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

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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)

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.

Suggested Citation

  • Fanjun Li & Amogh Kumar Baranwal & Sebastian J. Maerkl, 2025. "Continuous in situ synthesis of a complete set of tRNAs sustains steady-state translation in a recombinant cell-free system," Nature Communications, Nature, vol. 16(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-61671-8
    DOI: 10.1038/s41467-025-61671-8
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    References listed on IDEAS

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    1. K. Libicher & R. Hornberger & M. Heymann & H. Mutschler, 2020. "In vitro self-replication and multicistronic expression of large synthetic genomes," Nature Communications, Nature, vol. 11(1), pages 1-8, December.
    2. Suki Albers & Bertrand Beckert & Marco C. Matthies & Chandra Sekhar Mandava & Raphael Schuster & Carolin Seuring & Maria Riedner & Suparna Sanyal & Andrew E. Torda & Daniel N. Wilson & Zoya Ignatova, 2021. "Repurposing tRNAs for nonsense suppression," Nature Communications, Nature, vol. 12(1), pages 1-10, December.
    3. Barbora Lavickova & Nadanai Laohakunakorn & Sebastian J. Maerkl, 2020. "A partially self-regenerating synthetic cell," Nature Communications, Nature, vol. 11(1), pages 1-11, December.
    4. Akshay J. Maheshwari & Jonathan Calles & Sean K. Waterton & Drew Endy, 2023. "Engineering tRNA abundances for synthetic cellular systems," Nature Communications, Nature, vol. 14(1), pages 1-11, December.
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