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A Cell-Free Gene Expression Platform for Discovering and Characterizing Stop Codon Suppressing tRNAs

Authors :
Seki, Kosuke
Galindo, Joey L.
Karim, Ashty S.
Jewett, Michael C.
Source :
ACS Chemical Biology; June 2023, Vol. 18 Issue: 6 p1324-1334, 11p
Publication Year :
2023

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 colicrude 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.

Details

Language :
English
ISSN :
15548929 and 15548937
Volume :
18
Issue :
6
Database :
Supplemental Index
Journal :
ACS Chemical Biology
Publication Type :
Periodical
Accession number :
ejs63204679
Full Text :
https://doi.org/10.1021/acschembio.3c00051