Continuous in situ synthesis of a complete set of tRNAs sustains steady-state translation in a recombinant cell-free system

Fanjun Li, Amogh Kumar Baranwal and Sebastian J. Maerkl ()
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Fanjun Li: École Polytechnique Fédérale de Lausanne
Amogh Kumar Baranwal: École Polytechnique Fédérale de Lausanne
Sebastian J. Maerkl: École Polytechnique Fédérale de Lausanne

Nature Communications, 2025, vol. 16, issue 1, 1-12

Abstract: Abstract Construction of a self-regenerating biochemical system is critical for building a synthetic cell. An essential step in building a self-regenerative system is producing a complete set of tRNAs for translation, which remains a significant challenge. Here, we reconstitute a complete set of 21 in vitro transcribed tRNAs and optimize their abundance to improve protein yield. Next, we show that protein expression in the PURE transcription-translation system can be achieved by in situ transcribing tRNAs from 21 linear tRNA templates or a single plasmid template. To enable synthesis of mature tRNAs from a circular template, we employ either a nicked plasmid template or T. maritima tRNase Z to post-transcriptionally process the precursor tRNAs. We ultimately achieve continuous in situ synthesis of a complete set of tRNAs capable of supporting sustained, steady-state protein expression in PURE reactions running on microfluidic chemostats. Our findings advance the development of an autopoietic biochemical system.

Date: 2025
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DOI: 10.1038/s41467-025-61671-8

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