Difference between revisions of "Part:BBa K2933276"

 
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<partinfo>BBa_K2933276 parameters</partinfo>
 
<partinfo>BBa_K2933276 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, the thrombin restriction site and our target protein SHD. It encodes a protein which is SHD fused with His tag. The fusion protein is about 29.0 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.<br>
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<p style="text-align: center;">
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  [[File:SHD-PCR.png|500px]]<br>
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'''Figure 1.'''  Left: The PCR result of SHD. Right: 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 07:28, 24 September 2019


RBS+Linker h+His+Linker f+SHD+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker h+Sumo+Linker f+SHD) 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 946
  • 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 three basic parts, the HiS tag, the thrombin restriction site and our target protein SHD. It encodes a protein which is SHD fused with His tag. The fusion protein is about 29.0 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.

SHD-PCR.png
Figure 1. Left: The PCR result of SHD. 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.