Safety matters, and also does public opinion. To improve the safety of our P. agglomerans Trojan Horse, we devised a simple, proof-of-concept acid-dependent kill switch !
This promoter of our kill switch is pGadA , an E. coli promoter active under acidic conditions and previously characterized by iGEM Dundee 2016. This is regulating the protein MazF, an endoribonuclease toxin also from E.coli, well characterized for use in containment (1).
Our bacteria would be delivered in alginate capsules (2), both of which can have the pH regulated to be >7. If the bacteria ended up in the mosquito midgut, it would live in a pH between 8.5 and 11 happily (3) . On the other hand, the pH of normal rain (5-6) (4) would be enough to trigger the “self-destruct” mechanism and prevent unwanted spread.
However, we were not able to clone and test this construct. This could be due to low efficiency transformation, as leaky expression of the toxin MazF could inhibit E. coli growth. This might be circumvented by optimizind transformation protocols for less acidic conditions or increasing the degree of control of the system (1).
- Chan, Clement TY, et al. "'Deadman'and'Passcode'microbial kill switches for bacterial containment." Nature chemical biology 12.2 (2016): 82-86.
- Arora, Arinder K., et al. "A delivery system for field application of paratransgenic control." BMC biotechnology 15.1 (2015): 59.
- Linser, Paul J., et al. "Carbonic anhydrases and anion transport in mosquito midgut pH regulation." Journal of Experimental Biology 212.11 (2009): 1662-1671.
- Liljestrand, Howard M. "Average rainwater pH, concepts of atmospheric acidity, and buffering in open systems." Atmospheric Environment (1967) 19.3 (1985): 487-499.