Difference between revisions of "Part:BBa K2933019"

Revision as of 04:52, 8 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.

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

Sequence and Features

Usage and Biology

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.

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.

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.However, we failed.