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.

As an alternative to the traditional lysis cassette approach used by many iGEM teams, we decided to create a single-component kill switch. We redesigned the BBa_K1659002 part submitted by the 2015 Oxford iGEM team as an inducible construct as the basis for our kill switch. Intended as a method of generating novel antimicrobials in order to tackle antibiotic resistance, the 2015 team formed a composite of the artilysin Art-175 with a Dsb 2-19 secretion tag. They showed when inserted into an expression vector, host cell lysis could be induced and the supernatant could lyse P. putida cells.

To adapt this part, we inserted a bidirectional pTet promoter and RBS before the secretion tag, thus creating a self-contained inducible kill switch. The incorporation of a bidirectional promoter adds to the versatility of the part, enabling activity in bacterial strains which lack endogenous TetR expression.

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 .