Difference between revisions of "Part:BBa K3332021"

 
 
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<partinfo>BBa_K3332021 short</partinfo>
 
<partinfo>BBa_K3332021 short</partinfo>
  
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Subunit of phosphonate ABC transporter, permease protein phnE, from S.meliloti 1021.Use K823004 to construct a new part that can transport glyphosate to cytoplasm.
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Subunit of phosphonate ABC transporter, permease protein phnE, from ''S.meliloti'' 1021.Use <partinfo>K823004</partinfo> to construct a new part which can transport glyphosate to cytoplasm.
  
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<!-- Add more about the biology of this part here
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===Biology===
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===Usage and 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. ''Sinorhizobium meliloti'' 1021 uses PhnE1 and PhnE2 to construct the permease protein of ABC transporter, which includes PhnE1, PhnE2, PhnC and PhnD proteins, which can translate glyphosate to cytoplasm. The phnE2 gene encodes a subunit of permease protein which can transport glyphosate to cytoplasm.
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<span class='h3bb'>Sequence and Features</span>
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===Usage===
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;We ligased the strong promoter and RBS（BBa_J23100+BBa_B0034）and the parts (phnE2) on the expression vector pSB1C3 by standard assembly. Then the ligation mixture was transformed into ''E. coli'' DH5α & ''E. coli'' BL21(DE3), which enabled the ''E. coli'' to express PhnE2 protein.
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===Characterization===
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'''1. Agarose Gel Electrophoresis:'''
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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.
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<table><tr><th>[[File:T--XMU-China--BBa K3332066new.png|thumb|720px|Fig 1. The result of plasmid cut with enzyme ''EcoR''I and ''Pst''I. Plasmid: pSB1C3.]]</th><th></table>
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'''2.SDS-PAGE:'''
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The constructed plasmid was transformed into ''E. coli'' BL21 (DE3). Both of them were electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by silver staining.
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<table><tr><th>[[File:T--XMU-China2020--BBa K3332021 2.png|thumb|720px|Fig 2. SDS-PAGE analysis of protein in lysate of ''E. coli'' BL21 (DE3) cells. Target bands can be seen at about 36.1 kDa and 55.4kDa.]]</th><th></table>
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'''3. HPLC:'''
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Verify that phnEE enhances the transport of glyphosate by the chassis bacteria by HPLC. The result is shown in fig 3., phnEE enhance the transport of glyphosate by the chassis bacteria compared to the blank control obviously.
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<table><tr><th>[[File:T--XMU-China2020--BBa K3332067 2.png|thumb|500px|Fig 3. Relationship between elution peak area of glyphosate and culture time]]</th><th></table>
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===Sequence and Features===
 
<partinfo>BBa_K3332021 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3332021 SequenceAndFeatures</partinfo>
  

Latest revision as of 01:14, 28 October 2020


phnE2

Subunit of phosphonate ABC transporter, permease protein phnE, from S.meliloti 1021.Use BBa_K823004 to construct a new part which can transport glyphosate to cytoplasm.

Biology

        Phn system is a gene cluster for organophosphorus transportation and degradation in many microorganisms. Sinorhizobium meliloti 1021 uses PhnE1 and PhnE2 to construct the permease protein of ABC transporter, which includes PhnE1, PhnE2, PhnC and PhnD proteins, which can translate glyphosate to cytoplasm. The phnE2 gene encodes a subunit of permease protein which can transport glyphosate to cytoplasm.

Usage

        We ligased the strong promoter and RBS（BBa_J23100+BBa_B0034）and the parts (phnE2) on the expression vector pSB1C3 by standard assembly. Then the ligation mixture was transformed into E. coli DH5α & E. coli BL21(DE3), which enabled the E. coli to express PhnE2 protein.

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.SDS-PAGE:


        The constructed plasmid was transformed into E. coli BL21 (DE3). Both of them were electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by silver staining.

Fig 2. SDS-PAGE analysis of protein in lysate of E. coli BL21 (DE3) cells. Target bands can be seen at about 36.1 kDa and 55.4kDa.

3. HPLC:

        Verify that phnEE enhances the transport of glyphosate by the chassis bacteria by HPLC. The result is shown in fig 3., phnEE enhance the transport of glyphosate by the chassis bacteria compared to the blank control obviously.

Fig 3. Relationship between elution peak area 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
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1159
  • 1000
    COMPATIBLE WITH RFC[1000]