Counter‐Selection Method for Markerless Allelic Exchange in Bordetella bronchiseptica Based on sacB Gene From Bacillus subtilis

SISTI, FEDERICO (CORR. AUTHOR); SISTI, FEDERICO (CORR. AUTHOR); FERNÁNDEZ, JULIETA (CORR. AUTHOR); FERNÁNDEZ, JULIETA (CORR. AUTHOR); AMBROSIS, NICOLÁS; AMBROSIS, NICOLÁS

Current Protocols in Microbiology

Wiley

Año: 2020 vol. 59

1934-8525

Bordetella bronchiseptica is a gram-negative bacterium that causes respiratorytract infections. It is a natural pathogen of a wide variety of mammals, includingsome used as laboratory models. This makes B. bronchiseptica an ideal organismto study pathogen?host interactions in order to unveil molecular mechanismsbehind pathogenic processes. Even though genetic engineering is anessential tool in this area, there are just a few reports about genome manipulationtechniques in this organism. In this article we describe an allelic exchangeprotocol based on double crossover recombination facilitated by the Bacillussubtilis sacB gene that can be applied for partial or complete gene knockouts,single-nucleotide mutations, or even introduction of coding sequences for transcriptionalfusions. In contrast to previously employed techniques, this protocolrenders genetically manipulated chromosomes without foreign DNA andenables the construction of successive genome manipulation using the samevector backbone. The entire procedure has been developed for fast and reliablemanipulations with a total duration of 2 weeks