title: Establishment of a markerless mutation delivery system in Bacillus subtilis stimulated by a double-strand break in the chromosome.
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.
authors.affiliation: Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, People's Republic of China ; Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin, People's Republic of China ; Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, People's Republic of China ; Edinburgh-Tianjin Joint Research Centre for Systems Biology and Synthetic Biology, Tianjin University, Tianjin, People's Republic of China.
authors.forename: Ting
authors.initials: T
authors.lastname: Shi
authors.forename: Guanglu
authors.initials: G
authors.lastname: Wang
authors.forename: Zhiwen
authors.initials: Z
authors.lastname: Wang
authors.forename: Jing
authors.initials: J
authors.lastname: Fu
authors.forename: Tao
authors.initials: T
authors.lastname: Chen
authors.forename: Xueming
authors.initials: X
authors.lastname: Zhao
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics*; D002876:Chromosomes, Bacterial; D017353:Gene Deletion; D016680:Genome, Bacterial; D016296:Mutagenesis; D009154:Mutation; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction