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

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biochemistry

Background:

• Non-canonical amino acids (ncAAs) enable novel protein properties and functions.
• Site-specific ncAA incorporation relies on orthogonal translation systems (OTS).
• Current methods for discovering suppressor tRNAs are laborious and time-consuming.

Purpose of the Study:

• To develop a rapid cell-free gene expression system for functional characterization of suppressor tRNAs.
• To demonstrate the utility of this system for discovering novel suppressor tRNAs and enabling multiplexed ncAA incorporation.

Main Methods:

• Utilized an *Escherichia coli* crude extract-based cell-free system.
• Co-expressed orthogonal tRNAs with a superfolder green fluorescent protein (sfGFP) reporter containing a stop codon.
• Evaluated UAG and UAA suppressing activity and demonstrated co-transcription of two orthogonal tRNAs for dual ncAA incorporation.

Main Results:

• Successfully expressed and characterized functional suppressor tRNAs in a cell-free system.
• Demonstrated multiplexed ncAA incorporation into a single protein.
• Identified putative UAG-suppressor tRNAs from metagenomic data using the cell-free workflow.

Conclusions:

• The developed cell-free system significantly accelerates the discovery and characterization of functional suppressor tRNAs.
• This platform facilitates the rapid expansion of orthogonal translation systems for synthetic biology.
• Enables efficient engineering of proteins with novel functionalities through ncAA incorporation.