Difference between revisions of "Part:BBa K2933222"
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First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.We then extracted plasmids and performed double enzyme digestion verification.<br>
 
First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.We then extracted plasmids and performed double enzyme digestion verification.<br>
 
<p style="text-align: center;">
 
<p style="text-align: center;">
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   [[File:Fla.103-PCR.png]]<br>
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   [[File:Fla.103-PCR1.png]]<br>
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'''Figure 1.'''  (a) The PCR result of Fla.103. (b) The verification results by enzyme digestion.The 1 is the original plasmid.The 2 is the
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'''Figure 1.'''  The PCR result of Fla.103. <br>
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results verified by double enzyme digestion.<br>
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</p>
 
</p>
 
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification. <br>
 
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification. <br>

Revision as of 12:27, 23 September 2019


T7 promoter+RBS b+Linker h+His+Linker f+Fla.103+T7 terminator

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+Fla.103），and the biological module can be built into E.coli for protein expression.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 47
    Illegal PstI site found at 280
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 169
    Illegal PstI site found at 280
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 762
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
    Illegal PstI site found at 280
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
    Illegal PstI site found at 280
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology=

This composite part is made up with three basic parts, the His tag, T7 promoter, RBS and our target protein Fla.103. It encodes a protein which is Fla.103 fused with His tag. The fusion protein is about 28.7 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of Fla.103. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.We then extracted plasmids and performed double enzyme digestion verification.

Fla.103-PCR1.png
Figure 1. The PCR result of Fla.103.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.