Difference between revisions of "Part:BBa K3332024"
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1. Agarose Gel Electrophoresis
 
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.
 
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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<table><tr><th>[[File:T--XMU-China2020--BBa K3332024.png|thumb|300px|Fig.1 The result of plasmid cut with enzyme ''EcoR''I and ''Pst''I. Plasmid: pSB1C3]]</th><th></table>
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<table><tr><th>[[File:T--XMU-China2020--BBa K3332024.png|thumb|300px|Fig.1 The result of plasmid cut with enzyme ''EcoR''I and ''Pst''I. Plasmid: pSB1C3.]]</th><th></table>
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2. Enzyme activity
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We use Negative Control, experiment groups phnEE and phnEEJ to verify that phnJ enhances the degradation of glyphosate by the chassis bacteria by our detection system. phnJ-phnE1E2 gene cluster enhance the degration of glyphosate by the chassis bacteria compared to the blank control, but it has no different compare to the phnE1E2 group, indicate 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).
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<table><tr><th>[[File:T--XMU-China2020--BBa K3332024 2.png|thumb|300px|Fig.2 Relationship between concentration of glyphosate and culture time.]]</th><th></table>
  
 
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Revision as of 10:34, 27 October 2020


phnJ

Subunit Subunit 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.

Usage

We ligased the strong promoter (BBa_J23100) and the parts (RBS-phnJ-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.

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.

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 enhances the degradation of glyphosate by the chassis bacteria by our detection system. phnJ-phnE1E2 gene cluster enhance the degration of glyphosate by the chassis bacteria compared to the blank control, but it has no different compare to the phnE1E2 group, indicate 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 NotI site found at 113
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 597
    Illegal NgoMIV site found at 619
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 609