Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange.
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.
Department of Microbiology, University of Washington, Seattle, Washington, USA.
Laura R
LR
Hmelo
Colorado State University, College of Veterinary Medicine and Biomedical Sciences, A101 Regional Biocontainment Lab, Fort Collins, Colorado, USA.
Bradley R
BR
Borlee
Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Costerton Biofilm Center, University of Copenhagen, Copenhagen, Denmark.
Henrik
H
Almblad
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
Michelle E
ME
Love
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
Trevor E
TE
Randall
Department of Microbiology, University of Washington, Seattle, Washington, USA.
Boo Shan
BS
Tseng
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
Chuyang
C
Lin
Department of Molecular Biology, Umeå University, Umeå, Sweden.
Yasuhiko
Y
Irie
Department of Microbiology and Immunology, Stanford University, Stanford, California, USA.
Kelly M
KM
Storek
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
Jaeun Jane
JJ
Yang
Department of Microbiology, University of Washington, Seattle, Washington, USA.
Richard J
RJ
Siehnel
Department of Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, Canada.|Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
P Lynne
PL
Howell
Department of Microbiology, University of Washington, Seattle, Washington, USA.
Pradeep K
PK
Singh
Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, Costerton Biofilm Center, University of Copenhagen, Copenhagen, Denmark.
Tim
T
Tolker-Nielsen
Department of Microbiology, University of Washington, Seattle, Washington, USA.
Matthew R
MR
Parsek
Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, USA.
Herbert P
HP
Schweizer
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
Joe J
http://orcid.org/0000-0003-0368-9539
JJ
Harrison
D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering; D005822:Genetic Vectors; D016680:Genome, Bacterial; D059765:Homologous Recombination; D008967:Molecular Biology / Q000379:methods*; D011550:Pseudomonas aeruginosa / Q000235:genetics*