Difference between revisions of "Part:BBa K2933221"
 
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<partinfo>BBa_K2933221 parameters</partinfo>
 
<partinfo>BBa_K2933221 parameters</partinfo>
 
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==Usage and Biology===
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==Usage and Biology==
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This composite part is made up with three basic parts, the His tag, T7 promoter, RBS 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. In order to gain the highly purified target protein, we add His tag in N-terminal of IMP-71 .  It is convenient for us to purify our target protein.<br>
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This composite part is made up with seven basic parts，the His tag, T7 promoter, RBS ，Linker h ，Linker f，T7 terminatorand and our target protein IMP-71. It encodes a protein which is IMP-71 fused with His tag.Linker h is from the vector pET-28a, which connects the RBS to His tag sequence.Linker f from vector pGEX-6p-1, contains the thrombin restriction site and T7 tag. The fusion protein is about 27.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of IMP-71 .  It is convenient for us to purify our target protein.<br>
 
===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 pGEX-28a to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>
 
<p style="text-align: center;">
 
<p style="text-align: center;">
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   [[File:IMP-71-PCR.png|400px]]<br>
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   [[File:IMP-71-PCR1.png|400px]]<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>
 
</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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===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 11:40, 24 September 2019


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

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+IMP-71），and the biological module can be built into E.coli for protein expression

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 47
    Illegal PstI site found at 928
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 169
    Illegal PstI site found at 928
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
    Illegal PstI site found at 928
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
    Illegal PstI site found at 928
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with seven basic parts，the His tag, T7 promoter, RBS ，Linker h ，Linker f，T7 terminatorand and our target protein IMP-71. It encodes a protein which is IMP-71 fused with His tag.Linker h is from the vector pET-28a, which connects the RBS to His tag sequence.Linker f from vector pGEX-6p-1, contains the thrombin restriction site and T7 tag. The fusion protein is about 27.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of IMP-71 . It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pGEX-28a to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

IMP-71-PCR1.png
Figure 1. The PCR result of IMP-71.

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]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.