Strand Trine Aakvik, Lale Rahmi, Degnes Kristin Fløgstad, Lando Malin, Valla Svein
Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway.
Department of Biotechnology, SINTEF Materials and Chemistry, Trondheim, Norway.
PLoS One. 2014 Mar 3;9(3):e90372. doi: 10.1371/journal.pone.0090372. eCollection 2014.
Bacterial conjugation is a process that is mediated either by a direct cell-to-cell junction or by formation of a bridge between the cells. It is often used to transfer DNA constructs designed in Escherichia coli to recipient bacteria, yeast, plants and mammalian cells. Plasmids bearing the RK2/RP4 origin of transfer (oriT) are mostly mobilized using the E. coli S17-1/SM10 donor strains, in which transfer helper functions are provided from a chromosomally integrated RP4::Mu. We have observed that large plasmids were occasionally modified after conjugal transfer when using E. coli S17-1 as a donor. All modified plasmids had increased in size, which most probably was a result of co-transfer of DNA from the chromosomally located oriT. It has earlier also been demonstrated that the bacteriophage Mu is silently transferred to recipient cells by these donor strains, and both occurrences are very likely to lead to mutations within the recipient DNA. Here we report the construction of a new biological system addressing both the above mentioned problems in which the transfer helper functions are provided by a plasmid lacking a functional oriT. This system is compatible with all other replicons commonly used in conjugation experiments and further enables the use of diverse bacterial strains as donors. Plasmids containing large inserts were successfully conjugated and the plasmid modifications observed when E. coli S17-1 was used as donor were eliminated by the use of the new host-independent vector system.
细菌接合是一个由细胞间直接连接或细胞间形成桥梁介导的过程。它常用于将在大肠杆菌中设计的DNA构建体转移到受体细菌、酵母、植物和哺乳动物细胞中。携带RK2/RP4转移起点（oriT）的质粒大多使用大肠杆菌S17-1/SM10供体菌株进行转移，在这些菌株中，转移辅助功能由染色体整合的RP4::Mu提供。我们观察到，当使用大肠杆菌S17-1作为供体时，大质粒在接合转移后偶尔会发生修饰。所有修饰后的质粒大小都增加了，这很可能是位于染色体上的oriT的DNA共转移的结果。此前也已证明，噬菌体Mu会被这些供体菌株无声地转移到受体细胞中，这两种情况都很可能导致受体DNA内发生突变。在此，我们报告构建了一个新的生物系统，该系统解决了上述两个问题，其中转移辅助功能由缺乏功能性oriT的质粒提供。该系统与接合实验中常用的所有其他复制子兼容，还能使用多种细菌菌株作为供体。含有大插入片段的质粒成功进行了接合，并且通过使用新的不依赖宿主的载体系统，消除了使用大肠杆菌S17-1作为供体时观察到的质粒修饰。
