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Genetic code expansion (GCE) is a versatile tool to site‐specifically incorporate a noncanonical amino acid (ncAA) into a protein, for example, to perform fluorescent labeling inside living cells. To this end, an orthogonal aminoacyl‐tRNA‐synthetase/tRNA (RS/tRNA) pair is used to insert the ncAA in response to an amber stop codon in the protein of interest. One of the drawbacks of this system is that, in order to achieve maximum efficiency, high levels of the orthogonal tRNA are required, and this could interfere with host cell functionality. To minimize the adverse effects on the host, we have developed an inducible GCE system that enables us to switch on tRNA or RS expression when needed. In particular, we tested different promotors in the context of the T‐REx or Tet‐On systems to control expression of the desired orthogonal tRNA and/or RS. We discuss our result with respect to the control of GCE components as well as efficiency. We found that only the T‐REx system enables simultaneous control of tRNA and RS expression., Genetic code expansion on demand by combining the amber suppression technology with the T‐REx system. Amber suppression is regulated by the T‐REx system to switch on the tRNA production and/or the synthetase translation only when needed, thus minimizing the risk of adverse effects for the cells resulting from over‐production of unused tRNA or synthetase.

A bio-orthogonal and unnatural substance, such as an unnatural amino acid (Uaa), is an ideal regulator to control target gene expression in a synthetic gene circuit. Genetic code expansion technology has achieved Uaa incorporation into ribosomal synthesized proteins in vivo at specific sites designated by UAG stop codons. This site-specific Uaa incorporation can be used as a controller of target gene expression at the translational level by conditional read-through of internal UAG stop codons. Recent advances in optimization of site-specific Uaa incorporation for translational regulation have enabled more precise control over a wide range of novel important applications, such as Uaa-auxotrophy-based biological containment, live-attenuated vaccine, and high-yield zero-leakage expression systems, in which Uaa translational control is exclusively used as an essential genetic element. This review summarizes the history and recent advance of the translational control by conditional stop codon read-through, especially focusing on the methods using the site-specific Uaa incorporation.

Site-specific incorporation of noncanonical amino acids (ncAAs) into proteins expressed in E. coli using UAG-suppression competes with termination mediated by release factor 1 (RF1). Recently, unconditional deletion of RF1 was achieved in a genomically recoded E. coli (C321), devoid of all endogenous UAG stop codons. Here we evaluate the efficiency of ncAA incorporation in this strain using optimized suppression vectors. Even though the absence of RF1 does not benefit the suppression efficiency of a single UAG codon, multi-site incorporation of a series of chemically distinct ncAAs was significantly improved.

N-(e)-lysine-acetylation (short: lysine-acetylation) is a dynamic and powerful posttranslational modification to regulate protein function. Mutational approaches are often poor to access the real mechanistic impact of lysine-acetylation at the molecular level. Therefore, the ability to site-specifically incorporate N-(e)-acetyl-L-lysine (short: AcK) into proteins dramatically increased our understanding how lysine-acetylation regulates protein function by using diverse molecular mechanisms going far beyond neutralizing a positive charge at the lysine-side chain. Genetically encoding AcK is a powerful way to introduce AcK into proteins, resulting in homogenously, quantitatively, and site-specifically lysine-acetylated proteins. Thereby, lysine-acetylated proteins can be produced in their natively-folded state in a high quality and in a yield sufficient to perform biophysical studies, including X-ray crystallography. This protocol describes the expression and purification of site-specifically lysine-acetylated proteins in Escherichia coli using the genetic-code expansion concept (GCEC) and subsequent steps to assess the successful incorporation of AcK by immunoblotting and mass-spectrometry.

Expansion of the genetic code allows unnatural amino acids (Uaas) to be site-specifically incorporated into proteins in live biological systems, thus enabling novel properties selectively introduced into target proteins in vivo for basic biological studies and for engineering of novel biological functions. Orthogonal components including tRNA and aminoacyl-tRNA synthetase (aaRS) are expressed in live cells to decode a unique codon (often the amber stop codon UAG) as the desired Uaa. Initially developed in E. coli, this methodology has now been expanded in multiple eukaryotic cells and animals. In this Account, we focus on addressing various biological challenges for rewriting the genetic code, describing impacts of code expansion on cell physiology and discussing implications for fundamental studies of code evolution. Specifically, a general method using the type-3 polymerase III promoter was developed to efficiently express prokaryotic tRNAs as orthogonal tRNAs and a transfer strategy was devised to generate Uaa-specific aaRS for use in eukaryotic cells and animals. The aaRSs have been found to be highly amenable for engineering substrate specificity toward Uaas that are structurally far deviating from the native amino acid, dramatically increasing the stereochemical diversity of Uaas accessible. Preparation of the Uaa in ester or dipeptide format markedly increases the bioavailability of Uaas to cells and animals. Nonsense-mediated mRNA decay (NMD), an mRNA surveillance mechanism of eukaryotic cells, degrades mRNA containing a premature stop codon. Inhibition of NMD increases Uaa incorporation efficiency in yeast and Caenorhabditis elegans. In bacteria, release factor one (RF1) competes with the orthogonal tRNA for the amber stop codon to terminate protein translation, leading to low Uaa incorporation efficiency. Contradictory to the paradigm that RF1 is essential, it is discovered that RF1 is actually nonessential in E. coli. Knockout of RF1 dramatically increases Uaa incorporation efficiency and enables Uaa incorporation at multiple sites, making it feasible to use Uaa for directed evolution. Using these strategies, the genetic code has been effectively expanded in yeast, mammalian cells, stem cells, worms, fruit flies, zebrafish, and mice. It is also intriguing to find out that the legitimate UAG codons terminating endogenous genes are not efficiently suppressed by the orthogonal tRNA/aaRS in E. coli. Moreover, E. coli responds to amber suppression pressure promptly using transposon insertion to inactivate the introduced orthogonal aaRS. Persistent amber suppression evading transposon inactivation leads to global proteomic changes with a notable up-regulation of a previously uncharacterized protein YdiI, for which an unexpected function of expelling plasmids is discovered. Genome integration of the orthogonal tRNA/aaRS in mice results in minor changes in RNA transcripts but no significant physiological impairment. Lastly, the RF1 knockout E. coli strains afford a previously unavailable model organism for studying otherwise intractable questions on code evolution in real time in the laboratory. We expect that genetically encoding Uaas in live systems will continue to unfold new questions and directions for studying biology in vivo, investigating the code itself, and reprograming genomes for synthetic biology.

The pyrrolysine tRNA synthetase-tRNA pair is probably one of the most promiscuous tRNA-synthetase pairs found in nature, capable of genetically encoding a plethora of noncanonical amino acids through stop codon reassignment. Proteins containing reactive handles, post-translational modification mimics or both can be produced in practical quantities, allowing inter alia the probing of biological pathways, generating antibody-drug conjugates and enhancing protein function. This Minireview summarises the development of pyrrolysine amber stop-codon suppression, presents some of the considerations required to utilise this technique to its greatest potential, and showcases the creative ways in which this technique has led to a better understanding of biological systems.

By introducing engineered tRNA and aminoacyl-tRNA synthetase pairs into an organism, its genetic code can be expanded to incorporate nonstandard amino acids (nsAAs). The performance of these orthogonal translation systems (OTSs) varies greatly, however, with respect to the efficiency and accuracy of decoding a reassigned codon as the nsAA. To enable rapid and systematic comparisons of these critical parameters, we developed a toolkit for characterizing any Escherichia coli OTS that reassigns the amber stop codon (TAG). It assesses OTS performance by comparing how the fluorescence of strains carrying plasmids encoding a fused RFP-GFP reading frame, either with or without an intervening TAG codon, depends on the presence of the nsAA. We used this kit to (1) examine nsAA incorporation by seven different OTSs, (2) optimize nsAA concentration in growth media, (3) define the polyspecificity of an OTS, and (4) characterize evolved variants of amberless E. coli with improved growth rates.

Bioengineering advances have made it possible to fundamentally alter the genetic codes of organisms. However, the evolutionary consequences of expanding an organism's genetic code with a non-canonical amino acid are poorly understood. Here we show that bacteriophages evolved on a host that incorporates 3-iodotyrosine at the amber stop codon acquired neutral and beneficial mutations to this new amino acid in their proteins, demonstrating that an expanded genetic code increases evolvability.

Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting incorporation efficiency are largely unknown. This manuscript describes the use of green fluorescent protein (GFP) reporters to quantify the efficiency of amber codon reassignment using the Methanocaldococcus jannaschii orthogonal pair system, commonly employed for ncAA incorporation, and quantify the contribution of release factor 1 (RF1) to the overall efficiency of amino acid incorporation. The efficiencies of amber codon reassignments were quantified at eight positions in GFP and evaluated in multiple combinations. The quantitative contribution of RF1 competition to reassignment efficiency was evaluated through comparisons of amber codon suppression efficiencies in normal and genomically recoded Escherichia coli strains. Measured amber stop codon reassignment efficiencies for eight single stop codon GFP variants ranged from 51 to 117% in E. coli DH10B and 76 to 104% in the RF1 deleted E. coli C321.&Delta, A.exp. Evaluation of efficiency changes in specific sequence contexts in the presence and absence of RF1 suggested that RF1 specifically interacts with +4 Cs and that the RF1 interactions contributed approximately half of the observed sequence context-dependent variation in measured reassignment efficiency. Evaluation of multisite suppression efficiencies suggests that increasing demand for translation system components limits multisite incorporation in cells with competing RF1.

G protein-coupled receptor (GPCR) signaling complexes are important for mediating many different biological processes. Uncovering the mechanism for how a ligand triggers a GPCR to elicit a specific response is an active area of research. One step toward understanding this mechanism is through identifying a ligand's binding site on a GPCR. We have optimized a targeted photocrosslinking technology to detect the residues in a receptor that are within a precise distance from a bound ligand in the receptor-ligand complex. Here, we describe the method for introducing photoactivable crosslinkers into a GPCR using the amber stop codon suppression technology. In addition, we review the steps to identify the binding site of a fluorescein-tagged peptide ligand and a tritium-labeled small molecule ligand.

A lethal ccdB gene-based vector was developed for the construction of shotgun libraries in Escherichia coli. The mutated ccdB gene carries multiple cloning sites and the amber stop codon, which simultaneously allows for maintenance of the intact vector in supE(-) strains and removal of uncut and self-ligated plasmids in supE(+) strains.

Organisms that use the standard genetic code recognize UAA, UAG, and UGA as stop codons, whereas variant code species frequently alter this pattern of stop codon recognition. We previously demonstrated that a hybrid eRF1 carrying the Euplotes octocarinatus domain 1 fused to Saccharomyces cerevisiae domains 2 and 3 (Eo/Sc eRF1) recognized UAA and UAG, but not UGA, as stop codons. In the current study, we identified mutations in Eo/Sc eRF1 that restore UGA recognition and define distinct roles for the TASNIKS and YxCxxxF motifs in eRF1 function. Mutations in or near the YxCxxxF motif support the cavity model for stop codon recognition by eRF1. Mutations in the TASNIKS motif eliminated the eRF3 requirement for peptide release at UAA and UAG codons, but not UGA codons. These results suggest that the TASNIKS motif and eRF3 function together to trigger eRF1 conformational changes that couple stop codon recognition and peptide release during eukaryotic translation termination.

We show by sequence analysis of a 420-base-long region adjacent to the 3' polyadenylic acid of encephalomyocarditis viral RNA and by carboxy terminus analysis of protein E that the termination site of encephalomyocarditis virus polyprotein translation consists of two successive UAG codons located at positions 121 to 126 from the 3' polyadenylic acid.