Difference between revisions of "Part:BBa K2817006"

Revision as of 17:55, 17 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

When we use engineered bacteria to alleviate IBD in vivo, the growth of bacteria must be tightly regulated. Therefore, we designed a cold shock kill switch that based on the toxin-antitoxin system mazEF. The rational of this kill switch is relying on the mechanism that when E. coli is under stress, the maz-E will degrade faster than maz-F, so the relatively more maz-F will exert its toxicity. The cold shock promoter PcspA was linked with maz-F, so when the temperature is low, the maz-F will express while the maz-E stop expressing. Thus, we could kill our engineered E. coli when it escapes from human body. We inserted maz-F into the pColdI plasmid to build our kill switch.

Next, 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 OD600=0.02 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, the kill switch worked efficiently at low temperatures and indicated that mazF enable to cause cell death even at low expression level.

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

Figure 11. The effect of our killer gene under 16℃. A, the plate of BL21 with and without killer gene under induction. B, C The effect of mazF and Lysis on the growth of Escherichia coli at different temperature (C,16℃; D,37℃) or in different plasmids.

[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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <!-- Add more about the biology of this part here -->
 ===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.
+When we use engineered bacteria to alleviate IBD in vivo, the growth of bacteria must be tightly regulated. Therefore, we designed a cold shock kill switch that based on the toxin-antitoxin system mazEF. The rational of this kill switch is relying on the mechanism that when E. coli is under stress, the maz-E will degrade faster than maz-F, so the relatively more maz-F will exert its toxicity. The cold shock promoter PcspA was linked with maz-F, so when the temperature is low, the maz-F will express while the maz-E stop expressing. Thus, we could kill our engineered E. coli when it escapes from human body. We inserted maz-F into the pColdI plasmid to build our 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.
+Next, 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 OD600=0.02 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, the kill switch worked efficiently at low temperatures and indicated that mazF enable to cause cell death even at low expression level.
 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.
+Figure 11. The effect of our killer gene under 16℃. A, the plate of BL21 with and without killer gene under induction. B, C The effect of mazF and Lysis on the growth of Escherichia coli at different temperature (C,16℃; D,37℃) or in different plasmids.
 [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.