Difference between revisions of "Part:BBa K3332026"
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===Biology=== | ===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 16th threonine was mutated to serine，and the 40th arginine was mutated tyrosine. | + | 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 16th threonine was mutated to serine，and the 40th arginine was mutated tyrosine. |
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'''Enzyme activity''' | '''Enzyme activity''' | ||
| − | We use Negative Control, experiment groups phnEE and Mut16&40_phnJ-phnEE to analyze if Mut_16&40-phnJ enhances the degradation of glyphosate by the chassis bacteria by our detection system. Mut16&40_phnJ gene lowers the degradation rate of ''E.coli'' BL21(DE3) about 5.4%, indicate the mutant has higher binding ability to PhnHIK but lower degradation ability compare to endogenous PhnJ. | + | We use Negative Control, experiment groups phnEE and Mut16&40_phnJ-phnEE to analyze if Mut_16&40-phnJ enhances the degradation of glyphosate by the chassis bacteria by our detection system. Mut16&40_phnJ gene lowers the degradation rate of ''E.coli'' BL21(DE3) about 5.4%, indicate the mutant has higher binding ability to PhnHIK but lower degradation ability compare to endogenous PhnJ. |
The result is shown in Fig.2. | The result is shown in Fig.2. | ||
Revision as of 22:34, 27 October 2020
phnJ_mut40&16
The mutant of C-P lyase, which can degrade the glyphosate, from Enterobacterales. This subunit is essential to the activity of the whole enzyme.Use K823004 to construct a new part that can 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 16th threonine was mutated to serine，and the 40th arginine was mutated tyrosine.
Usage
We ligased the J23100-B0034-phnJ_mut16&40-B0015 (BBa_K3332026) 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), enabled 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 Mut16&40_phnJ-phnEE to analyze if Mut_16&40-phnJ enhances the degradation of glyphosate by the chassis bacteria by our detection system. Mut16&40_phnJ gene lowers the degradation rate of E.coli BL21(DE3) about 5.4%, 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).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 549
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 597
Illegal AgeI site found at 268
Illegal AgeI site found at 775 - 1000COMPATIBLE WITH RFC[1000]