Composite

Part:BBa_K3332100

Designed by: Shiyi Zhai   Group: iGEM20_XMU-China   (2020-10-18)
Revision as of 22:44, 27 October 2020 by Micoria (Talk | contribs)


RNAi sequence of phnF-RNAi sequence of phnJ-J23100-RBS-phnJ-T-J23100-RBS-phnO-T-J23100-RBS-phnE1E2

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Biology

        Phn system is a gene cluster for organophosphorus transport and degradation in many microorganisms. Enterobacterales uses phnHIJK genes to encode C-P lyase. PhnJ protein is an essential subunit that can crack C-P bond.

Usage

        We ligased the RNAi system (J23101-OmpA 5'UTR-phnF 0.97-Hfq binding sequence-J23101- OmpA 5'UTR-phnJ 0.69-Hfq binding sequence- BBa_J610480) and the phn system (RBS-phnJ-Terminator-RBS-phnO-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), which enables 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, digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure 1.

Fig.1 The result of plasmid cut with enzyme EcoRI and PstI. Plasmid: pSB1C3.

2. Enzyme activity

        We use Negative Control, experiment groups phnEE and phnEEJ to verify that phnJ can enhance the degradation of glyphosate by the chassis bacteria by our detection system. The phnJ-phnE1E2 gene cluster enhances the degration of glyphosate by the chassis bacteria compared to the blank control, but it has no difference with the phnE1E2 group, indicating that exogenous phnJ can’t bind to the endogenous PhnHIK well.

        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 438
    Illegal NheI site found at 461
    Illegal NheI site found at 869
    Illegal NheI site found at 892
    Illegal NheI site found at 1921
    Illegal NotI site found at 1036
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2134
    Illegal XhoI site found at 281
    Illegal XhoI site found at 712
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1520
    Illegal NgoMIV site found at 1542
    Illegal AgeI site found at 4812
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1532


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