Difference between revisions of "Part:BBa K2933021"

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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>
 
<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.
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Results verified by double enzyme digestion.<br>
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</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>

Revision as of 04:55, 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 digestion validation.

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.But we failed.