Bacillus SEVA siblings: A Golden Gate-based toolbox to create personalized integrative vectors for Bacillus subtilis.

Bacillus subtilis combines natural competence for genetic transformation with highly efficient homologous recombination. These features allow using vectors that integrate into the genome via double homologous recombination. So far, their utilization is restricted by the fixed combination of resistance markers and integration loci, as well as species- or strain-specific regions of homology. To overcome these limitations, we developed a toolbox for the creation of personalized Bacillus vectors in a standardized manner with a focus on fast and easy adaptation of the sequences specifying the integration loci. We based our vector toolkit on the Standard European Vector Architecture (SEVA) to allow the usage of their vector parts. The Bacillus SEVA siblings are assembled via efficient one-pot Golden Gate reactions from four entry parts with the choice of four different enzymes. The toolbox contains seven Bacillus resistance markers, two Escherichia coli origins of replication, and a free choice of integration loci. Vectors can be customized with a cargo, before or after vector assembly, and could be used in different B. subtilis strains and potentially beyond. Our adaptation of the SEVA-standard provides a powerful and standardized toolkit for the convenient creation of personalized Bacillus vectors.