Difference between revisions of "Part:BBa K2933220"
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[[File:BlaB-14-PCR1.png|300px]]<br> | [[File:BlaB-14-PCR1.png|300px]]<br> | ||
| − | '''Figure 1.''' | + | '''Figure 1.''' The PCR result of BlaB-14.<br> |
</p> | </p> | ||
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification. <br> | After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification. <br> | ||
Revision as of 12:25, 23 September 2019
T7 promoter+RBS b+Linker h+His+Linker f+BIaB-14+T7 terminator
This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+BIaB-14），and the biological module can be built into E.coli for protein expression
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 47
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 169
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 765
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 47
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 47
- 1000COMPATIBLE 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 BlaB-14. It encodes a protein which is BlaB-14 fused with His tag. The fusion protein is about 28.3 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of BlaB-14. It is convenient for us to purify our target protein.
Molecular cloning
First, 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.We then extracted plasmids and performed double enzyme digestion verification.
Figure 1. The PCR result of BlaB-14.
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.