Difference between revisions of "Part:BBa K2933258"
Line 18: | Line 18: | ||
<!-- --> | <!-- --> | ||
===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 VIM-66 and T7 terminator. It encodes a protein which is VIM-66 fused with His-Sumo tag. The fusion protein is about 40.3 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. | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | VIM- | + | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
===Molecular cloning=== | ===Molecular cloning=== | ||
− | + | 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. | |
[[File:VIM-66-PCR.jpeg|600px|center|]] | [[File:VIM-66-PCR.jpeg|600px|center|]] | ||
<p style="text-align: center;"> | <p style="text-align: center;"> | ||
'''Figure 1.''' Left: The result of PCR, Right:The result of double enzyme digestion verification.LaneM,Marker, Lane1, the plasmid with VIM-66, Lane2, after double enzyme verification | '''Figure 1.''' Left: The result of PCR, Right:The result of double enzyme digestion verification.LaneM,Marker, Lane1, the plasmid with VIM-66, Lane2, after double enzyme verification | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− |
Revision as of 13:47, 22 September 2019
RBS b+Linker h+His+Linker a+Sumo+Linker b+VIM-66+T7 terminator
This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+VIM-66) 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
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 298
Illegal NheI site found at 75
Illegal NheI site found at 1206 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 298
Illegal BglII site found at 187
Illegal BglII site found at 1135
Illegal BamHI site found at 386 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 298
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 298
- 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 VIM-66 and T7 terminator. It encodes a protein which is VIM-66 fused with His-Sumo tag. The fusion protein is about 40.3 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
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 result of PCR, Right:The result of double enzyme digestion verification.LaneM,Marker, Lane1, the plasmid with VIM-66, Lane2, after double enzyme verification