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 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. | ||
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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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| + | [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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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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]