Selection strategy for site-directed mutagenesis based on altered beta-lactamase specificity.
Conventional approaches to oligonucleotide-directed mutagenesis rely upon the application of a selection strategy to maximize mutagenesis efficiencies. We have developed a mutagenesis procedure that incorporates a novel antibiotic resistance for selection. The selection involves altering the substrate specificity of TEM-1 beta-lactamase, the enzyme responsible for bacterial resistance to beta-lactam antibiotics such as ampicillin. The gene encoding beta-lactamase is commonly found on cloning and shuttle vectors used in molecular biology. Amino acid substitutions in several active site residues of beta-lactamase result in increased hydrolytic activity against extended-spectrum penicillins and cephalosporins. This increased activity confers a novel resistance specific to the mutant and thus provides the basis of the selection strategy. We describe a simple and efficient mutagenesis procedure and its application to creating a range of oligonucleotide-directed mutants.
Andrews
C A
CA
Promega Corporation, Madison, WI 53711-5399, USA. candrews@promega.com
Lesley
S A
SA
D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity / Q000235:genetics; D018440:beta-Lactam Resistance / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D001618:beta-Lactamases; C034413:beta-lactamase TEM-1