Difference between revisions of "Part:BBa K3332025"

 
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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.
 
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.
  
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===Biology===
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===Usage and Biology===
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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 21th arginine was mutated to methionine.
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===Usage===
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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.
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===Characterization===
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1. Agarose Gel Electrophoresis
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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.
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Revision as of 03:33, 25 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 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.

Characterization

1. Agarose Gel Electrophoresis 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.


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]