Difference between revisions of "Part:BBa K2933200"
 
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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.<br>
 
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.<br>
 
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   [[File:IMP-71-PCR.png|400px]]<br>
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   [[File:IMP PCR.png|200px]]<br>
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'''Figure 1.'''  Left: The PCR result of IMP-71. Right: The verification results by enzyme digestion.<br>
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'''Figure 1.'''  The PCR result of IMP-71.<br>
 
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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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===References===
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[1]Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa.Furuyama T, Ishii Y, Ohya N, Tateda K, Hanson ND, Shimizu-Ibuka A.Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Dec;69(Pt 12):1397-400.

Latest revision as of 11:50, 24 September 2019


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

The part consists of T7 promoter,RBS and protein coding(His+Linker a+Sumo+Linker b+IMP-71)and the biological module can be built into E.coil for protein expression.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 390
    Illegal XbaI site found at 47
    Illegal PstI site found at 1213
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 390
    Illegal NheI site found at 167
    Illegal NheI site found at 1238
    Illegal PstI site found at 1213
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 390
    Illegal BglII site found at 279
    Illegal BamHI site found at 478
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 390
    Illegal XbaI site found at 47
    Illegal PstI site found at 1213
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 390
    Illegal XbaI site found at 47
    Illegal PstI site found at 1213
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with nine basic parts, T7 promoter, the RBS b， the linker h， His tag，the linker a, Sumo tag, linker b, the gene of IMP-71 and T7 terminator.It encodes a protein which is IMP-71 fused with His and Sumo tag. The fusion protein is about 39.2 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of IMP-71 and combine Sumo tag to increased protein solubility. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.

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.

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

References

[1]Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa.Furuyama T, Ishii Y, Ohya N, Tateda K, Hanson ND, Shimizu-Ibuka A.Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Dec;69(Pt 12):1397-400.