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Revision as of 19:05, 27 October 2020 by Master123 (Talk | contribs)


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None

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 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), 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, 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.

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Status: 500 Content-type: text/html

Software error:

Global symbol "$range" requires explicit package name at /websites/parts.igem.org/cgi/lib/Range.pm line 252.
Unmatched right curly bracket at /websites/parts.igem.org/cgi/lib/Range.pm line 253, at end of line
syntax error at /websites/parts.igem.org/cgi/lib/Range.pm line 253, near "}"
Can't use global @_ in "my" at /websites/parts.igem.org/cgi/lib/Range.pm line 263, near "= @_"
syntax error at /websites/parts.igem.org/cgi/lib/Range.pm line 284, near "}"
Can't use global @_ in "my" at /websites/parts.igem.org/cgi/lib/Range.pm line 288, near "= @_"
Global symbol "$course" requires explicit package name at /websites/parts.igem.org/cgi/lib/Range.pm line 290.
syntax error at /websites/parts.igem.org/cgi/lib/Range.pm line 316, near "}"
Compilation failed in require at /websites/parts.igem.org/cgi/lib/Part.pm line 16.
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