Difference between revisions of "Part:BBa K2933266"

 
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<partinfo>BBa_K2933266 parameters</partinfo>
 
<partinfo>BBa_K2933266 parameters</partinfo>
 
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===Usage and Biology===
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This composite part is made up with three basic parts(RBS，Linker h and T7 terminator)and a composite part(His+Linker f+SPG-1). It encodes a protein which is SPG-1 fused with His tag. The fusion protein is about 30.2 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:SPG-PCR.png]]<br>
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'''Figure 1.'''  a: The PCR result of SPG. 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>
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===References===
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The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance. Gudeta DD, Bortolaia V, Amos G, Wellington EM, Brandt KK, Poirel L, Nielsen JB, Westh H, Guardabassi L. Antimicrob Agents Chemother. 2015 Oct 19;60(1):151-60.

Revision as of 14:50, 23 September 2019


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

This part consists of RBS, protein coding sequence(His+Linker h+Sumo+Linker f+SPG-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 994
  • 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(RBS，Linker h and T7 terminator)and a composite part(His+Linker f+SPG-1). It encodes a protein which is SPG-1 fused with His tag. The fusion protein is about 30.2 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.

SPG-PCR.png
Figure 1. a: The PCR result of SPG. 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.

References

The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance. Gudeta DD, Bortolaia V, Amos G, Wellington EM, Brandt KK, Poirel L, Nielsen JB, Westh H, Guardabassi L. Antimicrob Agents Chemother. 2015 Oct 19;60(1):151-60.