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 single-component kill switches.
The kill switch was assayed by measuring the OD of cultures subjected to varying levels of the inducer. While not intended to be used in final probiotic, the tetracycline analogue, anhydrotetracycline (ATC), has been shown to have a lower antibiotic activity and affinity for TetR and was thus chosen for use in our experiments. By comparing the growth of cells transformed with a plasmid lacking the promoter and cells with a plasmid lacking the killswitch and the promoter, we showed that cell growth was not affected by the kill switch.
Cell growth was unaffected by leaking of the promoter in cells with the bidirectional kill switch construct, showing that our device will not restrict the ability for the chassis to colonise the target region of the gut.
Our plate reader assay shows that the bidirectional promoter/kill switch construct induces cell lysis in response to concentrations of ATC equal to or above 1nM. Higher concentrations increase the rate of cell lysis up to concentrations of 20nM. Above this concentration, the promoter is at maximum activity.
The comparison between our improved part and the 2015's old part is clearly shown in the graphs below. Our improved part shows a clear increase in cell lysis as a function of ATC concentration whereas the previous team's part show little fluctuation between the same concentrations.
The left graph displays the 2015 Art-175 with bidirectional promoter. The right graph displays the 2015 Art-175 without a promoter, this shows no significant difference in growth with the vector without Art-175 in the presence of ATC.