Team:Queens Canada/Proof Of Concept
Proof of Concept
Proof of Concept
Our team created two plasmids for bacterial expression in an E.Coli system. The first was to express just the OspA protein which we could then use to see if the ScFv we created was binding correctly. We plan to perform this proof of concept test by anchoring the OspA to a plate via a his-tag and then adding our ScFv with a chromogenic and fluorescent tag to the plate, then performing a wash step to ensure proper binding before our team performed further tests. We started this process by loading a PET28a plasmid backbone into Benchling to use as our vector. We then took the protein sequence from OspA (the same one used in the paper we used to build our ScFv) and codon-optimized it for E.Coli expression. Once we obtained that sequence, we added a T7 RNA Polymerase promoter site to the start of the sequence and used a BAMHI restriction site at the N-terminal end. At the end of the OspA protein sequence, we added a his-tag (6 histidine residues with non-repeating codons as repeating codons will mess with the RNA Polymerase activity) which we will use to purify the protein once expressed as well as we will use to bind OspA to the plate for the proof-of-concept test. Because we plan to use the his-tag as an anchor to the plate, we did not put a protease cut site between it and the OspA sequence as cleavage was not necessary. Following the his-tag, we added a termination codon to the sequence. At the end of the sequence, a SAL1 restriction site was added so the OspA sequence could be transformed into the plasmid and would not be flipped. We also made sure that both of our restriction sites had sticky ends.
For the second plasmid, we needed to add a few more elements as the protein expression is slightly more complex and has a few more elements at play. We started again by using a PET28a plasmid backbone. We then, in a separate Benchling document, added the ScFv sequence we had previously created but with a T7 RNA Polymerase promoter at the start of the sequence to promote RNA Polymerase binding. We attached a glycine linker to the end of the ScFv fragment which we the central portion from the paper (GGGGS)3 but added two serine's prior to the linker as our BLAST search for homologous regions indicated that this was a highly conserved feature of the ScFv linkers. We then used an alkaline phosphatase (BBa_J61032) which we used the sequence from the iGEM parts registry and then codon-optimized the protein for E.Coli expression. We added the alkaline phosphatase sequence immediately following the glycine linker region. Following the alkaline phosphatase, we added the same linker used prior followed by a TEV protease sequence. After the TEV protease sequence we added a green fluorescent protein (GFP), this way, we can use the GFP to see if the protein is folded correctly within the E.Coli cells prior to lysing them and then cleave the GFP segment off of the protein after we have verified it is folded correctly and purified it. To purify the protein, we added is his-tag at the end of the sequence followed by a stop codon. This way, we can purify the protein in a nickel column, use imidazole to wash the protein from the column, then cleave the GFP which, in turn, cleaves his tag as well. Thus, the fusion protein will not compete for binding on the plate with the OspA that will be bound. The GFP used in this experiment is from the iGEM parts registry and was codon-optimized for E.Coli expression (BBa_E0040). Our team used the BamH1 restriction site at the N-terminal side of the protein and the Sal1 restriction site at the C-terminal end of the protein. Using these restriction sites allows for our fusion protein to be transformed into the plasmid in our desired orientation (not flipped).
