Part:BBa_K2817012
PcspA-RBS-amilCP
CspA is a cold shock promoter and amilCP is a reporter.
Usage and Biology
We inserted this part into pColdI. Then we transformed the plasmid into DH5α and cultured overnight at 37℃. The overnight culture was diluted to OD = 0.2 and allowed to grow for 2 h at 37℃. It was then divided into different concentrations of IPTG at 16℃ and 37℃ for 6 h (Figure 1). It can be seen from the figure 1 that the reporter gene is efficiently expressed at low temperature, which indicates that the effective expression of the toxin gene mazF and the closed expression of the anti-toxin gene mazE at low temperature can kill our engineered bacteria in time. Although the cold shock promoter PcspA has a certain leakage at body temperature, the toxin is neutralized by the anti-toxin expressed at body temperature, so the effect is not significant. However, it is best to add a lacO site in the future to suppress leakage.
Figure 1. Pellets of bacteria transformed with constructed PcspA-amilCP plasmid after induction of 6h. From left to right: 37℃ without IPTG, 37℃ with 0.5mM IPTG, 37℃ with 1mM IPTG, 16℃ without IPTG, 16℃ with 0.5mM IPTG, 16℃ with 1mM IPTG.
[1] 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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