Difference between revisions of "Part:BBa K2933213"
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<partinfo>BBa_K2933213 parameters</partinfo>
 
<partinfo>BBa_K2933213 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 JOHN-1. It encodes a protein which is JOHN-1 fused with His tag. The fusion protein is about 28.1 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of JOHN-1 .  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 JOHN-1. It encodes a protein which is JOHN-1 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 28.1 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of JOHN-1 .  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:JOHN-1-6p.jpg|600px]]<br>
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   [[File:JOHN-1-6p1.jpg|600px]]<br>
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'''Figure 1.'''   Left: The PCR result of JOHN-1. Right: The verification results by enzyme digestion.<br>
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'''Figure 1.''' The PCR result of JOHN-1.<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]Naas Thierry,Bellais Samuel,Nordmann Patrice. Molecular and biochemical characterization of a carbapenem-hydrolysing beta-lactamase from Flavobacterium johnsoniae.[J]. Journal of Antimicrobial Chemotherapy,2003,51(2).<br>

Latest revision as of 14:03, 23 September 2019


T7 promoter+RBS b+Linker h+His+Linker f+JOHN-1+T7 terminator

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+JOHN-1），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 280
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 169
    Illegal PstI site found at 280
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
    Illegal PstI site found at 280
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
    Illegal PstI site found at 280
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 804


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 JOHN-1. It encodes a protein which is JOHN-1 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 28.1 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of JOHN-1 . 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.

JOHN-1-6p1.jpg
Figure 1. The PCR result of JOHN-1.

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]Naas Thierry,Bellais Samuel,Nordmann Patrice. Molecular and biochemical characterization of a carbapenem-hydrolysing beta-lactamase from Flavobacterium johnsoniae.[J]. Journal of Antimicrobial Chemotherapy,2003,51(2).