1039/c9ra09271d. PMID: 35493875; PMCID: PMC9049836.
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