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- W4378803022 abstract "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." @default.
- W4378803022 created "2023-06-01" @default.
- W4378803022 creator A5052300018 @default.
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- W4378803022 creator A5088165113 @default.
- W4378803022 date "2023-05-31" @default.
- W4378803022 modified "2023-09-28" @default.
- W4378803022 title "A Cell-Free Gene Expression Platform for Discovering and Characterizing Stop Codon Suppressing tRNAs" @default.
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- W4378803022 doi "https://doi.org/10.1021/acschembio.3c00051" @default.
- W4378803022 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/37257197" @default.
- W4378803022 hasPublicationYear "2023" @default.
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