Difference between revisions of "Part:BBa K2933012"
(→‎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>
 
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>
 
<p style="text-align: center;">
 
<p style="text-align: center;">
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   [[File:NDM-23-PCR.png|200px]]<br>
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   [[File:JOHN-1-6p.jpg|200px]]<br>
 
'''Figure 1.'''  Left: The PCR result of JOHN-1. Right: The verification results by enzyme digestion.<br>
 
'''Figure 1.'''  Left: The PCR result of JOHN-1. Right: The verification results by enzyme digestion.<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>

Revision as of 09:03, 8 September 2019


subclass B1 metallo-beta-lactamase JOHN-1, codon optimized in E. coli

This part encodes a protein called JOHN-1, which is a metallo-beta-lactamase of subclass B1.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 76
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 76
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 76
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 76
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 600


Usage and Biology

Flavobacterium johnsoniae (formerly Cytophaga johnsonae) is an environmental bacterium that can cause skin lesions in fish and is a plant pathogen.

References

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.