Difference between revisions of "Part:BBa K2933021"

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This part encodes a protein called Fla.103, which is a metallo-beta-lactamase of subclass B1.
 
This part encodes a protein called Fla.103, which is a metallo-beta-lactamase of subclass B1.
  
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<!-- Add more about the biology of this part here
 
  
  
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===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.We then extracted plasmids and performed double digestion validation.<br>
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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.We then extracted plasmids and performed double enzyme digestion verification.<br>
 
<p style="text-align: center;">
 
<p style="text-align: center;">
 
   [[File:Fla.103-PCR.png]]<br>
 
   [[File:Fla.103-PCR.png]]<br>
 
'''Figure 1.'''  (a) The PCR result of Fla.103. (b) The verification results by enzyme digestion.The 1 is the original plasmid.The 2 is the  
 
'''Figure 1.'''  (a) The PCR result of Fla.103. (b) The verification results by enzyme digestion.The 1 is the original plasmid.The 2 is the  
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Results verified by double enzyme digestion.<br>
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results verified by double enzyme digestion.<br>
 
</p>
 
</p>
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After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.But we failed. <br>
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After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification. <br>

Latest revision as of 14:34, 8 September 2019


subclass B1 metallo-beta-lactamase Fla.103, codon optimized in E. coli

This part encodes a protein called Fla.103, 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
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 558
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 76
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 76
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Fla.103 is a type of subclass B metal beta-lactamases. The beta lactamases can hydrolyze almost all available beta lactam antibiotics (except aztreonam) clinically, including the broad-spectrum antibiotic carbapenems. Because of the extensive substrate profile of this enzyme, the clinical strains carrying it become a great threat to human life and health.

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.We then extracted plasmids and performed double enzyme digestion verification.

Fla.103-PCR.png
Figure 1. (a) The PCR result of Fla.103. (b) The verification results by enzyme digestion.The 1 is the original plasmid.The 2 is the results verified by double 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.