Max Planck Institute for Terrestrial Microbiology &, LOEWE Center for Synthetic Microbiology (Synmikro), Karl-von-Frisch Strasse 16, 35043, Marburg, Germany.
Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152, Martinsried, Germany.
Chembiochem. 2019 Oct 15;20(20):2633-2642. doi: 10.1002/cbic.201900299. Epub 2019 Aug 28.
A biomimetic system capable of replication and segregation of genetic material constitutes an essential component for the future design of a minimal synthetic cell. Here we have used the simple T7 bacteriophage system and the plasmid-derived ParMRC system to establish in vitro DNA replication and DNA segregation, respectively. These processes were incorporated into biomimetic compartments providing an enclosed reaction space. The functional lifetime of the encapsulated segregation system could be prolonged by equipping it with ATP-regenerating and oxygen-scavenging systems. Finally, we showed that DNA replication and segregation processes could be coupled in vitro by using condensed DNA nanoparticles resulting from DNA replication. ParM spindles extended over tens of micrometers and could thus be used for segregation in compartments that are significantly longer than bacterial cell size. Overall, this work demonstrates the successful bottom-up assembly and coupling of molecular machines that mediate replication and segregation, thus providing an important step towards the development of a fully functional minimal cell.
一个能够复制和分离遗传物质的仿生系统是未来设计最小合成细胞的重要组成部分。在这里，我们分别使用简单的 T7 噬菌体系统和质粒衍生的 ParMRC 系统来建立体外 DNA 复制和 DNA 分离。这些过程被纳入提供封闭反应空间的仿生隔间中。通过配备 ATP 再生和氧气清除系统，可以延长封装分离系统的功能寿命。最后，我们通过使用 DNA 复制产生的浓缩 DNA 纳米颗粒证明了体外 DNA 复制和分离过程可以偶联。ParM 纺锤体延伸了数十微米，因此可以用于比细菌细胞尺寸明显更长的隔间中的分离。总的来说，这项工作展示了介导复制和分离的分子机器的成功自下而上组装和偶联，从而朝着开发完全功能的最小细胞迈出了重要一步。
