Difference between revisions of "Part:BBa K2933263"

 
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<partinfo>BBa_K2933263 parameters</partinfo>
 
<partinfo>BBa_K2933263 parameters</partinfo>
 
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===Usage and Biology===
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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.<br>
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===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 enzyme digestion verification.<br>
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<p style="text-align: center;">
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  [[File:Fla.103-PCR.png]]<br>
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'''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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</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. <br>

Revision as of 11:32, 21 September 2019


RBS b+Linker h+His+Linker a+Sumo+Linker b+Fla.103+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+Fla.103) 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 1158
    Illegal PstI site found at 473
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 298
    Illegal BglII site found at 187
    Illegal BglII site found at 955
    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
    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.

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.