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Nat Protoc. 2015 Nov;10(11):1820-41. doi: 10.1038/nprot.2015.115. Epub 2015 Oct 22.

Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange.

Author information

1
Department of Microbiology, University of Washington, Seattle, Washington, USA.
2
Colorado State University, College of Veterinary Medicine and Biomedical Sciences, A101 Regional Biocontainment Lab, Fort Collins, Colorado, USA.
3
Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Costerton Biofilm Center, University of Copenhagen, Copenhagen, Denmark.
4
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
5
Department of Molecular Biology, Umeå University, Umeå, Sweden.
6
Department of Microbiology and Immunology, Stanford University, Stanford, California, USA.
7
Department of Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, Canada.
8
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
9
Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, USA.

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 vitro and 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.

PMID:
26492139
PMCID:
PMC4862005
DOI:
10.1038/nprot.2015.115
[Indexed for MEDLINE]
Free PMC Article

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