Difference between revisions of "Part:BBa K2933220"

 
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<partinfo>BBa_K2933220 parameters</partinfo>
 
<partinfo>BBa_K2933220 parameters</partinfo>
 
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==Usage and Biology===
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This composite part is made up with three basic parts, the His tag, T7 promoter, RBS and our target protein BlaB-14. It encodes a protein which is BlaB-14 fused with His tag. The fusion protein is about 28.3 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of BlaB-14.  It is convenient for us to purify our target protein.<br>
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===Molecular cloning===
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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>
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<p style="text-align: center;">
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  [[File:BlaB-14-PCR.png|300px]]<br>
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'''Figure 1.'''  (a) The PCR result of BlaB-14. (b) The verification results by 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 14:06, 21 September 2019


T7 promoter+RBS b+Linker h+His+Linker f+BIaB-14+T7 terminator

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+BIaB-14），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
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 169
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 765
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
  • 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 BlaB-14. It encodes a protein which is BlaB-14 fused with His tag. The fusion protein is about 28.3 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of BlaB-14. 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.

BlaB-14-PCR.png
Figure 1. (a) The PCR result of BlaB-14. (b) The verification results by 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.