ArticleMolecular systems design & engineering2020
Reporter system architecture affects measurements of noncanonical amino acid incorporation efficiency and fidelity.
Article in Molecular systems design & engineering, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers.
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Who cites it
18 citing papers in PubMed, 26 citations in OpenAlex.
- A functional surface display system iniScience · 2025Article
- High throughput screening of eukaryotic release factor 1 variants to enhance noncanonical amino acid incorporation.bioRxiv : the preprint server for biology · 2025Article
- Directed evolution of aminoacyl-tRNA synthetases through in vivo hypermutation.Nature communications · 2025Article
- Reaching New Heights in Genetic Code Manipulation with High Throughput Screening.Chemical reviews · 2024Review
- Genome-Wide Screen for Enhanced Noncanonical Amino Acid Incorporation in Yeast.Methods in molecular biology (Clifton, N.J.) · 2024Article
- Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast.Biochemistry · 2023Article
- Yeast Display Enables Identification of Covalent Single-Domain Antibodies against Botulinum Neurotoxin Light Chain A.ACS chemical biology · 2022Article
- Genome-Wide Screen for Enhanced Noncanonical Amino Acid Incorporation in Yeast.ACS synthetic biology · 2022Article
- High-Throughput Aminoacyl-tRNA Synthetase Engineering for Genetic Code Expansion in Yeast.ACS synthetic biology · 2022Article
- Exploration ofACS synthetic biology · 2022Article
- Incorporating, Quantifying, and Leveraging Noncanonical Amino Acids in Yeast.Methods in molecular biology (Clifton, N.J.) · 2022Article
- A CHO-Based Cell-Free Dual Fluorescence Reporter System for the Straightforward Assessment of Amber Suppression and scFv Functionality.Frontiers in bioengineering and biotechnology · 2022Article
- Engineering Proteins Containing Noncanonical Amino Acids on the Yeast Surface.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Broadening the Toolkit for Quantitatively Evaluating Noncanonical Amino Acid Incorporation in Yeast.ACS synthetic biology · 2021Article
- Development of mammalian cell logic gates controlled by unnatural amino acids.Cell reports methods · 2021Article
- Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells.Nucleic acids research · 2021Article
- Chemical Diversification of Simple Synthetic Antibodies.ACS chemical biology · 2021Article
- Directed Evolution of theInternational journal of molecular sciences · 2021Article
Corrections and comments
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
The ability to genetically encode noncanonical amino acids (ncAAs) within proteins supports a growing number of applications ranging from fundamental biological studies to enhancing the properties of biological therapeutics. Currently, our quantitative understanding of ncAA incorporation systems is confounded by the diverse set of characterization and analysis approaches used to quantify ncAA incorporation events. While several effective reporter systems support such measurements, it is not clear how quantitative results from different reporters relate to one another, or which details influence measurements most strongly. Here, we evaluate the quantitative performance of single-fluorescent protein reporters, dual-fluorescent protein reporters, and cell surface-displayed protein reporters of ncAA insertion in response to the TAG (amber) codon in yeast. While different reporters support varying levels of apparent readthrough efficiencies, flow cytometry-based evaluations with dual reporters yielded measurements exhibiting consistent quantitative trends and precision across all evaluated conditions. Further investigations of dual-fluorescent protein reporter architecture revealed that quantitative outputs are influenced by stop codon location and N- and C-terminal fluorescent protein identity. Both dual-fluorescent protein reporters and a "drop-in" version of yeast display support quantification of ncAA incorporation in several single-gene knockout strains, revealing strains that enhance ncAA incorporation efficiency without compromising fidelity. Our studies reveal critical details regarding reporter system performance in yeast and how to effectively deploy such reporters. These findings have substantial implications for how to engineer ncAA incorporation systems-and protein translation apparatuses-to better accommodate alternative genetic codes for expanding the chemical diversity of biosynthesized proteins.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.