Difference between revisions of "Part:BBa K2933210"
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[[File:CPS-1-PCR.png|500px]]<br> | [[File:CPS-1-PCR.png|500px]]<br> | ||
| − | '''Figure 1.''' Left: The PCR result of CPS-1 | + | '''Figure 1.''' Left: The PCR result of CPS-1. <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 12:15, 23 September 2019
T7 promoter+RBS b+Linker h+His+Linker f+CPS-1+T7 terminator
This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+CPS-1），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
Illegal PstI site found at 741 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 169
Illegal PstI site found at 741 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1023
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 47
Illegal PstI site found at 741 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 47
Illegal PstI site found at 741
Illegal AgeI site found at 642 - 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 CPS-1. It encodes a protein which is CPS-1 fused with His tag. The fusion protein is about 33.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of CPS-1 . 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. Left: The PCR result of CPS-1.
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.