Difference between revisions of "Part:BBa K3332070"
 
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Use <partinfo>BBa_K823004</partinfo>-<partinfo>BBa_B0034</partinfo> to express the mutant of C-P lyase, which can degrade the glyphosate, from ''Enterobacterales''. This subunit is essential to the activity of the whole enzyme, improving the capability of chassis bacteria to degrade glyphosate.
 
Use <partinfo>BBa_K823004</partinfo>-<partinfo>BBa_B0034</partinfo> to express the mutant of C-P lyase, which can degrade the glyphosate, from ''Enterobacterales''. This subunit is essential to the activity of the whole enzyme, improving the capability of chassis bacteria to degrade glyphosate.
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===Biology===
 
===Biology===
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Phn system is a gene cluster for organophosphorus transportation 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. The 16th threonine was mutated to serine，and the 40th arginine was mutated tyrosine.
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Phn system is a gene cluster for organophosphorus transportation 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. The 16th threonine was mutated to serine，and the 40th arginine was mutated tyrosine.
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===Usage===
 
===Usage===
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We ligased the J23100-B0034-phnJ_mut16&40-B0015 (<partinfo>BBa_K3332070</partinfo>) and the part J23100-B0034-phnE<sub>1</sub>-B0034-phnE<sub>2</sub>(<partinfo>BBa_K3332067</partinfo>) 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 improves the binding ability with the endogenous PhnHIK.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We ligased the J23100-B0034-phnJ_mut16&40-B0015 (<partinfo>BBa_K3332026</partinfo>) and the part J23100-B0034-phnE<sub>1</sub>-B0034-phnE<sub>2</sub>(<partinfo>BBa_K3332067</partinfo>) 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 improves the binding ability with the endogenous PhnHIK.
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===Characterization===
 
===Characterization===
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'''Enzyme activity'''
 
'''Enzyme activity'''
  
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We use Negative Control, experiment groups phnEE and Mut16&40_phnJ-phnEE to verify that Mut16&40_phnJ can enhance the degradation of glyphosate by the chassis bacteria by our detection system. The Mut16&40_phnJ-phnEE 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.  
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We use Negative Control, experiment groups phnEE and Mut16&40_phnJ-phnEE to analyze if Mut_16&40-phnJ enhances the degradation of glyphosate by the chassis bacteria by our detection system. Mut16&40_phnJ gene lowers the degradation rate of ''E.coli'' BL21(DE3) about 5.4%, indicate the mutant has higher binding ability to PhnHIK but lower degradation ability compare to endogenous PhnJ.  
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The result is shown in Fig.2(Experiment groups in Fig.2
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The result is shown in Fig.1(Experiment groups in Fig.1
  
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Negative Control: J23100-B0034_pSB1C3
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Negative Control: J23100-B0034_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).
 
&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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<table><tr><th>[[File:T--XMU-China2020--BBa K3332024 3.png|thumb|500px|Fig.1 Relationship between concentration of glyphosate and culture time.]]</th><th></table>
  
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===Sequence and Features===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3332070 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3332070 SequenceAndFeatures</partinfo>
  

Latest revision as of 02:34, 28 October 2020


J23100-RBS-phnJ_mut40&16-terminator

Use BBa_K823004-BBa_B0034 to express the mutant of C-P lyase, which can degrade the glyphosate, from Enterobacterales. This subunit is essential to the activity of the whole enzyme, improving the capability of chassis bacteria to degrade glyphosate.

Biology

        Phn system is a gene cluster for organophosphorus transportation 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. The 16th threonine was mutated to serine，and the 40th arginine was mutated tyrosine.

Usage

        We ligased the J23100-B0034-phnJ_mut16&40-B0015 (BBa_K3332070) and the part J23100-B0034-phnE1-B0034-phnE2(BBa_K3332067) 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 improves the binding ability with the endogenous PhnHIK.

Characterization

Enzyme activity

        We use Negative Control, experiment groups phnEE and Mut16&40_phnJ-phnEE to analyze if Mut_16&40-phnJ enhances the degradation of glyphosate by the chassis bacteria by our detection system. Mut16&40_phnJ gene lowers the degradation rate of E.coli BL21(DE3) about 5.4%, indicate the mutant has higher binding ability to PhnHIK but lower degradation ability compare to endogenous PhnJ.          The result is shown in Fig.1(Experiment groups in Fig.1

       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.1 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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 610
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 658
    Illegal AgeI site found at 329
    Illegal AgeI site found at 836
  • 1000
    COMPATIBLE WITH RFC[1000]