In vitro constitution of biological functions is a useful strategy to understand the physicochemical principles underlying biological functions (Luisi and Stano, 2013). To date, various cellular functions have been artificially constituted, including self- replication of genetic information, a gene expression, and so on. As a self-replication system of genetic information, our group has reconstituted an RNA genome replication system coupled with translation (Ichihashi, et al. 2013). However, all living organisms have the DNA genomes, which replicate with DNA replicases translated from the genomes via mRNA transcription. A plan for the construction of such a DNA replication system coupled with transcription and translation has been proposed using rolling-circle-type replication scheme approximately 10 years ago (Fig.1, Forster and Church, 2006), although it has not been realized yet. In this study, we attempted to constitute the transcription-and translation-coupled DNA replication (TTcDR) system and perform an evolution experiment to make the system recursive. The aim of the present work is to show a method of the construction of a DNA genome replication system which leads to the in vitro construction of an artificial cell.

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