Difference between revisions of "Part:BBa K2933279"

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<partinfo>BBa_K2933279 short</partinfo>
 
<partinfo>BBa_K2933279 short</partinfo>
  
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This part consists of RBS, protein coding sequence(His+Linker h+Sumo+Linker f+VIM-66) 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+VIM-66) 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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===Usage and Biology===
 
===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+VIM-66). It encodes a protein which is VIM-66 fused with His tag. The fusion protein is about 28.3 kD. It is convenient for us to purify our target protein.
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This composite part is made up with six basic parts(RBS , Linker h, His, Linker f ,  TMB-2 and T7 terminator ). It encodes a protein which is VIM-66 fused with His tag. The fusion protein is about 28.3 kD. It is convenient for us to purify our target protein.
  
 
===Molecular cloning===
 
===Molecular cloning===

Latest revision as of 14:22, 24 September 2019


RBS+Linker h+His+Linker f+VIM-66+T7 terminator

This part consists of RBS, Linker h, protein coding sequence(His+Linker f+VIM-66) 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 937
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 866
  • 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 , TMB-2 and T7 terminator ). It encodes a protein which is VIM-66 fused with His tag. The fusion protein is about 28.3 kD. It is convenient for us to purify our target protein.

Molecular cloning

We insert VIM-66 gene into the standard vector then transfer it into E.coli.

VIM-66-PCR.jpeg

Figure 1. Left: The result of PCR, Right:The result of double enzyme digestion verification.LaneM,Marker, Lane1, the plasmid with VIM-66, Lane2, after double enzyme verification

References

1. Yoshihiro Yamaguchi. Wanchun Jin. Kazuyo Matsunaga. Crystallographic investigation of the inhibition mode of a VIM-2 metallo-beta-lactamase from Pseudomonas aeruginosa by a mercaptocarboxylate inhibitor. J. Med. Chem.200750266647-6653

2. Biochemical, Mechanistic, and Spectroscopic Characterizationof Metallo-β-lactamase VIM‑2[J]. Biochemistry, 2014, 53(46):7321-7331.

3. Christopeit T , Carlsen T J , Helland R , et al. Discovery of novel inhibitor scaffolds against the metallo-β-lactamase VIM-2 by SPR based fragment screening[J]. Journal of Medicinal Chemistry, 2015:151017114758002.

4. Christopeit T , Yang K W , Yang S K , et al. The structure of the metallo-β-lactamase VIM-2 in complex with a triazolylthioacetamide inhibitor[J]. 2016.