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

The construction of synthetic cells is of increasing interest for the understanding of cellular function and the evolution of biological complexity, as well as for the development of engineering and design tools. A fundamental criterion for the construction of synthetic cells is the ability to replicate and control DNA-encoded information within the underlying in vitro transcription-translation (IVTT) system. As a proof of concept, we constructed a genetic circuit based on TetR from Escherichia coli to regulate transcription-translation coupled DNA replication (TTcDR) in a modified Protein synthesis Using Recombinant Elements (PURE) system. We first characterized a TTcDR system based on the commercially available PUREfrex 1.0 system and two TTcDR-compatible energy mix compositions for their TTcDR and IVTT activity, as well as for nuclease contamination. We then constructed a TetR-based genetic circuit with IVTT (sfGFP-Pepper fluorescence) and TTcDR (Φ29 DNA polymerase-driven DNA amplification) outputs by producing TetR from plasmid and the reporter from linear DNA. We observed increased gene expression from plasmid DNA under resource-limited conditions in the modified PURE system. Based on these findings, we further improved the genetic circuit-regulated TTcDR system to achieve 62 ± 5.7-fold DNA amplification, 19 ± 2.8-fold repression by TetR co-production, and a 2.3-fold response to the ligand aTc. Finally, we tested Φ29 DNA polymerase mutants and further improved TTcDR output to ~1000-fold, >100-fold repression, and further improved derepression. Our results demonstrate the potential and challenges of genetically encoded TTcDR control for the integration and evolution of in vitro systems and the evolution of allosteric transcription factors.