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<p>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 <i>T. maritima</i> 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.</p>

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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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  • 出版模式：

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  • 发表日期：

    2025年7月5日

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  • Fanjun Li,
  • Amogh Kumar Baranwal,
  • Sebastian J. Maerkl

摘要

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.