Design
For the development of our project we ensured that we provided sufficient evidence that our system is functional. During the whole duration of our experiments we ensured that we keep safety as the number one priority. We also tried to keep results for each one of our experimental procedures to strengthen the transparency of our concept.
The beginning of the plan
After countless brainstorming sessions we ended up in our project idea, ATROPA, which is basically a diagnostic tool utilizing LRRs for TSWV diagnosis. The first step was to test in silico that our idea has a potential and this is where our project modeling came in.
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Our predictions really showed us that there is actually interaction between the LRR domain of Sw-5b resistance gene and the Nsm115-135. This was the confirmation we needed to proceed with the experimental part of our project.
Experimental planing
Before the lab work was initiated, we shared our plans with both our supervisor and our advisors. After getting critical feedback and insights from them we started planning our experimental procedures. When the planning was over we again consulted them and optimized every step of the process. This is when the actual experiments were ready to begin.
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Results and analysis
As mentioned before results from all of the steps of our experiments were collected to properly display the progress of our project. The final experiment showed positive results and the proof of concept for our project was established. In particular, after incubating the E.coli cells which were surface displaying the LRR and the NB-LRR of SW-5b resistance gene of Solanum Peruvianum , with the protein extract from N. Sylvestris leaves expressing the Nsm115-135 viral protein fused with YFP, we observed fluorescence in the samples. This means that our E.colicells have the ability to bind this protein.
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Conclusion
Overall we managed to show that our system of surface displaying cells are capable of binding our target effector protein, while E.coli surface displaying random peptides did not. In our opinion this is a novel approach to reconsider diagnostic tool’s basic elements. We think that whole-cell based diagnostic tools can significantly lower the price of production while providing the affinity and specificity of antibody-based systems.