Difference between revisions of "Part:BBa K2933262"
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===Usage and Biology===
 
===Usage and Biology===
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Bacteria with IMP-type enzymes have spread through out the world, and the IMP group now has more than 50 variants
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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 IMP-71 and T7 terminator. It encodes a protein which is IMP-71 fused with His-Sumo tag. The fusion protein is about 39.2 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.
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These enzymes possess a broad substrate specificity and a high affinity for cephalosporins and carbapenems but a low activity toward temocillin<br>
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==References==
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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 pET-28bs 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:IMP-71-PCR.png|400px]]<br>
 
   [[File:IMP-71-PCR.png|400px]]<br>

Revision as of 14:01, 22 September 2019


RBS b+Linker h+His+Linker a+Sumo+Linker b+IMP-71+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+IMP-71) 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
    Illegal PstI site found at 1121
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 298
    Illegal NheI site found at 75
    Illegal NheI site found at 1146
    Illegal PstI site found at 1121
  • 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
    Illegal PstI site found at 1121
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 298
    Illegal PstI site found at 1121
  • 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 IMP-71 and T7 terminator. It encodes a protein which is IMP-71 fused with His-Sumo tag. The fusion protein is about 39.2 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.

IMP-71-PCR.png
Figure 1. Left: The PCR result of IMP-71. 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.