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Abstract:
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.
