Difference between revisions of "Part:BBa K2933171"
Line 18: | Line 18: | ||
<partinfo>BBa_K2933171 parameters</partinfo> | <partinfo>BBa_K2933171 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
+ | ===Usage and Biology=== | ||
+ | <br> | ||
+ | |||
+ | ===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.<br> | ||
+ | <p style="text-align: center;"> | ||
+ | [[File:JOHN-1-6p.jpg|600px]]<br> | ||
+ | '''Figure 1.''' Left: The PCR result of JOHN-1. Right: The verification results by enzyme digestion.<br> | ||
+ | </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> |
Revision as of 12:39, 21 September 2019
Tac promoter+RBS a+Linker g+GST+Linker e+JOHN-1
This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+JOHN-1),and the biological module can be built into E.coli for protein expression.
Sequence and Features
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 870
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 870
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 870
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 870
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 181
Illegal SapI.rc site found at 1394
Usage and Biology
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 JOHN-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.