Combining Genes from Multiple Phages for Improved Cell Lysis and DNA Transfer from <i>Escherichia coli</i> to <i>Bacillus subtilis</i>

The ability to efficiently and reliably transfer genetic circuits between the key synthetic biology chassis, such as Escherichia coli and Bacillus subtilis, constitutes one of the major hurdles of the rational genome engineering. Using lambda Red recombineering we integrated the thermosensitive lambda repressor and the lysis genes of several bacteriophages into the E. coli chromosome. The lysis of the engineered autolytic cells is inducible by a simple temperature shift. We improved the lysis efficiency by introducing different combinations of lysis genes from bacteriophages lambda, ΦX174 and MS2 under the control of the thermosensitive lambda repressor into the E. coli chromosome. We tested the engineered autolytic cells by transferring plasmid and bacterial artificial chromosome (BAC)-borne genetic circuits from E. coli to B. subtilis. Our engineered system combines benefits of the two main synthetic biology chassis, E. coli and B. subtilis, and allows reliable and efficient transfer of DNA edited in E. coli into B. subtilis.能够在大肠杆菌（Escherichia coli）与枯草芽孢杆菌（Bacillus subtilis）等核心合成生物学底盘（synthetic biology chassis）之间高效且可靠地转移遗传回路，是理性基因组工程面临的主要挑战之一。我们借助λ Red重组工程技术，将热敏λ阻遏蛋白与多种噬菌体的裂解基因整合至大肠杆菌染色体中。该工程化自溶细胞的裂解可通过简单的温度转换进行诱导。我们通过在大肠杆菌染色体中引入受热敏λ阻遏蛋白调控的、来自噬菌体λ、ΦX174与MS2的不同组合裂解基因，提升了裂解效率。我们通过将质粒与细菌人工染色体（BAC）携带的遗传回路从大肠杆菌转移至枯草芽孢杆菌，对该工程化自溶细胞进行了测试。我们构建的工程化系统融合了大肠杆菌与枯草芽孢杆菌这两种主流合成生物学底盘的优势，能够将在大肠杆菌中编辑完成的DNA可靠且高效地转移至枯草芽孢杆菌中。