Part:BBa_K3332070

J23100-RBS-phnJ_mut40&16-terminator

Use BBa_K823004-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 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_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
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 610
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 658
  Illegal AgeI site found at 329
  Illegal AgeI site found at 836
  
• 1000
  COMPATIBLE WITH RFC[1000]