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Appl. Environ. Microbiol. doi:10.1128/AEM.01156-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Cre/lox System and PCR Based Genome Engineering in Bacillus subtilis

Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University, 6 Tongwei Road, Nanjing, Jiangsu 210095, People's Republic of China

^* To whom correspondence should be addressed. Email: lsp{at}njau.edu.cn.

	Abstract

We have developed a fast and accurate method to engineer the Bacillus subtilis genome that involves fusing by PCR two flanking homology regions with an antibiotic resistance gene cassette bordered by two mutant lox sites (lox71 and lox66). The resulting PCR products were used directly to transform B. subtilis; and, then, transient Cre recombinase expression in the transformants was used to recombine lox71 and lox66 into a double mutant lox72 site, thereby excising the marker gene. The mutation process could also be accomplished in two days by using a strain containing a cre IPTG-inducible expression cassette in the chromosome as recipient or using the lox sites flanked cassette containing both the cre (IPTG)-inducible expression cassette and resistance marker. The in vivo recombination efficiencies of different lox pairs were compared; the lox72 site that remains in the chromosome after Cre recombination had a low affinity for Cre and did not interfere with subsequent rounds of Cre/lox mutagenesis. We used this method to inactivate a specific gene, to delete a long fragment, to realize the in-frame deletion of a target gene, to introduce a gene of interest, and to carry out multi-manipulations in a same background. Furthermore, it should also be applied to large genome rearrangement.