Genetically Encoded Control of <i>In Vitro</i> Transcription–Translation Coupled DNA Replication

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 <i>in vitro</i> transcription-translation (IVTT) systems. Here, we constructed a transcription-translation coupled DNA replication (TTcDR) system that is based on a modified PURE (<u>P</u>rotein synthesis <u>U</u>sing <u>R</u>ecombinant <u>E</u>lements) 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-encoded 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 <i>in vitro</i> DNA replication, for example, for the evolution of <i>in vitro</i> systems.