Difference between revisions of "Part:BBa K3332021"
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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), enabled the ''E. coli'' to express PhnE2 protein.
 
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), enabled the ''E. coli'' to express PhnE2 protein.
 
===Characterization===
 
===Characterization===
−
1. Agarose Gel Electrophoresis
+
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.
 
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.
 
<table><tr><th>[[File:T--XMU-China2020--BBa K3332021 2.png|thumb|500px|Fig.1 The result of plasmid cut with enzyme ''EcoR''I and ''Pst''I. Plasmid: pSB1C3.]]</th><th></table>
 
<table><tr><th>[[File:T--XMU-China2020--BBa K3332021 2.png|thumb|500px|Fig.1 The result of plasmid cut with enzyme ''EcoR''I and ''Pst''I. Plasmid: pSB1C3.]]</th><th></table>
−
2.SDS-PAGE
+
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.
 
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.
 
<table><tr><th>[[File:T--XMU-China2020--BBa K3332067 1.png|thumb|500px|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>
 
<table><tr><th>[[File:T--XMU-China2020--BBa K3332067 1.png|thumb|500px|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>
−
3. HPLC
+
3. HPLC:
 
Verify that phnEE enhances the transport of glyphosate by the chassis bacteria by HPLC. The result is shown in fig.2, phnEE enhance the transport of glyphosate by the chassis bacteria compared to the blank control obviously.  
 
Verify that phnEE enhances the transport of glyphosate by the chassis bacteria by HPLC. The result is shown in fig.2, phnEE enhance the transport of glyphosate by the chassis bacteria compared to the blank control obviously.  
 
<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>
 
<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>

Revision as of 16:50, 27 October 2020


phnE2

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.

Biology

Phn system is a gene cluster for organophosphorus transport and degradation in many microorganisms. Sinorhizobium meliloti 1021 use PhnE1 and PhnE2 to construct the permease protein of ABC transporter, which includes PhnE1, PhnE2, PhnC and PhnD proteins, this transporter can transport 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), enabled the E. coli to express PhnE2 protein.

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.

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.2, 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]