CDPS enzymes use aatRNA molecules as substrates, hijacking the already aminoacylated tRNA within cells for cyclodipeptide synthesis. Purified tRNA was previously synthesised by us and others using a time consuming in vitro transcription reaction which includes an initial PCR of the desired tRNA sequence to amplify the DNA template encoding the desired tRNA sequence, followed by in vitro transcription using a mutant T7 RNA polymerase (Δ172-173) to ensure homogeneous 3'-end in the tRNA, finishing with a phenol-chloroform extraction to yield purified tRNA. This method, whilst reliable, is costly, lengthy and requires specialised materials.