Part:BBa_K3317007
Inducible p3B5C promoter
Sensitive promoter against protocatechuic acid and presents its maximum induction in the presence of 1000mM RPU 1.6 of vanillinic acid with a leak of 4.5x10-3 RPU in absence of induction(Meyer, A. J et. al 2019) this part needs its regulatory protein (BBa_K3317005)
Improve the p3B5C
p3b5b was obtained by point mutation of p3b5c,iGEM 2020 RDFZ-China performed a site-specific mutation on the promoter of p3b5c.This part improves the existing Part p3b5c.
Characterization
The pcauam_p3B5B system is constructed with two parts, upstream activator, and downstream reporter.p3b5b was obtained by point mutation of p3b5c,iGEM 2020 RDFZ-China performed a site-specific mutation on the promoter of p3b5c.This part improves the existing Part p3b5c. The target of this system is to test the sensitivity of the PcauAM activator and p3b5b promoter combination. The upstream sequence is composed of pLacIQ promoter, PcauAM and tonB terminator . The upstream sequence will express PcauAM protein, which is not stable, and denature itself. The downstream sequence is composed of p3b5b promoter, eYFP reporter and L3S3P21(terminator). This downstream system will present high expression when PcauAM protein is generated. We use eYFP to show the sensitivity of this combination.
Design
We constructed the promoter into the low copy p15A plasmid with YFP downstream its regulatory protein PcauAM. We transformed pET28b_p3B5B vector into E .coli BL21 for testing.
Experimenting
Activity test: Sensor activity of PcaU-pPCA, PcaUAM-p3B5B, PcaUAM-p3B5C system under different emission light.
The data in the right of figure are the tested data of PcaUAM-YFP under emission light 525nm. When PCA molecular is not added, the fluorescence intensity divided by bacterial concentration of pcauAM-p3b5b and pcauAM 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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