Team:Oxford/Improve

Part Improvement

As part of our research on GMO biosafety, the possibility of therapeutic side-effects in patients was highlighted as a common occurrence. Thus, there was a clear need for a method to rapidly eliminate the engineered bacteria from the body. In order to achieve this, we decided to develop a probiotic kill switch which would be activated by an external supplement.

The 2015 Oxford team designed the Art-175 protein with a Dsb 2-19 secretion tag (BBa_K1659002) to hydrolyse the cell walls of pathogenic bacteria to tackle rising antibiotic resistances. They showed when inserted into an expression vector, host cell lysis could be induced and the supernatant could lyse P. putida cells. We have inserted a bidirectional pTet promoter and RBS before the secretion tag improving the part. The part has never been used as a kill switch before The part now shows activity without alteration in any TetR expressing strain Insertion of TetR on the opposite side of the promoter gives a standalone system which has been well characterised in E.coli allowing predictable and tunable expression The new part acts as a platform for future kill switches in probiotics due to the small size of the sequence. No auxiliary transport proteins are required for the inducible response, reducing strain of the device on cell growth and impact on colonisation efficacy .