Part:BBa_K2933215

T7 promoter+RBS b+Linker h+His+Linker f+BCll-194+T7 terminator

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+NDM-23），and the biological module can be built into E.coli for protein expression.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 47
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 169
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 47
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 47
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology=

This composite part is made up with three basic parts, the His tag, T7 promoter, RBS and our target protein BcII-194. It encodes a protein which is BcII-194 fused with His tag. The fusion protein is about 28.1 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of BcII-194. It is convenient for us to purify our target protein.

Molecular cloning

First,we obtained BcII by PCR.

Figure 1. The PCR result of BcII.
Then we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.

Figure 2. Left:The plasmid of BcII.Right:The verification results by enzyme digestion.

Expression and purification

Exploration of expression condition:
Take monoclone in the culture plate into LB tube and cultivate in shaking incubator overnight(10-12h) to activate bacteria. Test the OD600 number of bacteria, then pipe 5-10ul into each new 5 mL LB tube. Don’t forget to add antibiotic into tubes and mark them.Cultivate in shaking incubator for 3-4 hours until the OD600 of bacteria range from 0.6 to 0.8.Set the gradient of condition to explore how to express it best.We use 0.2mM IPTG, 16°C/0.2mM IPTG, 37°C/0.4mM IPTG, 16°C/0.4mM IPTG, 37°C/0.6mM IPTG, 16°C/0.6mM IPTG, 37°C/0.8mM IPTG, 16°C/0.8mM IPTG, 37°Cas different conditions.