Difference between revisions of "Part:BBa K2933284"

 
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<partinfo>BBa_K2933284 parameters</partinfo>
 
<partinfo>BBa_K2933284 parameters</partinfo>
 
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===Usage and Biology===
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This composite part is made up with four basic parts(RBS,Linker h , T7 terminator and His+Linker f+Fla.103). It encodes a protein which is Fla.103 fused with His tag. The fusion protein is about 28.7 kD. It is convenient for us to purify our target protein.
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===Molecular cloning===
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First, we used the vector pET28a 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>
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<p style="text-align: center;">
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  [[File:Fla.103-PCR.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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results verified by double enzyme digestion.<br>
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</p>
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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 07:55, 24 September 2019


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

This part consists of RBS, protein coding sequence(His+Linker h+Sumo+Linker f+Fla.103) and T7 terminator,and the biological module can be build into E.coli for protein expression. This part can be prefaced with promoters of different strengths and types to regulate expression function.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 204
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 93
    Illegal NheI site found at 889
    Illegal PstI site found at 204
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 686
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 204
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 204
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with four basic parts(RBS,Linker h , T7 terminator and His+Linker f+Fla.103). It encodes a protein which is Fla.103 fused with His tag. The fusion protein is about 28.7 kD. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pET28a 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-PCR.png
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 results verified by double enzyme digestion.

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