Difference between revisions of "Part:BBa K2817012"

Latest revision as of 17:45, 17 October 2018

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 PcspA-amilCP plasmid into DH5α and cultured overnight at 37 ℃. The overnight culture was diluted to OD600 = 0.2 and then grow for 2 h at 37 ℃. It was then induced for 6 hours at 16 ℃ or 37 ℃ under different concentrations of IPTG conditions (Figure 1). From Figure 1, the reporter gene was efficiently expressed at low temperature, which indicated that the effective expression of the toxin gene mazF and the blocking expression of the anti-toxin gene mazE at low temperature can kill our engineered bacteria in time. Although the cold shock promoter PcspA can prevent the most of the leakage expression at 37 ℃, the weak leakage expression of mazF may still cause the functioning engineered probiotics being killed in human. Therefore, the introduction of the anti-toxin protein mazE is necessary to effectively antagonize the potential leakage expression of the cold shock promoter PcspA.

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

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===
-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.
+We inserted this part into pColdI.
+Then we transformed the PcspA-amilCP plasmid into DH5α and cultured overnight at 37 ℃. The overnight culture was diluted to OD600 = 0.2 and then grow for 2 h at 37 ℃. It was then induced for 6 hours at 16 ℃ or 37 ℃ under different concentrations of IPTG conditions (Figure 1). From Figure 1, the reporter gene was efficiently expressed at low temperature, which indicated that the effective expression of the toxin gene mazF and the blocking expression of the anti-toxin gene mazE at low temperature can kill our engineered bacteria in time. Although the cold shock promoter PcspA can prevent the most of the leakage expression at 37 ℃, the weak leakage expression of mazF may still cause the functioning engineered probiotics being killed in human. Therefore, the introduction of the anti-toxin protein mazE is necessary to effectively antagonize the potential leakage expression of the cold shock promoter PcspA.
 https://static.igem.org/mediawiki/2018/5/5a/T--NEU_China_A--results-10.png
 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.
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