Genetically encoded control of in vitro transcription-translation coupled DNA replication

Abstract The bottom-up reconstruction of cellular functions has gained increasing attention for studying biological complexity, and for developing advanced biotechnological tools, including synthetic cells. A fundamental challenge is the ability to control and replicate DNA-encoded information within basic in vitro transcription-translation (IVTT) systems. Here, we constructed a transcription-translation coupled DNA replication (TTcDR) system that is based on a modified PURE (Protein synthesis Using Recombinant Elements) IVTT system and Φ29 DNA polymerase, which is controlled by external signals. To this end, we first established and characterized a PUREfrex 1.0-based TTcDR system. We then constructed and optimized TetR-based control of TTcDR activity, either by DNA-encoding TetR or by supplying purified TetR. Our final DNA-encoded TetR circuit allows ∼1000-fold DNA replication, ∼100-fold repression, and ∼4-fold induction with anhydrotetracycline. Our results demonstrate the potential and challenges of controlling in vitro DNA replication, for example for the evolution of in vitro systems. Competing Interest Statement The authors have declared no competing interest.