Minimization of the Escherichia coli Genome Using the Tn5-Targeted Cre/loxP Excision System

Efficient genome-engineering tools have been developed for use in whole-genome essentiality studies. In this chapter, we describe a powerful genomic deletion tool, the Tn5-targeted Cre/loxP excision system, for determining genetic essentiality and minimizing bacterial genomes on a genome-wide scale. This tool is based on the Tn5 transposition system, phage P1 transduction, and the Cre/loxP excision system. We have generated two large pools of independent transposon insertion mutants in Escherichia coli using random transposition of two modified Tn5 transposons (TnKloxP and TnCloxP) with two different selection markers, kanamycin-resistance gene (Km(R)) or chloramphenicol-resistance gene (Cm(R)), and a loxP site. Transposon integration sites are identified by direct genome sequencing of the genomic DNA. By combining a mapped transposon mutation from each of the mutant pools into the same chromosome using phage P1 transduction and then excising the nonessential genomic regions flanked by the two loxP sites using Cre-mediated loxP recombination, we can obtain numerous E. coli deletion strains from which nonessential regions of the genome are deleted. In addition to the combinatorial deletion of the E. coli genomic regions, we can create a cumulative E. coli deletion strain from which all the individual deleted regions are excised. This process will eventually yield an E. coli strain in which the genome is reduced in size and contains only regions that are essential for viability.