Integrative Vectors for Gene Deletion and Replacement

A b s t r a c t An improved method for gene deletion or replacement in Escherichia coli was developed. It employs a set of integrative vectors and two helper plasmids, as a temporary source of RecA and Flp activities. #e integrative vectors combine several useful features including three di%erent selection markers placed between two parallel oriented Flp recombinase target ( FRT ) sites. Each marker is $anked by two MCSs, for cloning the chosen homologous fragments of DNA to gene targeting. #e vectors contain two properly oriented E. coli Chi sites for recombination enhancement. When required, selection markers can be excised from the chromosome resulting in unmarked strains. K e y w o r d s: gene deletion; gene replacement, integrative rectors