Title

Precision-Engineering the Pseudomonas aeruginosa Genome with Two-Step Allelic Exchange

Document Type

Article

Publication Date

10-2015

Publication Source

Nature Protocols

Abstract

Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions and substitutions, in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selections are enabled solely by suicide vector–encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitroand then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic-resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires ~2 weeks.

Inclusive pages

1820–1841

ISBN/ISSN

1754-2189

Comments

Permission documentation is on file.

Publisher

Macmillan Publishers

Volume

10

Issue

11

Peer Reviewed

yes