Part of our research on GMO biosafety focussed on designing the system in such a way to minimise the impact of therapeutic side-effects in patients. 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: BBa_K2757001. 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 that 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 an inducible system as shown below:

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 results in a self-contained kill switch which has been well characterised in E. coli, allowing predictable and tunable expression. No auxiliary transport proteins are required for the inducible response, thereby reducing strain of the device on cell growth and colonisation efficacy.

In view of these factors, we hope our part can provide a framework to help future teams develop singe-component kill switches.