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

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. We reconstituted a complete set of 21 in vitro transcribed (IVT) tRNAs by improving the transcription yield of four tRNAs and optimized their abundance to improve protein yield. Next, we showed 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 encoding all 21 tRNAs. To enable synthesis of mature tRNAs from a circular template, we employed either a nicked plasmid template or T. maritima tRNase Z to post-transcriptionally process the precursor tRNAs. We ultimately achieved 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.