Difference between revisions of "Part:BBa K2933246"
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<partinfo>BBa_K2933246 short</partinfo>
 
<partinfo>BBa_K2933246 short</partinfo>
  
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This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+ElBla2-1) 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.
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This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+ElBlaII) 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.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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===Usage and Biology===
 
===Usage and Biology===
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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 ElBla2-1 and T7 terminator. It encodes a protein which is ElBla2-1 fused with His-Sumo tag. The fusion protein is about 39.5 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.<br>
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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 ElBlaII and T7 terminator. It encodes a protein which is ElBlaII fused with His-Sumo tag. The fusion protein is about 39.5 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.<br>
  
 
===Molecular cloning===
 
===Molecular cloning===
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<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]]<br>
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'''Figure 1.'''  Left: The PCR result of Elbla2-1. Right: The verification results by enzyme digestion.<br>
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'''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>
 
===References===
 
===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]
 
[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]

Revision as of 14:51, 28 September 2019


RBS b+Linker h+His+Linker a+Sumo+Linker b+ElBlaII+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+ElBlaII) 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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 298
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 298
    Illegal NheI site found at 75
    Illegal NheI site found at 1191
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 298
    Illegal BglII site found at 187
    Illegal BamHI site found at 386
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 298
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 298
    Illegal AgeI site found at 881
  • 1000
    COMPATIBLE 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 ElBlaII and T7 terminator. It encodes a protein which is ElBlaII fused with His-Sumo tag. The fusion protein is about 39.5 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

First, 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.

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.

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]