Part:BBa_K2933279
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 93
Illegal NheI site found at 937 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 866
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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.| None |