Difference between revisions of "Part:BBa K2933268"

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<partinfo>BBa_K2933268 short</partinfo>
 
<partinfo>BBa_K2933268 short</partinfo>
  
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This part consists of RBS, protein coding sequence(His+Linker h+Sumo+Linker f+AFM-1) 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.
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This part consists of RBS,Linker h, protein coding sequence(His+Linker f+AFM-1) 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.
  
  
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===Molecular cloning===
 
===Molecular cloning===
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First, we used the vector pET28Ba to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>
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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.<br>
 
<p style="text-align: center;">
 
<p style="text-align: center;">
 
   [[File:AFM-1-PCR.png|500px]]<br>
 
   [[File:AFM-1-PCR.png|500px]]<br>

Latest revision as of 11:15, 25 September 2019


RBS+Linker h+His+Linker f+AFM-1+T7 terminator

This part consists of RBS,Linker h, protein coding sequence(His+Linker f+AFM-1) 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
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 93
    Illegal NheI site found at 940
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with six basic parts(RBS , Linker h, His, Linker f , AFM-1 and T7 terminator ). It encodes a protein which is AFM-1 fused with His tag. The fusion protein is about 28.2 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.

AFM-1-PCR.png
Figure 1. Left: The PCR result of AFM-1. Right: 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.