We then adapted a procedure by Mechulam et al. to produce a 'pool' of tRNA naturally found in E. coli. Using this method, a highly concentrated stock of all the tRNAs required for our experiments was purified and used with any CDPS/aaRSs (aminoacyl-tRNA synthetase) combination of choice. More specifically, ParaCDPS and ParcuCDPS could use this tRNA pool to generate their respective products -cHF and cHP -in relatively high yield (Fig. ). In comparison, we also performed reactions using in vitro transcribed tRNA to confirm that the same cyclodipeptides were produced, however the tRNA pool method remains significantly cheaper, easier, and faster than previously described methods and has become a staple in our research with CDPSs. This method could be used for other enzymes that use aatRNAs as substrates, such as Fem transferases and LanB-like dehydratases. Overall, the tRNA pool was a far superior alternative to produce high quality tRNA for use and therefore was employed in subsequent reactions.