LAUSR

Non-canonical amino acids (ncAAs) can be incorporated into peptides and proteins to create new properties and functions. Site-specific ncAA incorporation is typically enabled by orthogonal translation systems comprising a stop codon suppressing tRNA (typically UAG), an aminoacyl-tRNA synthetase, and an ncAA of interest. Unfortunately, methods to discover and characterize suppressor tRNAs are limited because of laborious and time-consuming workflows in living cells. In this work, we develop anEscherichia coli crude extract-based cell-free gene expression system to rapidly express and characterize functional suppressor tRNAs. Our approach co-expresses orthogonal tRNAs using endogenous machinery alongside a stop-codon containing superfolder green fluorescent protein (sfGFP) reporter, which can be used as a simple read-out for suppression. As a model, we evaluate the UAG and UAA suppressing activity of several orthogonal tRNAs. Then, we demonstrate that co-transcription of two mutually orthogonal tRNAs can direct the incorporation of two unique ncAAs within a single modified sfGFP. Finally, we show that the cell-free workflow can be used to discover putative UAG-suppressor tRNAs found in metagenomic data, which are nonspecifically recognized by endogenous aminoacyl-tRNA synthetases. We anticipate that our cell-free system will accelerate the development of orthogonal translation systems for synthetic biology.