Protocol for Constructing a Gene Knockout in <i>Pseudomonas</i> Using pT18mobsacB

ABSTRACT Gene knockout via homologous recombination is a powerful approach for investigating gene function in bacteria. Here, we present a streamlined protocol for constructing an unmarked gene knockout in Pseudomonas using the suicide plasmid pT18mobsacB. The method involves the insertion of upstream and downstream homologous arms into the plasmid, followed by two‐step allelic exchange using tetracycline selection and sucrose counter‐selection. Unlike commonly used vectors, pT18mobsacB confers tetracycline resistance, which offers broader inhibitory coverage across bacterial species and expands the host range of this system. The method enables precise marker‐free knockout without leaving any antibiotic resistance cassette, allowing unrestricted downstream applications. A complete knockout can be achieved in as little as 1 week. Despite its versatility, the use of pT18mobsacB has been rarely detailed in previous literature. This protocol fills that gap by providing a fully annotated, reproducible guide that covers every experimental detail—from vector construction to mutant verification. It offers a valuable tool for functional genomics, microbial physiology, and applications ranging from food microbiology to biotechnology.