Difference between revisions of "Part:BBa K2933208"
(→Molecular cloning) |
(→Usage and Biology) |
||
| Line 19: | Line 19: | ||
<!-- --> | <!-- --> | ||
==Usage and Biology== | ==Usage and Biology== | ||
| − | This composite part is made up with seven 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.<br> | + | This composite part is made up with seven basic parts, the His tag, T7 promoter, RBS ，Linker h ，Linker f，T7 terminatorand 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.<br> |
===Molecular cloning=== | ===Molecular cloning=== | ||
First, we used the vector pGEX-28a 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-28a to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br> | ||
Revision as of 13:49, 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 seven basic parts, the His tag, T7 promoter, RBS ，Linker h ，Linker f，T7 terminatorand 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-28a 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.