Difference between revisions of "Part:BBa K3332068"
 
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<partinfo>BBa_K3332068 short</partinfo>
 
<partinfo>BBa_K3332068 short</partinfo>
  
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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.
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Use <partinfo>BBa_K823004</partinfo>-<partinfo>BBa_B0034</partinfo> 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===
 
===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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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Phn system is a gene cluster for organophosphorus transport and degradation in many microorganisms. ''Enterobacterales'' use phnHIJK gene to encode C-P lyase.PhnJ protein is an essential subunit that can crack C-P bond.
 
===Usage===
 
===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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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We ligased the strong promoter、RBS、Terminator(<partinfo>BBa_J23100</partinfo>、<partinfo>BBa_B0034</partinfo>、<partinfo>BBa_B0015</partinfo>) and the parts (phnJ) 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===
 
===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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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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'''1. Agarose Gel Electrophoresis'''
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<span class='h3bb'>Sequence and Features</span>
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 +
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;After receiving the synthesized DNA, digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure 1.
 +
<table><tr><th>[[File:T--XMU-China2020--BBa K3332024.png|thumb|500px|Fig.1 The result of plasmid cut with enzyme ''Eco''R I and ''Pst'' I  . Plasmid: pSB1C3.]]</th><th></table>
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'''2. Enzyme activity'''
 +
 
 +
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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.
 +
 
 +
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The result is shown in Fig.2(Experiment groups in Fig.2
 +
 
 +
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Negative Control: J23100-B0034_pSB1C3
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;phnE1E2: J23100-B0034-phnE1-B0034-phnE2_pSB1C3,
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;phnJ-phnE1E2: J23100-B0034-phnJ-B0015-J23100-B0034-phnE1-B0034-phnE2_pSB1C3,
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Mut21-phnJ-phnE1E2: J23100-B0034-phnJ_mutR21M-B0015- J23100-B0034-phnE1-B0034-phnE2_pSB1C3
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Mut1640-phnJ-phnE1E2: J23100-B0034-phnJ_mutT16S&R40Y-B0015-J23100-B0034-phnE1-B0034-phnE2_pSB1C3
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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 3.png|thumb|500px|Fig.2 Relationship between concentration of glyphosate and culture time.]]</th><th></table>
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 +
 
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===Sequence and Features===
 
<partinfo>BBa_K3332068 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3332068 SequenceAndFeatures</partinfo>
  

Latest revision as of 02:28, 28 October 2020


J23100-RBS-phnJ-terminator

Use BBa_K823004-BBa_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 gene to encode C-P lyase.PhnJ protein is an essential subunit that can crack C-P bond.

Usage

        We ligased the strong promoter、RBS、Terminator(BBa_J23100、BBa_B0034、BBa_B0015) and the parts (phnJ) 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, 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 EcoR I and Pst I . 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 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