Difference between revisions of "Part:BBa K2933260"
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===Usage and Biology=== | ===Usage and Biology=== | ||
This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein ARL-1 and T7 terminator. It encodes a protein which is ARL-1 fused with His-Sumo tag. The fusion protein is about 43.0 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein. | This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein ARL-1 and T7 terminator. It encodes a protein which is ARL-1 fused with His-Sumo tag. The fusion protein is about 43.0 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein. | ||
| − | Molecular cloning | + | ===Molecular cloning=== |
First, we used the vector pET-28bs to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely. | First, we used the vector pET-28bs to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely. | ||
<p style="text-align: center;"> | <p style="text-align: center;"> | ||
Latest revision as of 13:54, 22 September 2019
RBS b+Linker h+His+Linker a+Sumo+Linker b+ARL-1+T7 terminator
This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+ARL-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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 298
Illegal PstI site found at 824
Illegal PstI site found at 938
Illegal PstI site found at 989 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 298
Illegal NheI site found at 75
Illegal NheI site found at 1254
Illegal PstI site found at 824
Illegal PstI site found at 938
Illegal PstI site found at 989 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 298
Illegal BglII site found at 187
Illegal BamHI site found at 386 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 298
Illegal PstI site found at 824
Illegal PstI site found at 938
Illegal PstI site found at 989 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 298
Illegal PstI site found at 824
Illegal PstI site found at 938
Illegal PstI site found at 989 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein ARL-1 and T7 terminator. It encodes a protein which is ARL-1 fused with His-Sumo tag. The fusion protein is about 43.0 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.
Molecular cloning
First, we used the vector pET-28bs 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 ARL-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.