Difference between revisions of "Part:BBa K2933222"
(→Usage and Biology) |
(→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 ，Linker h ，Linker f，T7 terminatorand our target protein Fla.103. It encodes a protein which is Fla.103 fused with His tag. The fusion protein is about 28.7 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of Fla.103. 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 our target protein Fla.103. It encodes a protein which is Fla.103 fused with His tag.Linker h is from the vector pET-28a, which connects the RBS to His tag sequence.Linker f from vector pGEX-6p-1, contains the thrombin restriction site and T7 tag. The fusion protein is about 28.7 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of Fla.103. 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> | ||
Latest revision as of 14:06, 23 September 2019
T7 promoter+RBS b+Linker h+His+Linker f+Fla.103+T7 terminator
This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+Fla.103），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 280 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 169
Illegal PstI site found at 280 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 762
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 47
Illegal PstI site found at 280 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 47
Illegal PstI site found at 280 - 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 our target protein Fla.103. It encodes a protein which is Fla.103 fused with His tag.Linker h is from the vector pET-28a, which connects the RBS to His tag sequence.Linker f from vector pGEX-6p-1, contains the thrombin restriction site and T7 tag. The fusion protein is about 28.7 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of Fla.103. 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 Fla.103.
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.