Difference between revisions of "Part:BBa K2933225"
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<partinfo>BBa_K2933225 short</partinfo> | <partinfo>BBa_K2933225 short</partinfo> | ||
− | This part consists of RBS a, protein coding sequence(GST+Linker e+ | + | This part consists of RBS a, protein coding sequence(GST+Linker e+ElBlaII), the RBS and the protein coding sequence can be connected by linker g. ElBlaII is a MBLs isolated from B that we temporarily named it as ElBlaII. 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. |
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===Usage and Biology=== | ===Usage and Biology=== | ||
− | This composite part is made up with five basic parts, the RBSa, the linker g and the GST tag, the cutting site of Prescission Protease and our target protein | + | This composite part is made up with five basic parts, the RBSa, the linker g and the GST tag, the cutting site of Prescission Protease and our target protein ElBlaII. It encodes a protein which is ElBlaII fused with GST tag. The fusion protein is about 53.5 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of ElBlaII 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> |
===References=== | ===References=== | ||
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===Molecular cloning=== | ===Molecular cloning=== | ||
− | First, we used the vector | + | First, we used the vector pET28b-sumo 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;"> | ||
− | [[File:TJUSLS China--Elbla2-1-PCR.png]]<br> | + | [[File:TJUSLS China--Elbla2-1-PCR.png|600px]]<br> |
− | '''Figure 1.''' Left: The PCR result of | + | '''Figure 1.''' Left: The PCR result of ElblaII. Right: The verification results by enzyme digestion.<br> |
</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> | ||
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Latest revision as of 13:04, 20 October 2019
RBS a+Linker g+GST+Linker e+ElBlaII
This part consists of RBS a, protein coding sequence(GST+Linker e+ElBlaII), the RBS and the protein coding sequence can be connected by linker g. ElBlaII is a MBLs isolated from B that we temporarily named it as ElBlaII. 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1210
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 113
Usage and Biology
This composite part is made up with five basic parts, the RBSa, the linker g and the GST tag, the cutting site of Prescission Protease and our target protein ElBlaII. It encodes a protein which is ElBlaII fused with GST tag. The fusion protein is about 53.5 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of ElBlaII 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.
References
[1] Girlich D, Poirel L, Nordmann P, Diversity of naturally occurring Ambler class B metallo-β-lactamases in Erythrobacter spp. The Journal of Antimicrobial Chemotherapy [31 Jul 2012, 67(11):2661-2664]
Molecular cloning
First, we used the vector pET28b-sumo 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 ElblaII. 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.