Part:BBa_K2933201

T7 promoter+RBS b+linker h+His+Linker a+Sumo+Linker b+Fla.103+T7 terminator

The part consists of T7 promoter,RBS and protein coding(His+Linker a+Sumo+Linker b+Fla.103)and the biological module can be built into E.coil for protein expression.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 390
  Illegal XbaI site found at 47
  Illegal PstI site found at 565
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 390
  Illegal NheI site found at 167
  Illegal NheI site found at 1250
  Illegal PstI site found at 565
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 390
  Illegal BglII site found at 279
  Illegal BglII site found at 1047
  Illegal BamHI site found at 478
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 390
  Illegal XbaI site found at 47
  Illegal PstI site found at 565
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 390
  Illegal XbaI site found at 47
  Illegal PstI site found at 565
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

This composite part is made up with nine basic parts, T7 promoter, the RBS b， the linker h， His tag，the linker a, Sumo tag, linker b, the gene of Fla.103 and T7 terminator.It encodes a protein which is Fla.103 fused with His and Sumo tag. The fusion protein is about 40.7 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of Fla.103 and combine Sumo tag to increased protein solubility. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pET28B-Sumo to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.We then extracted plasmids and performed double enzyme digestion verification.

Figure 1. (a) The PCR result of Fla.103. (b) The verification results by enzyme digestion.The 1 is the original plasmid.The 2 is the results verified by double 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.