Revision as of 09:27, 19 October 2019

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, Moreover, in order to test the efficiency of mazF. 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 37℃. After diluting to OD600=0.02 on the next day, the cells were cultured at 16℃(Figure 1B) and 37 ℃(Figure 1C), and the OD value was measured every hour for 9 hours (Figure 1B). From Figure 11, the kill switch worked efficiently at low temperatures and indicated that mazF enable to cause cell death even at low expression level.

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Figure 11. Figure 11. The effect of our killer gene under different conditions. 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 (B,16℃; C,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.

NEU_China 2019

Improve the Characterization of BBa_K2817006

OVERVIEW

Due to time constraints, last year we only measured the survival rate of the cells transformed with PcspA-mazF plasmid once and we did not get the ideal results that demonstrated a high survival rate of those cells cultured at 37 ℃. Therefore, this year we did some repetitive experiments to remeasure the survival rate of the cells at first. Moreover, we introduced the toxin-antitoxin system - MazEF system to soothe the serious leakage problem of MazF in order to optimize the effect of kill switch.

Parts for this section: BBa_K2817012 BBa_K2817006

METHOD

We transformed the constructed PcspA-mazF plasmid constructed last year into E. coli BL21 and cultured at 16°C for 11 hours. We then cultured respectively overnight at 16 °C and 37°C. After diluting to OD₆₀₀=0.02 on the next day, the cells were cultured at 16 °C (Fig. 1A) and 37°C (Fig. 1B) and the OD₆₀₀ value was measured every hour for 11 hours. Finally, we got the growth curves and calculated the survival rates at these two temperatures.

Figure 1. The effect of our killer gene under different conditions. A, B. The effect of MazF on the growth of E. coli at different temperature (A,16°C; B,37°C) within different plasmids

Optimization of kill switch

Last year, we constructed the engineered bacteria and found the serious leakage problem of mazF. This year we did some repetitive experiments to remeasure the survival rate of the cells.By analyzing the growth curve, it showed the survival rate of cells reaching 9.12 percent at 16℃ and the survival rate of cells reaching 34.15 percent at 37℃ this year. (Fig.2) The data showed that there was not a distinctive difference between the two temperatures., which indicated that a severe leakage problem of toxic protein leading to the cell death. Therefore, we introduced mazEF system to soothe the serious leakage problem of mazF in order to maximize the effect of the kill switch.

Figure2.   The effect of our killer gene under different conditions. The effect of mazF and Lysis on the growth of Escherichia coli at different temperature (A,37℃; B,16℃) or in different plasmids.

Sequence and Features