Composite

Part:BBa_K3332102

Designed by: Meihao Ou   Group: iGEM20_XMU-China   (2020-10-27)


J23100-RBS-phnJ_mut16&40-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 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

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.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]


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