Towards Synthetic Life: Establishing a Minimal Segrosome for the Rational Design of Biomimetic Systems

DNA segregation is a fundamental life process, crucial for renewal, reproduction and propagation of all forms of life. Hence, a dedicated segregation machinery, a segrosome, must function reliably also in the context of a minimal cell. Conceptionally, the development of such a minimal cell follows a minimalistic approach, aiming at engineering a synthetic entity only consisting of the essential key elements necessary for a cell to survive. In this thesis, various prokaryotic segregation systems were explored as possible candidates for a minimal segrosome. Such a minimal segrosome could be applied for the rational design of biomimetic systems including, but not limited to, a minimal cell. DNA segregation systems of type I (ParABS) and type II (ParMRC) were compared for ensuring genetic stabilities in vivo using vectors derived from the natural secondary chromosome of Vibrio cholerae. The type II segregation system R1-ParMRC was chosen as the most promising candidate for a minimal segrosome, and it was characterized and reconstituted in vitro. This segregation system was encapsulated into biomimetic micro-compartments and its lifetime prolonged by coupling to ATP-regenerating as well as oxygen-scavenging systems. The segregation process was coupled to in vitro DNA replication using DNA nanoparticles as a mimic of the condensed state of chromosomes. Furthermore, another type II segregation system originating from the pLS20 plasmid from Bacillus subtilis (Alp7ARC) was reconstituted in vitro as a secondary orthogonal segrosome. Finally, a chimeric RNA segregation system was engineered that could be applied for an RNA-based protocell. Overall, this work demonstrates successful bottom-up assemblies of functional molecular machines that could find applications in biomimetic systems and lead to a deeper understanding of living systems