Difference between revisions of "Part:BBa K2933283"
 
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<partinfo>BBa_K2933283 short</partinfo>
 
<partinfo>BBa_K2933283 short</partinfo>
  
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This part consists of RBS, protein coding sequence(His+Linker h+Sumo+Linker f+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.
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This part consists of RBS, Linker h, protein coding sequence(His+Linker f+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.
  
  
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===Usage and Biology===
 
===Usage and Biology===
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This composite part is made up with three basic parts, the HiS tag, the thrombin restriction site and our target protein IMP-71. It encodes a protein which is IMP-71 fused with His tag. The fusion protein is about 27.2 kD. It is convenient for us to purify our target protein.
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This composite part is made up with six basic parts(RBS , Linker h, His, Linker f ,  IMP-71 and T7 terminator ). It encodes a protein which is IMP-71 fused with His tag. The fusion protein is about 27.2 kD. It is convenient for us to purify our target protein.
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===Molecular cloning===
 
===Molecular cloning===
 
First, we used the vector pET28a 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 pET28a to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>

Latest revision as of 14:29, 24 September 2019


RBS+Linker h+His+Linker f+IMP-71+T7 terminator

This part consists of RBS, Linker h, protein coding sequence(His+Linker f+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 PstI site found at 852
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 93
    Illegal NheI site found at 877
    Illegal PstI site found at 852
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 852
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 852
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with six basic parts(RBS , Linker h, His, Linker f , IMP-71 and T7 terminator ). It encodes a protein which is IMP-71 fused with His tag. The fusion protein is about 27.2 kD. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pET28a 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.