Difference between revisions of "Part:BBa K2933207"
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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:T--TJUSLS China--MYX-1 | + | [[File:T--TJUSLS China--MYX-1 PCRmeiqie1.png|300px]] [[File:T--TJUSLS China--MYX-1 meiqie.png|300px]]<br> |
| − | '''Figure 1.''' Left: The PCR result of MYX-1 | + | '''Figure 1.''' Left: The PCR result of MYX-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 12:12, 23 September 2019
T7 promoter+RBS b+Linker h+His+Linker f+MYX-1+T7 terminator
This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+Myxo），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 284 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 169
Illegal PstI site found at 284 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 47
Illegal PstI site found at 284 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 47
Illegal PstI site found at 284 - 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 Myxo. It encodes a protein which is Myxo fused with His tag. The fusion protein is about 27.9 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of Myxo . 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.
300px 
Figure 1. Left: The PCR result of MYX-1.
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.