Part:BBa_K2933278

RBS+Linker h+His+Linker f+TMB-2+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker h+Sumo+Linker f+TMB-2) 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 874
  
• 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 TMB-2. It encodes a protein which is TMB-2 fused with His tag. The fusion protein is about 27.2 kD. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pET-28a to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

Figure 1. The PCR result of TMB-2.

References

[1]Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding.Skagseth S, Christopeit T, Akhter S, Bayer A, Samuelsen Ø, Leiros HS.Antimicrob Agents Chemother. 2017 Jul 25;61(8).