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.