Replacement and amplification of bacterial genes with sequences altered in vitro.

Abstract

An efficient method for the replacement of chromosomal DNA by segments altered in vitro has been developed for bacteria. The method requires (i) a recombinant plasmid with a ColE1-like replicon and (ii) a strain defective in DNA polymerase I (polA), which is unable to replicate the plasmid extrachromosomally. This method is of general use since there are a number of suitable vectors and polA strains are available in both Escherichia coli and Salmonella typhimurium, the two most widely studied bacterial species. Using the method, we have constructed two chromosomal deletions in the chemotaxis gene region of S. typhimurium. In addition, plasmid sequences integrated into the chromosome have been amplified up to 30-fold by varying the concentration of ampicillin or tetracycline in the growth medium.