Describe the improvements your team made of an existing part.
To fulfil this requirement, iGEM Guelph planned to compare the toxic effects of Cyt1Aa (BBa_K2938003) and our Cyt1Aa cassette (BBa_K4321007). This would involve cloning these biobricks into our E.coli to Bacillus subtilis shuttle plasmid and performing a toxicity assay on Drosophila melanogaster to see if our cassette is more effective in killing these insects. Considering that P20 can improve the stability and expression of Cyt1Aa, we decided that it would be a meaningful improvement on the Cyt1Aa biobrick (Manasherob et al., 2001).
Although this would show an improvement to the Cyt1Aa biobrick, co-expression of P20 with this cytotoxic protein protects recombinant E.coli and host cells from Cyt1Aa’s lethal effects (Manasherob et al., 2001). For this reason, iGEM Guelph could not determine and compare the toxicity effects of Cyt1Aa to our cassette. Despite the fact that we could not physically observe and test the effect of BBa_K2938003, we have successfully transformed our Cyt1Aa cassette into E.coli and Bacillus subtilis cells. Following the transformation into Bacillus subtilis, we were able to detect over 60% of Drosophila melanogaster deaths in our toxicity trials href="https://gitlab.igem.org/2022/guelph/-/blob/main/wiki/pages/proof-of-concept.html">Proof of Concept. Further proving the addition of P20 to Cyt1Aa increases the toxicity.
Manasherob, Robert, et al. “Effect of Accessory Proteins P19 and P20 on Cytolytic Activity of cyt1aa from Bacillus Thuringiensis Subsp. Israelensis in Escherichia Coli.” Current Microbiology, vol. 43, no. 5, 3 Apr. 2001, pp. 355–364.