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Applied and Environmental Microbiology, September 2008, p. 5556-5562, Vol. 74, No. 17
0099-2240/08/$08.00+0     doi:10.1128/AEM.01156-08
Copyright © 2008, American Society for Microbiology. 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

Received 23 May 2008/ Accepted 9 July 2008

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 2 days by using a strain containing a cre isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible expression cassette in the chromosome as the recipient or using the lox site-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 multiple manipulations in the same background. Furthermore, it should also be applicable to large genome rearrangement.

Published ahead of print on 18 July 2008.

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