The initial literature research for our project began in March 2022 and our final project idea was formed in May. Minor modifications were made throughout the summer months, as a result of extensive literature revisits. The designing module of the project involved deciding which parts, organisms, technics, and reagents will be required. Specifically, we used an iGEM part, OmpA BBa_K1489002, to surface display the LRR and NB-LRR domains of the Sw-5b resistance gene of Solanum peruvianum on E. coli DH10B cells. We designed primers for gene acquisition, specific domains of gene acquisition, coding DNA synthesis, and primers for cloning compatibility. A small region of the movement protein of TSWV (Nsm115-135) is required for NLR recognition thus only this 21-amino acid coding sequence was utilized in our experiments. Our novel binding molecule consisted of an existing part(BB) and the plasmids used are: plCH86988, pBluescript, pCHW1383 for cloning and pET-26b for protein expression.
It is well documented in the literature that the Sw-5b resistance gene directly binds the movement protein of several tospoviruses, also our in silico investigation for the predicted structure and binding potential of our novel chimeric protein indicated it was a gene well-chosen. In addition, OmpA fusion proteins are abundant in the literature which makes it a less risky option for a fusion partner.
Alphafold is a recent AI system able to predict protein structures when only given protein residue sequences. During our project design, we frequently advised protein modeling predictions using this program. Our Alphafold projections showed a high probability of correct folding of both fusion partners and an adequate probability of interaction with the movement protein of the tomato-spotted wilt virus. The proof-of-concept demonstration experiment will include the introduction of Agrobacterium tumefaciens transformed with NSm115-135:YFP which is the conserved effector domain of the movement protein of the viruses recognized by Sw-5b fused with YFP into Nicotiana sylvestris. Leaves expressing this construct will be subdued to protein extraction with various methods and the protein extracts will be used for E.coli treatment to test the binding activity of these surface-displaying cells based on fluorescence after several washes with TBS. On the other hand, E.coli surface-displaying random peptides will be tested as a negative control. Our final results given that our model is working as intended will be: fluorescent E.coli cells that display our novel chimeric protein and not the negative controls.
In order to assemble the desired constructs, we proceeded in planning out various experiments. Our end goal was to create bacterial cells that surface display binding proteins for viral effectors and a fusion protein of a “detectable” effector protein. Our effector fusion protein is detectable with fluorescence.
Primer design to include suitable restriction enzyme recognition sites was key to our successful build. Primers for the amplification of our preferred part (OmpA) right from iGEM’s 2021 Distribution Kit was designed so the amplicon was Golden Gate compatible with BsaI recognition sites and ensured that its C-terminal fusion partners remain in frame. Primer design for NB-LRR and LRR domains of S.peruvianum were designed to be golden gate compatible for C-terminal fusion with OmpA. Forward primer for OmpA’s amplification as well as a reverse primer for the NB-LRR and LRR domains were made so that after BsaI restrictive digestion, they can ligate to the pLCH86988 vector. Primers for amplification of pLCH86988:NB-LRR and pLCH86988:LRR were designed for cloning with restriction enzymes (SstI and NdeI) and insertion into expression vector pET-2b.
The movement protein (Nsm115-135) of tomato spotted wilt virus is recognized via a small (21 amino acid) sequence and due to the difficulty in viral gene acquisition, we proceeded to utilize only this small protein sequence that we synthesized using oligo-nucleotides. We designed primers to cover the entire cDNA of this sequence with an overlap of 20-nt for annealing and used PCR to create and amplify the dsDNA. The amplicon had no stop codon for C-terminal fusion with YFP for detection.
After the acquisition of E.coli surface-displaying NB-LRR and LRR domains of resistant protein Sw-5b and plant tissue expressing Nsm115-135:YFP (effector recognition domain fused with yellow fluorescent protein), our system is ready for testing. The surface-displaying cells will be pelleted and resuspended in 100μl PBS and the plant tissue expressing our construct will be subjected to total protein extraction. 100μl of cells suspended in PBS will be mixed with 100μl of protein extraction and incubated at 4 degrees Celsius rotating. Incubation time will be 20 minutes, 30 minutes, 1 hour, and 2 hours. After the incubation cells will be subjected to 3x washes with PBS and OD600 being measured after the third step. Different cell concentrations were tested to find the optimal number of cells for the detection of our target protein using a spectrophotometer. Finally, samples will be tested for fluorescent activity using a Biomolecular imager.
If our system works as intended, fluorescence will be observed in samples of E.coli surface-displaying our binder protein and not in E.coli surface-displaying random peptides (negative control). The observance of fluorescent activity will indicate that our binding proteins interacted with Nsm115-135 and are able to be utilized as a novel component of diagnostic tests.
Our initial plan was to develop a project able to evolve through constant troubleshooting, re-evaluation, and improving our design. As intended, our early failures to transform E.coli Dh10b using iGEM part DNA from the 2021 Distribution kit led us to begin our troubleshooting rampage with different iGEM parts and various DNA quantities. We also tested our competent cells using intact plasmid DNA from our lab and checked our antibiotic stocks. Realizing that none of the above were responsible for our unexpected results, we proceeded to amplify the part of interest with PCR which was successful. The process of taking a step back, trying to figure out the problem, and reassessing our strategy was a huge learning curve.
Another setback that made us reconsider and redesign was our inability to clone our PCR products to pBluescript vector. After A-tailing of the PCR products, we ligated them to T-tailed linear pBluescript vector which yielded incorrect products. So, we changed our objective and proceeded to Golden Gate cloning with the PCR products changing the ratio insert: vector as instructed by our advisors. This procedure was enlightening and contributed to a solution to our mishap. In retrospect, this alternative process was more time-efficient than the original plan and from now on it will be our go-to strategy.
In our final experiment where we provide evidence of our binder efficiency, we had to rearrange our protocol. Initially, we incubated our E.coli surface-displaying the binding proteins with the total protein extraction for 20 minutes and we failed to detect any fluorescent signal. Thus, we had to change the incubation time, as well as try other cell concentrations and temperatures. Finally, we optimized the conditions necessary for our effector-binding cells to thrive.