Efficient cloning of a mutant adenylate-kinase-encoding gene from Escherichia coli

Abstract

An optimized system has been developed for the transfer of a mutant gene from the Escherichia coli chromosome to a plasmid carrying the wild type (wt) allele.The wt allele was first cloned into a low-copy-number, self-transmissible plasmid with a single Eco RI, Hind111,and Bam HI site. The plasmid was then transferred to a mutant strain that had been previously transformed with a high-copy-number plasmid carrying the recA + gene to allow efficient homologous recombination.A 15% frequency of homogenotization was obtained during cloning of an adk gene that encodes a temperature-sensitive adenylate kinase (AK).The mutant AK had decreased mobility on sodium dodecyi sulfate-polyacrylamide gels compared with the wt enzyme.This was due to a point mutation that changed leucine-107 in the wt enzyme to glutamine-107 in the mutant enzyme as determined by nucleotide sequencing.