Difference between revisions of "Part:BBa K2933019"

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This part encodes a protein called BlaB-14, which is a metallo-beta-lactamase of subclass B1.
 
This part encodes a protein called BlaB-14, which is a metallo-beta-lactamase of subclass B1.
  
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BlaB is a chromosome-encoded MBL produced by Elizabethkingia meningoseptica. It has a broad substrate profile and is produced constitutively. C. meningosepticum is an ubiquitous Gramnegative rod bacterium of clinical relevance because it can cause neonatal meningitis, adult septicemia, and nosocomial infections and is resistant to most β-lactams, including carbapenems.
 
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This pathogen's multidrug-resistant phenotype and attendant lack of adequate molecular mechanistic data limit the current approaches for its effective management in hospitals and public health settings
 
  
  
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===Usage and Biology===
 
===Usage and Biology===
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BlaB-14 is a type of subclass B metal beta-lactamases. The beta lactamases of the blaB family 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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BlaB is a chromosome-encoded MBL produced by Elizabethkingia meningoseptica. It has a broad substrate profile and is produced constitutively. C. meningosepticum is an ubiquitous Gramnegative rod bacterium of clinical relevance because it can cause neonatal meningitis, adult septicemia, and nosocomial infections and is resistant to most β-lactams, including carbapenems.
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==References==
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This pathogen's multidrug-resistant phenotype and attendant lack of adequate molecular mechanistic data limit the current approaches for its effective management in hospitals and public health settings.<br>
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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.<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;">
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   [[File:BlaB-14-PCR.png]]<br>
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   [[File:BlaB-14-PCR.png|300px]]<br>
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'''Figure 1.'''  (a) The PCR result of BlaB-14. (b) The verification results by enzyme digestion.
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'''Figure 1.'''  (a) The PCR result of BlaB-14. (b) The verification results by 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>
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===References===
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[1]González LJ, Vila AJ. Carbapenem resistance in Elizabethkingia meningoseptica is mediated by metallo-β-lactamase BlaB. Antimicrob Agents Chemother. 2012;56(4):1686–1692. doi:10.1128/AAC.05835-11<br>
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[2]Yum, J.H., Lee, E.Y., Hur, SH. et al. J Microbiol. (2010) 48:358.https://doi.org/10.1007/s12275-010-9308-5

Latest revision as of 08:44, 24 September 2019


subclass B1 metallo-beta-lactamase BlaB-14, codon optimized in E. coli

This part encodes a protein called BlaB-14, which is a metallo-beta-lactamase of subclass B1.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 561
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

BlaB is a chromosome-encoded MBL produced by Elizabethkingia meningoseptica. It has a broad substrate profile and is produced constitutively. C. meningosepticum is an ubiquitous Gramnegative rod bacterium of clinical relevance because it can cause neonatal meningitis, adult septicemia, and nosocomial infections and is resistant to most β-lactams, including carbapenems. This pathogen's multidrug-resistant phenotype and attendant lack of adequate molecular mechanistic data limit the current approaches for its effective management in hospitals and public health settings.


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.

BlaB-14-PCR.png
Figure 1. (a) The PCR result of BlaB-14. (b) 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.

References

[1]González LJ, Vila AJ. Carbapenem resistance in Elizabethkingia meningoseptica is mediated by metallo-β-lactamase BlaB. Antimicrob Agents Chemother. 2012;56(4):1686–1692. doi:10.1128/AAC.05835-11
[2]Yum, J.H., Lee, E.Y., Hur, SH. et al. J Microbiol. (2010) 48:358.https://doi.org/10.1007/s12275-010-9308-5