Difference between revisions of "Part:BBa K2933254"
 
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===Usage and Biology===
 
===Usage and Biology===
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Flavobacterium johnsoniae (formerly Cytophaga johnsonae) is an environmental bacterium that can cause skin lesions in fish and is a plant pathogen.
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This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein JOHN-1 and T7 terminator. It encodes a protein which is JOHN-1 fused with His-Sumo tag. The fusion protein is about 40.1 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.
  
  
 
===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 PET-28bs 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;">
 
   [[File:JOHN-1-6p.jpg|600px]]<br>
 
   [[File:JOHN-1-6p.jpg|600px]]<br>

Latest revision as of 13:28, 22 September 2019


RBS b+Linker h+His+Linker a+Sumo+Linker b+JOHN-1+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+JOHN-1) 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 EcoRI site found at 298
    Illegal PstI site found at 473
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 298
    Illegal NheI site found at 75
    Illegal NheI site found at 1152
    Illegal PstI site found at 473
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 298
    Illegal BglII site found at 187
    Illegal BamHI site found at 386
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 298
    Illegal PstI site found at 473
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 298
    Illegal PstI site found at 473
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 997


Usage and Biology

This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein JOHN-1 and T7 terminator. It encodes a protein which is JOHN-1 fused with His-Sumo tag. The fusion protein is about 40.1 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.


Molecular cloning

First, we used the vector PET-28bs 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.