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- Title
A new method for multiple gene inactivations in 168, producing a strain free of selectable markers.
- Authors
Zhang, Chong; Zhang, Xiaohui; Yao, Zhengying; Lu, Yaping; Lu, Fengxia; Lu, Zhaoxin
- 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 P lysA promoter with the P spac promoter. The homologous recombination integration vector PLC-T was constructed to contain lacI, which encodes a P spac 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.
- Subjects
GENE silencing; BACILLUS subtilis genetics; GENETIC markers; PROMOTERS (Genetics); GENETIC recombination; GENETIC code; NUCLEOTIDE sequence; LYSINE
- Publication
Canadian Journal of Microbiology, 2011, Vol 57, Issue 5, p427
- ISSN
0008-4166
- Publication type
Article
- DOI
10.1139/w11-035