A Robust and Quantitative Reporter System To Evaluate Noncanonical Amino Acid Incorporation in Yeast

Stieglitz, Jessica T; Kehoe, Haixing P.; Lei, Ming; Deventer, James A. Van

doi:10.1021/acssynbio.8b00260

Cited by 50 publications

(189 citation statements)

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“…The preparation of liquid and solid media was performed as described previously. 28 The strain RJY100 was constructed using standard homologous recombination approaches and has been described in detail previously. 37 The strain BY4741 (YSC1048) was purchased from Dharmacon.…”

Section: Methodsmentioning

confidence: 99%

“…The pCTCON2-FAPB2.3.6, pCTCON2-FAPB2.3.6L1TAG, pCTCON2-RYG, and pCTCON2-RXG reporter constructs have been previously described. 28 The pCTCON2-BXG reporter was cloned by replacing the RFP segment in pCTCON2-RXG with a BFP gene amplified from pBAD-mTagBFP2, obtained from the laboratory of Nikhil U. Nair at Tufts University and originally purchased from Addgene (Addgene plasmid # 34632; http://n2t.net/addgene:34632; RRID:Addgene_34632), by digesting both pCTCON2-RXG and the PCR-amplified BFP gene with EcoRI-HF and BamHI-HF (NEB), then ligating with T4 DNA ligase (NEB) to insert BFP. The pCTCON2-BXG-2TAG and pCTCON2-BXG-altTAG constructs were cloned by Gibson assembly with EcoRI-HF-and PstI-HF-digested pCTCON2-BXG.…”

Section: Methodsmentioning

confidence: 99%

“…Several reporter strategies for evaluating ncAA incorporation efficiency and fidelity have been described in the literature. 28, 29 By far the most common approach is single-fluorescent protein reporters. 14, 15 The primary advantage of these reporters is the easy-to-read fluorescent output, which in most cases is strongly correlated to the level of ncAA incorporation.…”

Section: Introductionmentioning

confidence: 99%

“…Recent reports have demonstrated the use of cell surface display systems for evaluating codon suppression events. 28, 30 We recently showed that detection of the N- and C-termini of yeast-displayed constructs facilitates the use of the rigorous relative readthrough efficiency and maximum misincorporation frequency metrics described above. In addition, the surface accessibility of the reporter enables the use of chemical modifications to confirm the presence of a ncAA containing a specified reactivity, reminiscent of earlier residue-specific ncAA incorporation engineering work.…”

Section: Introductionmentioning

confidence: 99%

“…In this study, we investigated the performance of three types of reporter systems that support fluorescence-based measurements of ncAA incorporation efficiency and fidelity. Our work here is conducted in S. cerevisiae , the only organism in which quantitative measurements of ncAA incorporation efficiency and fidelity with single-fluorescent protein reporters, 15, 35, 36 dual-fluorescent protein reporters, 28 and display-based reporters 28 have all previously been performed (Fig. 1).…”

Section: Introductionmentioning

confidence: 99%

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Reporter system architecture affects measurements of noncanonical amino acid incorporation efficiency and fidelity

Potts

Stieglitz

Lei

et al. 2019

Preprint

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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 readthough 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.Design, System, Application ParagraphOn earth, the genetic code provides nearly invariant instructions for generating the proteins present in all organisms using 20 primary amino acid building blocks. Scientists and engineers have long recognized the potential power of altering the genetic code to introduce amino acids that enhance the chemical versatility of proteins. Proteins containing such “noncanonical amino acids” (ncAAs) can be used to elucidate basic biological phenomena, discover new therapeutics, or engineer new materials. However, tools for measuring ncAA incorporation during protein translation (reporters) exhibit highly variable properties, severely limiting our ability to engineer improved ncAA incorporation systems. In this work, we sought to understand what properties of these reporters affect measurements of ncAA incorporation events. Using a series of ncAA incorporation systems in yeast, we evaluated reporter architecture, measurement techniques, and alternative data analysis methods. We identified key factors contributing to quantification of ncAA incorporation in all of these categories and demonstrated the immediate utility of our approach in identifying genomic knockouts that enhance ncAA incorporation efficiency. Our findings have important implications for how to evolve cells to better accommodate alternative genetic codes.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Reporter system architecture affects measurements of noncanonical amino acid incorporation efficiency and fidelity

Potts

Stieglitz

Lei

et al. 2019

Preprint

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Creation of a Yeast Strain with Co‐Translationally Acylated Nucleosomes

Liu

Zhang

et al. 2022

Angewandte Chemie

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Structurally diverse acylations have been identified as post-translational modifications (PTMs) on histone lysine residues, but their functions and regulations remain largely unknown. Interestingly, in nature, a lysine acylation analog, pyrrolysine, is introduced as a co-translational modification (CTM) through genetic encoding. To explore this alternative life form, we created a model organism Saccharomyces cerevisiae containing site-specific lysine CTMs (acetyllysine, crotonyl-lysine, or another synthetic analog) at histone H3K56 using non-canonical amino acid mutagenesis to afford a chemically modified nucleosome in lieu of their own in vivo. We further demonstrated that acetylation of histone H3K56 partly tends to provide a more favorable chromatin environment for DNA repair in yeast compared to crotonylation and crosstalk with other PTMs differently. This study provides a potentially universal approach to decipher the consequences of different histone lysine PTMs in eukaryotes.

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