Difference between revisions of "Part:BBa K2933165"
(→Usage and Biology) |
(→Usage and Biology) |
||
| Line 19: | Line 19: | ||
<!-- --> | <!-- --> | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
| − | This composite part is made up with | + | This composite part is made up with six basic parts(Tac promoter，RBS a , Linker g, GST, Linker e and MYX-1). It encodes a protein which is MYX-1 fused with GST tag. The fusion protein is about 53.9 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of MYX-1 and combine the two parts with the cutting site of Prescission Protease. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.<br> |
===Molecular cloning=== | ===Molecular cloning=== | ||
Latest revision as of 13:43, 24 September 2019
Tac promoter+RBS a+Linker g+GST+Linker e+MYX-1
This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+MYX-1),and the biological module can be built into E.coli for protein expression.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 874
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 874
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 874
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 874
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 181
Usage and Biology
This composite part is made up with six basic parts(Tac promoter，RBS a , Linker g, GST, Linker e and MYX-1). It encodes a protein which is MYX-1 fused with GST tag. The fusion protein is about 53.9 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of MYX-1 and combine the two parts with the cutting site of Prescission Protease. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.
Molecular cloning
First, we used the vector pGEX-6p-1 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 MYX-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.
References
[1]Correction for Goldman et al., Evolution of sensory complexity recorded in a myxobacterial genome. Proc Natl Acad Sci U S A. 2006;103(51):19605. doi:10.1073/pnas.0609567103.