Difference between revisions of "Part:BBa K2933206"
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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.<br> | 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.<br> | ||
<p style="text-align: center;"> | <p style="text-align: center;"> | ||
| − | [[File:AFM-1- | + | [[File:AFM-1-PCR1.png|500px]]<br> |
'''Figure 1.''' The PCR result of AFM-1.<br> | '''Figure 1.''' The PCR result of AFM-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 12:51, 23 September 2019
T7 promoter+RBS b+Linker h+His+Linker f+AFM-1+T7 terminator
This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+AFM-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
- 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 seven basic parts, the His tag, T7 promoter, RBS and our target proteinAFM-1 NDM-23. It encodes a protein which is AFM-1 fused with His tag. The fusion protein is about 28.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of AFM-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. The PCR result of AFM-1.
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.