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

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Abstract
An efficient method for the replacement of chromosomal DNA by segments altered in vitro was developed for bacteria. The method requires a recombinant plasmid with a ColE1-like replicon and a strain defective in DNA polymerase I (polyA), which is unable to replicate the plasmid extrachromosomally. This method is of general use since there are a number of suitable vectors and polyA strains are available in Escherichia coli and Salmonella typhimurium, the 2 most widely studied bacterial species. Using the method, one constructed 2 chromosomal deletions in the chemotaxis gene region of S. typhimurium. Plasmid sequences integrated into the chromosome were amplified up to 30-fold by varying the concentration of ampicillin or tetracycline in the growth medium.