Work

Genetically Encoded Control of In Vitro Transcription–Translation Coupled DNA Replication

Year: 2025

Type: article

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 ( P rotein synthesis U sing R ecombinant E 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-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. Graphical Abstract

Source: ACS Synthetic Biology

DOI: 10.1021/acssynbio.5c00477

Authors: Sebastian Barthel, Maximilian Hoffmann-Becking, Islomjon G Karimov, Tobias J Erb

Institutions: Max Planck Institute for Terrestrial Microbiology, Philipps University of Marburg, Loewe Center for Synthetic Microbiology

Language: English

Topic: RNA and protein synthesis mechanisms

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $4,500

Funders: Max-Planck-Gesellschaft, Max-Planck-Institut für Terrestrische Mikrobiologie