Efficient dual-negative selection for bacterial genome editing

• DOI: 10.1186/s12866-020-01819-2
• Publication Date: 2020-05-24T11:02:52Z
• Authors: Francesca Romana ...; Olivier Cunrath; Dirk Bumann
• Keywords: Parasitology; Negative selection

Gene editing is key for elucidating gene function. Traditional methods, such as consecutive single-crossovers, have been widely used to modify bacterial genomes. However, cumbersome cloning and limited efficiency of negative selection often make this method slower than other methods recombineering.Here, we established a time-effective variant single-crossovers. This exploits rapid plasmid construction using Gibson assembly, convenient E. coli donor strain, efficient dual-negative improved suicide vector resolution. We generate in-frame deletions, insertions point mutations in Salmonella enterica with hands-on time. Adapted versions enabled also Pseudomonas aeruginosa multi-drug resistant (MDR) Escherichia clinical isolates.Our allows facile manipulation multiple species including MDR isolates. anticipate that might be broadly applicable additional species, those which recombineering has difficult implement.