Difference between revisions of "Part:BBa K2933268"
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===Molecular cloning=== | ===Molecular cloning=== | ||
| − | First, we used the vector | + | First, we used the vector pET28a 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-PCR.png|500px]]<br> | [[File:AFM-1-PCR.png|500px]]<br> | ||
Latest revision as of 11:15, 25 September 2019
RBS+Linker h+His+Linker f+AFM-1+T7 terminator
This part consists of RBS,Linker h, protein coding sequence(His+Linker f+AFM-1) and T7 terminator,and the biological module can be build into E.coli for protein expression. This part can be prefaced with promoters of different strengths and types to regulate expression function.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 93
Illegal NheI site found at 940 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composite part is made up with six basic parts(RBS , Linker h, His, Linker f , AFM-1 and T7 terminator ). It encodes a protein which is AFM-1 fused with His tag. The fusion protein is about 28.2 kD. It is convenient for us to purify our target protein.
Molecular cloning
First, we used the vector pET28a 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 AFM-1. Right: The verification results by enzyme digestion.
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.