Part:BBa_K3332069
J23100-RBS-phnJ_mut21-terminator
Use BBa_K823004-BBa_B0034 to express the mutant of C-P lyase, which can degrade the glyphosate, from Enterobacterales. This subunit is essential to the activity of the whole enzyme.Improve the capability of chassis bacteria to degrade glyphosate.
Biology
Phn system is a gene cluster for organophosphorus transport and degradation in many microorganisms. Enterobacterales use phnHIJK genes to encode C-P lyase. PhnJ protein is an essential subunit that can crack C-P bond. The 21th arginine was mutated to methionine.
Usage
We ligased the J23100-B0034-phnJ_mut21-B0015 (BBa_K3332025) and the part J23100-B0034-phnE1-B0034-phnE2(BBa_K3332067) on the expression vector pSB1C3 by standard assembly. Then the ligation mixture was transformed into E. coli DH5α & E. coli BL21(DE3), which enables the E. coli to degrade glyphosate at higher efficiency and improved the binding ability with the endogenous PhnHIK.
Characterization
Enzyme activity
We use Negative Control, experiment groups phnEE and Mut21_phnJ-phnEE to analyze if Mut21_phnJ enhances the degradation of glyphosate by the chassis bacteria by our detection system. Mut21_phnJ gene lowers the degradation rate of E.coli BL21(DE3) about 16.4%, indicate the mutant has higher binding ability to PhnHIK but lower degradation ability compare to endogenous PhnJ.
The result is shown in Fig.2(Experiment groups in Fig.2
Negative Control: J23100-B0034_pSB1C3
phnE1E2: J23100-B0034-phnE1-B0034-phnE2_pSB1C3,
phnJ-phnE1E2: J23100-B0034-phnJ-B0015-J23100-B0034-phnE1-B0034-phnE2_pSB1C3,
Mut21-phnJ-phnE1E2: J23100-B0034-phnJ_mutR21M-B0015- J23100-B0034-phnE1-B0034-phnE2_pSB1C3
Mut1640-phnJ-phnE1E2: J23100-B0034-phnJ_mutT16S&R40Y-B0015-J23100-B0034-phnE1-B0034-phnE2_pSB1C3
RNAi system-phnJ-phnO-phnE1E2: J23101-OmpA 5'UTR-phnF 0.97-Hfq binding sequence-J23101- OmpA 5'UTR-phnJ 0.69-Hfq binding sequence- BBa_J61048-J23100-B0034-phnJ-B0015-J23100-phnO-B0015-J23100-B0034-phnE1-B0034-phnE2_pSB1C3).
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