A self-replicating artificial module-genome that generates bacterial chromosome replication system in vitro

Autonomous self-reproduction is a major goal of bottom-up synthetic biology aimed at building artificial cells. This requires that the genome be replicated by its self-encoded replication machinery. While the reconstituted Escherichia coli chromosomal replication system, termed the Replication-Cycle Reaction (RCR) system, offers a promising platform for genome-scale replication, its generation from genetic information has not yet been achieved. Here we show that a 53 kb circular DNA, termed RCR module-genome, encoding all 26 RCR proteins, can self-replicate in a one-pot reaction when expressed using the protein synthesis using recombinant elements (PURE) system. We first built a prototype of the RCR module-genome and then optimized reaction conditions and solved expression bottlenecks to achieve robust self-replication. This artificial module-genome supports more than 28 doublings of recursive self-replication. This system, termed PRIMES (PURE-driven RCR for In-vitro Module-gEnome Self-replication), represents a milestone toward constructing self-reproducing artificial cells.