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- Title
Establishment of a Markerless Mutation Delivery System in <i>Bacillus subtilis</i> Stimulated by a Double-Strand Break in the Chromosome.
- Authors
Shi, Ting; Wang, Guanglu; Wang, Zhiwen; Fu, Jing; Chen, Tao; Zhao, Xueming
- Abstract
Bacillus subtilis has been a model for gram-positive bacteria and it has long been exploited for industrial and biotechnological applications. However, the availability of facile genetic tools for physiological analysis has generally lagged substantially behind traditional genetic models such as Escherichia coli and Saccharomyces cerevisiae. In this work, we have developed an efficient, precise and scarless method for rapid multiple genetic modifications without altering the chromosome of B. subtilis. This method employs upp gene as a counter-selectable marker, double-strand break (DSB) repair caused by exogenous endonuclease I-SceI and comK overexpression for fast preparation of competent cell. Foreign dsDNA can be simply and efficiently integrated into the chromosome by double-crossover homologous recombination. The DSB repair is a potent inducement for stimulating the second intramolecular homologous recombination, which not only enhances the frequency of resolution by one to two orders of magnitude, but also selects for the resolved product. This method has been successfully and reiteratively used in B. subtilis to deliver point mutations, to generate in-frame deletions, and to construct large-scale deletions. Experimental results proved that it allowed repeated use of the selectable marker gene for multiple modifications and could be a useful technique for B. subtilis.
- Subjects
GENETIC mutation; BACILLUS subtilis; CHROMOSOMES; GRAM-positive bacteria; BIOTECHNOLOGY; GENETIC recombination; GENETIC markers
- Publication
PLoS ONE, 2013, Vol 8, Issue 11, p1
- ISSN
1932-6203
- Publication type
Article
- DOI
10.1371/journal.pone.0081370