Team:Michigan/Demonstrate
Proof of Concept
In Tunable thermal bioswitches for in vivo control of microbial therapeutics (Piraner et. al., 2016), a library of tunable bioswitches that were highly sensitive to changes in temperature were reported by the researchers. This work sparked our idea to try and develop a proof of concept for a genetic kill switch using a holin/endolysin/antiholin lysis system for the containment of bacteria in research labs. Our goal was to develop a switch in E. coli that would execute when temperature dropped below 34C for longer than two hours. We designed a plasmid that contains T4 holin and T4 endolysin expressed constitutively. At temperatures above 34 C, these enzymes would be prevented from lysing the cell by T4 antiholin, expressed under the control of TlpA36, a temperature sensitive repressor.
Once we designed and synthesized our plasmid we tested it via a series of experiments that were intended to see whether or not significant cell death occurred after exposure to temperatures below 34C for extended periods of time, as outlined on our Experiments page. We tested three constitutive promoters of varying strength in order to find a degree of holin and endolysin expression that would only kill the cells after a significant exposure time, as working with the strains containing the plasmid would be impossible if death were too rapid.
Unfortunately, none of the three promoters we tested were strong enough to outright kill the cells, even after 6 hours at room temperature. However, as seen in our data analysis, one of the promoters did have a negative effect on growth. This gives us hope that if a strong enough promoter were used, our system would be viable.
