[3S00m] 01. ASBA - Part 7

10:45 AM - 11:00 AM JST(1:45 AM - 2:00 AM UTC)

[3ASBA-05] A Self-Replicating Artificial Genome and Darwinian Evolution of a Replication Protein In Vitro Using the Bacterial Genome Replication Reconstitution System

○Yuta Yamagishi¹, Yoshiki Sonoyama¹, Naoki Kawakami¹, Tomonori Hasebe¹, Masayuki Su'etsugu¹ (1. Rikkyo University (Japan))

Keywords:

Artificial Cells,Cell-free,reconstitution system,self-replication,DNA replication

Reconstituting genomic self-replication and evolution in vitro is essential for artificial cell construction. Using the reconstituted E. coli chromosomal replication (RCR) system, capable of amplifying up to 2 Mb DNA, we designed an artificial genome encoding 26 essential RCR enzymes. Coupled with the PURE system for cell-free protein synthesis, this genome achieved 50-fold amplification in one pot, sustaining recursive self-replication over 28 generations through serial transfers. This system, termed PRIMES (PURE-driven RCR for In-vitro Molecular gEnome Self-replication), represents a milestone toward constructing self-reproducing artificial cells.

We further demonstrated Darwinian evolution of a replication gene driven by differential self-replication activity. We constructed a simplified system in which only a single replication protein is expressed from DNA, with all remaining RCR enzymes supplied as purified proteins. By encapsulating a mutation library in water-in-oil emulsions, faster-replicating variants were enriched across serial transfers. These results demonstrate Darwinian evolution in vitro, providing a foundation toward evolutionary optimization of artificial cell genomes.