Difference between revisions of "Part:BBa K2817006"

Revision as of 16:12, 16 October 2018

PcspA-RBS-mazF

The mRNA mediated by the cold-acting promoter CspA can only be efficiently translated at a low temperature of, for example, 16 ℃, so we use it to activate the expression of mazF at low temperatures. In this way, when the engineered bacteria leaves the intestine and enters the environment, it will be killed by our cold shock kill switch.

Usage and Biology

It is necessary for us to do biocontainment work to avoid the release of engineered bacteria into the environment. In our project, we designed a cold shock kill switch based on the toxin-antitoxin system-mazEF- a natural toxin-antitoxin system found in E. coli. MazF is a stable toxin protein, and mazE is an unstable antitoxin protein. When the bacteria are inhibited by environmental stress and the expression of mazEF is inhibited, the unstable antitoxin protein is preferentially degraded, and the relative content of the stable toxin protein increases to a certain extent, which will lead to the death of bacterial. The mRNA mediated by the cold-acting promoter CspA can only be efficiently translated at a low temperature of, for example, 16 ° C, so we use it to activate the expression of mazF at low temperatures. In this way, when the engineered bacteria leave the intestine and enters the environment, it will be killed by our cold shock kill switch.

We transformed the constructed PcspA-mazF plasmid into BL21, added 1 mM IPTG to the plate, and cultured at 16℃ for 16 h (Figure 1A). We then cultured BL21 transformed with the PcspA-mazF plasmid overnight at 16℃. After diluting to OD=0.2 on the next day, the cells were cultured at 16℃, and the OD value was measured every hour for 9 hours (Figure 1B). From Figure 1 below, you can see that our kill switch can work at low temperatures. Although some engineered bacteria still survive, we believe they will be effectively killed in the absence of nutritional support.

http://219.216.82.193/cache/4/04/2018.igem.org/c5f91b2ae7df1be5dabd856e6b8dd194/T--NEU_China_A--results-11.png

Figure 1. The effect of our killer gene under 16℃. A, the plate of BL21 with and without killer gene under induction. B, the growth curve of BL21 with and without killer gene under induction.

[1] Aizenman E, Engelberg-Kulka H, Glaser G, et al. An Escherichia coli chromosomal “addiction module” regulated by guanosine 3′5′-bispyrophosphate: a model for programmed bacterial cell death. Proc Natl Acad Sci, 1996, 93(12): 6059−6063.

[2] Pandey DP, Gerdes K. Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes. Nucleic Acids Res, 2005, 33(3): 966−976.

[3] Amitai S, Yassin Y, Engelberg-Kulk H. MazF-mediated cell death in Escherichia coli: A point of no return. J Bacteriol, 2004, 186(24): 8295−8300.

[4] Stirling F, Bitzan L, O'Keefe S, et al. Rational Design of Evolutionarily Stable Microbial Kill Switches[J]. Molecular Cell, 2017, 68(4):686-697.

Sequence and Features