In:
Canadian Journal of Microbiology, Canadian Science Publishing, Vol. 57, No. 5 ( 2011-05), p. 427-436
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.
Type of Medium:
Online Resource
ISSN:
0008-4166
,
1480-3275
Language:
English
Publisher:
Canadian Science Publishing
Publication Date:
2011
detail.hit.zdb_id:
280534-0
detail.hit.zdb_id:
1481972-7
SSG:
12
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