Difference between revisions of "Part:BBa K3332025"

Revision as of 19:03, 27 October 2020

phnJ_mut21

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 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), 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 Mut21_phnJ-phnEE to verify that Mut21_phnJ can enhance the degradation of glyphosate by the chassis bacteria by our detection system. The Mut21_phnJ-phnEE gene cluster enhances the degration of glyphosate by the chassis bacteria compared to the blank control, but it has no difference with the phnE1E2 group, indicating that exogenous phnJ can’t bind to the endogenous PhnHIK well.

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).

Fig.2 Relationship between concentration of glyphosate and culture time.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 549
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 597
Illegal AgeI site found at 268
Illegal AgeI site found at 775
1000
COMPATIBLE WITH RFC[1000]

@@ Line 8: / Line 8: @@
 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 strong promoter (BBa_J23100) and the parts (RBS-phnJ_mut21-Terminator) 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.
+&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We ligased the J23100-B0034-phnJ_mut21-B0015 (<partinfo>BBa_K3332025</partinfo>) and the part J23100-B0034-phnE<sub>1</sub>-B0034-phnE<sub>2</sub>(<partinfo>BBa_K3332067</partinfo>) 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'''