Difference between revisions of "Part:BBa K2933210"
(→Molecular cloning) |
(→Usage and Biology) |
||
| (2 intermediate revisions by the same user not shown) | |||
| Line 18: | Line 18: | ||
<partinfo>BBa_K2933210 parameters</partinfo> | <partinfo>BBa_K2933210 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
| − | ==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 CPS-1. It encodes a protein which is CPS-1 fused with His tag. The fusion protein is about 33.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of CPS-1 . 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 CPS-1. It encodes a protein which is CPS-1 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 33.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of CPS-1 . 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> | ||
| Line 27: | Line 27: | ||
</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> | ||
| + | ===References=== | ||
| + | [1]Dereje Dadi Gudeta, Valeria Bortolaia,The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance[J]，Antimicrobial Agents and chemotherapy,January 2016<br> | ||
Latest revision as of 14:02, 23 September 2019
T7 promoter+RBS b+Linker h+His+Linker f+CPS-1+T7 terminator
This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+CPS-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
Illegal PstI site found at 741 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 169
Illegal PstI site found at 741 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1023
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 47
Illegal PstI site found at 741 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 47
Illegal PstI site found at 741
Illegal AgeI site found at 642 - 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 CPS-1. It encodes a protein which is CPS-1 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 33.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of CPS-1 . 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 CPS-1.
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.
References
[1]Dereje Dadi Gudeta, Valeria Bortolaia,The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance[J]，Antimicrobial Agents and chemotherapy,January 2016