Difference between revisions of "Part:BBa K2933225"
 
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===Molecular cloning===
 
===Molecular cloning===
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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>
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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;">
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   [[File:TJUSLS China--Elbla2-1-PCR.png]]<br>
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   [[File:TJUSLS China--Elbla2-1-PCR.png|600px]]<br>
 
'''Figure 1.'''  Left: The PCR result of ElblaII. Right: The verification results by enzyme digestion.<br>
 
'''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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===Expression and purification===
 
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'''Pre-expression:'''<br>
 
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The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br>
 
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===References===
 
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[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]
 

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1210
  • 1000
    INCOMPATIBLE 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.

TJUSLS China--Elbla2-1-PCR.png
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.