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We describe a new set of tools for inserting DNA into the bacterial chromosome. The system uses site-specific recombination reactions carried out by bacteriophage integrases to integrate plasmids at up to eight phage attachment sites in E . coli MG1655. The introduction of mutan loxP sites in the integrating plasmids allows repeated removal of antibiotic resistance genes and other plasmid sequences without danger of inducing chromosomal rearrangements. The protocol for Cre-mediated antibiotic resistance gene removal is greatly simplified by introducing the Cre plasmid by phage infection. Finally, we have also developed a set of four independently inducible expression modules with tight control and high dynamic range which can be inserted at specific chromosomal locations.

The pIT5 Plasmid Series, an Improved Toolkit for Repeated Genome Integration in E. coli