title: A new method for multiple gene inactivations in Bacillus subtilis 168, producing a strain free of selectable markers.
abstract: This study describes a novel method for repeated gene inactivation in Bacillus subtilis 168. A B. subtilis strain (BS-PS) that is conditionally auxotrophic for lysine was obtained by replacing the PlysA promoter with the Pspac promoter. The homologous recombination integration vector PLC-T was constructed to contain lacI, which encodes a Pspac promoter repressor, and the chloromycetin resistance gene. Target genes were manipulated by generating an insertion sequence with two homologous arms and the target gene in PLC-T to create a specific integrating vector. Integration into the BS-PS chromosome occurred by a single crossover at either of the two homologous arms. The resulting transitional strain (BS-PS-PI) was chloromycetin resistant and lysine auxotrophic and had an unstable genome structure because of the duplication. Excision of lacI and chloromycetin resistance gene was achieved by a second single crossover at the duplication. Recovery of a lysine prototroph functioned as counter-selection and was identified by PCR. In this work, we inactivated nprE and aprE, two protease genes secreted by B. subtilis 168 free of selectable markers.
authors.affiliation: Laboratory of Enzyme Engineering, College of Food Science and Technology, Nanjing Agriculture University, the People's Republic of China.
authors.forename: Chong
authors.initials: C
authors.lastname: Zhang
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Zhang
authors.forename: Zhengying
authors.initials: Z
authors.lastname: Yao
authors.forename: Yaping
authors.initials: Y
authors.lastname: Lu
authors.forename: Fengxia
authors.initials: F
authors.lastname: Lu
authors.forename: Zhaoxin
authors.initials: Z
authors.lastname: Lu
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D020868:Gene Silencing; D018390:Gene Targeting; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D059765:Homologous Recombination; D008239:Lysine / Q000378:metabolism; D011401:Promoter Regions, Genetic
chemical_list: D008239:Lysine