ArticleNature protocols2015
Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange.
Article in Nature protocols, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 373 papers.
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Who cites it
373 citing papers in PubMed.
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- Structure and conformational dynamics of the Pseudomonas CbrA transceptor.Protein science : a publication of the Protein Society · 2026Article
- Altered regulation of septal peptidoglycan synthesis modulates β-lactam susceptibility in Pseudomonas aeruginosa.PLoS genetics · 2026Article
- Identification of iron and zinc responsive TonB-dependent transporters in Pseudomonas aeruginosa.Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine · 2026Article
- The Caenorhabditis elegans microbiome impacts microsporidia infection through nutrient limitation and spore inactivation.Nature communications · 2026Article
- Oxygen-dependent partitioning of acetate assimilation via Acs and AckA-Pta pathways inJournal of bacteriology · 2026Article
- Markerless fliC Knockout inBiology · 2026Article
- Characterizing the V516M penicillin-binding protein 2 (PBP2) mutation causing zidebactam resistance inAntimicrobial agents and chemotherapy · 2026Article
- END nucleases are antiphage defence systems targeting multiple phages with modified genomes.Nature microbiology · 2026Article
- The two-component system ColRS discriminates metal signals to regulate virulence and resistance in Pseudomonas aeruginosa.PLoS pathogens · 2026Article
- Structural basis of biofilm formation mediated by thebioRxiv : the preprint server for biology · 2026Article
- L-type pyocins inhibit the BAM complex to kill without cell entry.Nature communications · 2026Article
- Altered distribution of chronicity-deficientMicrobiology (Reading, England) · 2026Article
313 more citing papers are in PubMed but not listed here.
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
17 authors.
Funding
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.
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