Continuous in situ synthesis of a complete set of tRNAs sustains steady-state translation in a recombinant cell-free system
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...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-07-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-61671-8 |
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| author | Fanjun Li Amogh Kumar Baranwal Sebastian J. Maerkl |
| author_facet | Fanjun Li Amogh Kumar Baranwal Sebastian J. Maerkl |
| author_sort | Fanjun Li |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-c2af97969fa44521ace34b401a672c2f |
| institution | DOAJ |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-c2af97969fa44521ace34b401a672c2f2025-08-20T03:03:38ZengNature PortfolioNature Communications2041-17232025-07-0116111210.1038/s41467-025-61671-8Continuous in situ synthesis of a complete set of tRNAs sustains steady-state translation in a recombinant cell-free systemFanjun Li0Amogh Kumar Baranwal1Sebastian J. Maerkl2Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de LausanneInstitute of Bioengineering, School of Engineering, École Polytechnique Fédérale de LausanneInstitute of Bioengineering, School of Engineering, École Polytechnique Fédérale de LausanneAbstract 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.https://doi.org/10.1038/s41467-025-61671-8 |
| spellingShingle | Fanjun Li Amogh Kumar Baranwal Sebastian J. Maerkl Continuous in situ synthesis of a complete set of tRNAs sustains steady-state translation in a recombinant cell-free system Nature Communications |
| title | Continuous in situ synthesis of a complete set of tRNAs sustains steady-state translation in a recombinant cell-free system |
| title_full | Continuous in situ synthesis of a complete set of tRNAs sustains steady-state translation in a recombinant cell-free system |
| title_fullStr | Continuous in situ synthesis of a complete set of tRNAs sustains steady-state translation in a recombinant cell-free system |
| title_full_unstemmed | Continuous in situ synthesis of a complete set of tRNAs sustains steady-state translation in a recombinant cell-free system |
| title_short | Continuous in situ synthesis of a complete set of tRNAs sustains steady-state translation in a recombinant cell-free system |
| title_sort | continuous in situ synthesis of a complete set of trnas sustains steady state translation in a recombinant cell free system |
| url | https://doi.org/10.1038/s41467-025-61671-8 |
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