A broad-host-range shuttle system for gene insertion into the chromosomes of gram-negative bacteria

Abstract

A deletion derivative of transposon Tn7 containing the Escherichia coli lacZY genes as a selectable marker for insertion of foreign DNA into the chromosomes of soil bacteria was improved to facilitate the cloning of additional genes and their insertion by this element. This report describes a series of plasmid vectors that enable this cloning to be carried out in small, high-copy, narrow host-range plasmids. The final Tn element can then be easily moved (by transposition) without further use of restriction enzymes, to plasmids suitable for delivering it to the bacterial chromosome. The very high specificity for insertion of Tn7 into single locations in bacterial chromosomes has been exploited in the construction of a shuttle system for delivering these Tn7 elements.