Difference between revisions of "Part:BBa K2933208"
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[[File:T--TJUSLS China--IND-10 PCR.png|250px]] | [[File:T--TJUSLS China--IND-10 PCR.png|250px]] | ||
| − | '''Figure 1.''' | + | '''Figure 1.''' The PCR result of IND-10. <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 12:23, 23 September 2019
T7 promoter+RBS b+Linker h+His+Linker f+IND-10+T7 terminator
This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+IND-10），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
- 21COMPATIBLE WITH RFC[21]
- 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 IND-10. It encodes a protein which is IND-10 fused with His tag. The fusion protein is about 26.9 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of IND-10 . 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.
Figure 1. The PCR result of IND-10.
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.