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bioRxiv · 10.1101/2025.07.08.663768

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

Barthel, S.·Hoffmann-Becking, M.·Karimov, I. G.·Erb, T. J.
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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 {Phi}29 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 O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/663768v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@cff8fcorg.highwire.dtl.DTLVardef@1e3751dorg.highwire.dtl.DTLVardef@1e037corg.highwire.dtl.DTLVardef@76f330_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Barthel, S., Hoffmann-Becking, M., Karimov, I. G., Erb, T. J.. 2025-07-08. Genetically encoded control of in vitro transcription-translation coupled DNA replication. https://doi.org/10.1101/2025.07.08.663768

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