Altered β-Lactamase Selection Approach for Site-Directed Mutagenesis.

Many protocols exist to perform site-directed mutagenesis, and here we present one of the more commonly used ones-site-directed mutagenesis by altered β-lactamase selection. β-Lactamase is an enzyme that cleaves ampicillin, rendering it impotent to bacteria. Certain mutations in the active site of β-lactamase can alter the substrate specificity of the enzyme and allow it to have increased hydrolytic activity for the cephalosporin family of antibiotics, a property not shared by wild-type lactamases. E. coli cells carrying the β-lactamase triple mutant G238S:E240:R241G show increased resistance to cefotaxime and ceftriaxone, two cephalosporins, compared with wild-type cells. This protocol takes advantage of this property to select for plasmids that have undergone site-directed mutagenesis.