Part:BBa_K3332101

J23100-RBS-phnJ_mut21-B0015-J23100-RBS-phnE1-RBS-phnE2

        Subunits of phosphonate ABC transporter, permease protein phnE, from S.meliloti 1021. We use K823004 to construct a new part that can transport glyphosate to cytoplasm.

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-Mut21_phnJ-B0015 (BBa_K3332069) and 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.

Characterization

Enzyme activity

        We use Negative Control, experiment groups phnEE and Mut21_phnJ-phnEE to analyze if Mut21_phnJ enhances the degradation of glyphosate by the chassis bacteria by our detection system. Mut21_phnJ gene lowers the degradation rate of E.coli BL21(DE3) about 16.4%, 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).

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

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
  Illegal NheI site found at 1057
  Illegal NheI site found at 2583
  Illegal NheI site found at 2606
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3186
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 3234
  Illegal AgeI site found at 2209
  Illegal AgeI site found at 2905
  Illegal AgeI site found at 3412
  
• 1000
  COMPATIBLE WITH RFC[1000]