Difference between revisions of "Part:BBa K2817012"
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CspA is a cold shock promoter and amilCP is a reporter. | CspA is a cold shock promoter and amilCP is a reporter. | ||
| − | <!-- Add more about the biology of this part here | + | <!-- Add more about the biology of this part here --> |
===Usage and Biology=== | ===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. | ||
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| + | https://static.igem.org/mediawiki/2018/5/5a/T--NEU_China_A--results-10.png | ||
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| + | 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. | ||
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Revision as of 15:59, 16 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 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.
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]