Difference between revisions of "Part:BBa K3332068"

 
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Use BBa_K823004-B0034 to express the subunit of C-P lyase, which can degrade the glyphosate, from Enterobacterales. This subunit is essential to the activity of the whole enzyme.Improve the capability of chassis bacteria to degrade glyphosate.
 
Use BBa_K823004-B0034 to express the subunit of C-P lyase, which can degrade the glyphosate, from Enterobacterales. This subunit is essential to the activity of the whole enzyme.Improve the capability of chassis bacteria to degrade glyphosate.
  
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===Biology===
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===Usage and Biology===
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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.
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===Usage===
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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.
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===Characterization===
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1. Agarose Gel Electrophoresis
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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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Revision as of 07:20, 25 October 2020


J23100-RBS-phnJ-terminator

Use BBa_K823004-B0034 to express the subunit of C-P lyase, which can degrade the glyphosate, from Enterobacterales. This subunit is essential to the activity of the whole enzyme.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

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 NotI site found at 174
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 658
    Illegal NgoMIV site found at 680
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 670