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.
Highlights
Bacillus subtilis has been applied as a model system for researches on the aspects of biochemistry, genetics and physiology of Grampositive bacteria, and has long been used as an important cell factory for industrial applications [1]
We report on the development of an easy-toimplement and highly efficient cloning-independent genetic manipulation system in B. subtilis
This system exploits upp as a counter-selectable marker in concert with double-strand break (DSB) stimulation repair caused by I-SceI endonuclease
Summary
Bacillus subtilis has been applied as a model system for researches on the aspects of biochemistry, genetics and physiology of Grampositive bacteria, and has long been used as an important cell factory for industrial applications [1]. Programmed competence coordinates the expression of proteins involved in DNA uptake and translocation with expression of some proteins of the recombination machinery. One of the early competence induced genes encodes the master regulator (ComK) that subsequently regulates the expression of the ‘‘late’’ genes to drive expression a set of genes in wild type cells that are necessary for building up the DNA uptake apparatus and recombination [7,8,9,10]. Overexpression of comK gene or deletion of rok gene (encoding the negative regulator of comK) is a valuable method for improvement of natural transformation efficiency and has been applied in B. subtilis [11,12,13]. Induction of comK combined with positive auto-stimulation of native comK is sufficient for the transcriptional activation of the late competence genes, which results in an increased percentage of competent cells in the population [14] and leads to establishment of the competent state when it enters the stationary growth phase [15]
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