Difference between revisions of "Part:BBa K3332026"
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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.
 
===Usage===
 
===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.
+
We ligased the strong promoter (BBa_J23100) and the parts (RBS-phnJ_mut16&40-Terminator, RBS-phnE1-RBS-phnE2) 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===
 
===Characterization===
−
1. Agarose Gel Electrophoresis
+
1. Enzyme activity
−
After receiving the synthesized DNA, restriction digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure 1.
+
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.1(Experiment groups in Fig.1: 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).
 +
<table><tr><th>[[File:T--XMU-China2020--BBa K3332024 3.png|thumb|300px|Fig.1 Relationship between concentration of glyphosate and culture time.]]</th><th></table>
  
  

Revision as of 15:27, 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 strong promoter (BBa_J23100) and the parts (RBS-phnJ_mut16&40-Terminator, RBS-phnE1-RBS-phnE2) 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

1. 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.1(Experiment groups in Fig.1: 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.1 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]