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.
Highlights
The creation of cell-mimicking systems relies on combining a minimal set of functionalities that are inspired by essential cellular features and are either directly derived from biological systems or reconstituted by using unnatural parts.[1,2] The capabilities to replicate and subsequently to segregate replicated genetic information are among these essential features.DNA replication systems of various degrees of complexity have been successfully reconstituted.[3,4,5,6,7,8] In particular, mesophilic bacteriophages offer attractive choices when developing a replication apparatus for a minimal cell, due to their robustness and the small set of components required
We successfully reconstituted modules for DNA replication and DNA segregation by using bacteriophage and plasmid systems, encapsulated these reactions in microcompartments, and showed that they could be coupled through the formation of DNA nanoparticles
We used rolling-circle amplification (RCA) replication mediated by T7 DNA polymerase, in which replication could be initiated in a primer-free system by T7 RNA polymerase,[49] providing an attractive alternative for minimal self-replication systems
Summary
The creation of cell-mimicking systems relies on combining a minimal set of functionalities that are inspired by essential cellular features and are either directly derived from biological systems or reconstituted by using unnatural parts.[1,2] The capabilities to replicate and subsequently to segregate replicated genetic information are among these essential features. Phi29-mediated RCA synthesizes long concatamers of linear double-stranded DNA, which could be recircularized through Cre-lox recombination.[12] The T7 replisome is another well understood system suitable for highly processive RCA of DNA. It requires four core proteins, including DNA polymerase Gp5, which complexes with Escherichia coli host protein thioredoxin, the bifunctional helicase-primase Gp4, and single-stranded DNA-binding protein Gp2.5.[13]. We showed that DNA replication could be coupled to the segregation process by using condensed DNA nanoparticles These minimal replisome and segrosome machineries were further incorporated into biomimetic compartments
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