This summer, I am working with the probiotic bacteria Escherichia coliNissle 1917. Probiotic bacteria pose a self-replicating and versatile drug-delivery platform. The Lesser lab has introduced into Nissle 1917 the Type III Secretion System (T3SS), a complex nanomachine capable of secreting many proteins outside the cell. It is unknown whether the levels of secretion observed in this modified strain are the maximum possible. Specifically, I am investigating the essential T3SS genes Spa13, Spa33, andMxiA. Interestingly, they contain transcriptional slippage sites, sequences that cause RNA polymerase to integrate additional nucleotides not encoded by DNA. We hypothesized modifying the DNA sequence to prevent slippage would increase secretion. To test this, I will knockout Spa13, Spa33and MxiAin our strain and complement with expression plasmids encoding genes where the slippage site has been repaired. I will then compare secretion of each strain using liquid secretion assays and western blot. This work will inform the process of generating an optimized strain that better secretes therapeutic payloads.
