(228ci) in Vitro DNA Assembly for Marker-Less Gene Replacement in Bacillus Subtilis

Bacillus subtilis, the best-characterized Gram-positive bacterium, is the model organism for studying Gram-positive pathogens such as Bacillus anthracis and biotechnologically important species such as Bacillus licheniformis and B. subtilis for the production of proteins and food additives. It is also a model organism for studying cell biology including sporulation, bacterial chromosome replication, cell development and differentiation. Along with the development of functional genomics, B. subtilis is now one of the most advanced model organisms for systems biology study and host for synthetic modules. Despite of its importance and wide use as research or industrial host, genetic engineering of B. subtilis is not very efficient. Though a genetic toolkit is available for modifying its genome, almost all of them rely on E. coli-B. subtilis shuttle vectors and genetic modification takes much longer time compared with E. coli. In addition, a resistance marker is often created for either gene deletion or insertion. The limitation of available resistance markers and long turnover time significantly impede the research related to B. subtilis. In this work, we developed an E. coli independent method for marker-less gene deletion or insertion. Four pieces of DNA for deletion or five pieces for insertion, including the homologous fragments and a resistance marker, are assembled using Gibson assembly and transform B. subtilis. Double crossover homologous recombination followed by linearizing the chromosome at a designed I-sceI site will force the removal of the resistance marker. Compared with previous method, our design does not require cloning in E. coli and this greatly shortens the overall process. In addition, the efficiency of resistance marker removal is 100% and all colonies obtained have the desired design, which is much better than a 50% from a previously reported method, and no further experiment is needed for confirmation. We have demonstrated this method by deleting the amyE gene or integrating a gfp expression operon into the amyE site. It takes about one day to get the deletion or insertion and another 2-3 days to remove the resistance marker, which is comparable to genome modification in E. coli. The development of the E. coli independent marker-less gene replacement method will greatly facilitate the genome editing in B. subtilis and expedite relevant research including cell biology study, strain development, and system/synthetic study.