Difference between revisions of "Part:BBa K2933171"

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===Usage and Biology===
 
===Usage and Biology===
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This composite part is made up with three basic parts(Tac promoter，RBS a and Linker g)and a composite part(GST+Linker e+JOHN-1). It encodes a protein which is JOHN-1 fused with GST tag. The fusion protein is about 54.1 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of JOHN-1 and combine the two parts with the cutting site of Prescission Protease. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.<br>
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This composite part is made up with six basic parts(Tac promoter，RBS a ,  Linker g, GST, Linker e and JOHN-1). It encodes a protein which is JOHN-1 fused with GST tag. The fusion protein is about 54.1 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of JOHN-1 and combine the two parts with the cutting site of Prescission Protease. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.<br>
  
 
===Molecular cloning===
 
===Molecular cloning===

Latest revision as of 13:47, 24 September 2019


Tac promoter+RBS a+Linker g+GST+Linker e+JOHN-1

This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+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 PstI site found at 870
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 870
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 870
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 870
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 181
    Illegal SapI.rc site found at 1394


Usage and Biology

This composite part is made up with six basic parts(Tac promoter，RBS a , Linker g, GST, Linker e and JOHN-1). It encodes a protein which is JOHN-1 fused with GST tag. The fusion protein is about 54.1 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of JOHN-1 and combine the two parts with the cutting site of Prescission Protease. 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 pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

JOHN-1-6p.jpg
Figure 1. Left: The PCR result of JOHN-1. 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.

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).