Institute of Microbiology, Technische Universität (TU) Dresden, 01062, Dresden, Germany.
Department of Biology I, Ludwig-Maximilians-Universität (LMU) München, 82152, Planegg-Martinsried, Germany.
Sci Rep. 2017 Oct 26;7(1):14134. doi: 10.1038/s41598-017-14329-5.
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.
枯草芽孢杆菌将自然转化的遗传转化能力与高效的同源重组相结合。这些特性允许使用通过双同源重组整合到基因组中的载体。到目前为止，它们的利用受到抗性标记和整合位点的固定组合以及种属或菌株特异性同源区域的限制。为了克服这些限制，我们开发了一个工具盒，用于以标准化的方式创建个性化的枯草芽孢杆菌载体，重点是快速、轻松地适应指定整合位点的序列。我们的载体工具包基于标准欧洲载体架构（SEVA），以允许使用它们的载体部分。枯草芽孢杆菌 SEVA 兄弟姐妹通过从带有四种不同酶的四个入口部分进行高效的一锅式 Golden Gate 反应组装而成。该工具盒包含七种枯草芽孢杆菌抗性标记物、两种大肠杆菌复制起点和一个自由选择的整合位点。载体可以在载体组装之前或之后进行定制，并可用于不同的枯草芽孢杆菌菌株，可能还可以用于其他菌株。我们对 SEVA 标准的适应为方便地创建个性化枯草芽孢杆菌载体提供了一个强大且标准化的工具包。
