Journal: ACS synthetic biology

Article Title: Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli

doi: 10.1021/acssynbio.5b00197

Figure Lengend Snippet: Biochemical characterization of the aminoacylation efficiencies and dissociation constants of IFRS for each tRNAPyl anticodon variant. (a) The aminoacylation efficiency of IFRS for each of the anticodon variants was measured via an aminoacylation assay, where 3′-end-labeled tRNAPyl anticodon variants were incubated with IFRS and 3-I-Phe to measure the amount of product formed over 60 min at 37 °C. (b) Binding affinities were determined via filter binding assay, where varying concentrations of IFRS were incubated on ice with 3′-end-labeled tRNAPyl and the fraction of bound IFRS was measured to obtain dissociation constants.

Article Snippet: The aminoacylation assays were carried out for 60 min at 37 °C in aminoacylation buffer containing 100 mM Tris-HCl (pH 7.0), 10 mM MgCl 2 , 50 mM potassium chloride, and 5 mM DTT, with 1 mM 3-I-Phe (ChemImpex), 2 mM ATP, 500 nM IFRS, and 5 µM 32 P 3′-end-labeled transcript.

Techniques: Variant Assay, Aminoacylation Assay, Labeling, Incubation, Binding Assay, Filter-binding Assay

Journal: ACS synthetic biology

Article Title: Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli

doi: 10.1021/acssynbio.5b00197

Figure Lengend Snippet: Northern blot analyses of intracellular aminoacylation of tRNAPyl isoacceptors. Total RNA was obtained from E. coli cells grown in the presence or absence of 3-I-Phe (1 mM) expressing IFRS and (A) either one or five gene copies of tRNAPylACU or (B) one gene copy of tRNAPylCAG, from the pCAM plasmid. Total RNA was suspended in either 10 mM sodium acetate, pH 4.5 (acylated), or 200 mM Tris, pH 8.0 (deacylated, OH−). Positions of acylated and deacylated tRNAPyl isoacceptors are indicated.

Article Snippet: The aminoacylation assays were carried out for 60 min at 37 °C in aminoacylation buffer containing 100 mM Tris-HCl (pH 7.0), 10 mM MgCl 2 , 50 mM potassium chloride, and 5 mM DTT, with 1 mM 3-I-Phe (ChemImpex), 2 mM ATP, 500 nM IFRS, and 5 µM 32 P 3′-end-labeled transcript.

Techniques: Northern Blot, Expressing, Plasmid Preparation

Journal: ACS synthetic biology

Article Title: Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli

doi: 10.1021/acssynbio.5b00197

Figure Lengend Snippet: Positions of successful 3-I-Phe incorporation in sfGFP, as detected by LC-MS/MS of chymotrypsin/trypsin-digested samples. E. coli cultures expressing each individual tRNAPyl anticodon variant (tRNAPylACU, tRNAPylCGA, or tRNAPylCAG) were grown in the presence of 1 mM 3-I-Phe, and sfGFP was overexpressed, purified, and chymotrypsin/trypsin-digested, followed by analysis via tandem mass spectrometry. Triangles denote targeted codons; empty triangles indicate the failure of 3-I-Phe incorporation, and filled triangles indicate the successful incorporation of 3-I-Phe.

Article Snippet: The aminoacylation assays were carried out for 60 min at 37 °C in aminoacylation buffer containing 100 mM Tris-HCl (pH 7.0), 10 mM MgCl 2 , 50 mM potassium chloride, and 5 mM DTT, with 1 mM 3-I-Phe (ChemImpex), 2 mM ATP, 500 nM IFRS, and 5 µM 32 P 3′-end-labeled transcript.

Techniques: Liquid Chromatography with Mass Spectroscopy, Expressing, Variant Assay, Purification, Mass Spectrometry

Journal: ACS synthetic biology

Article Title: Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli

doi: 10.1021/acssynbio.5b00197

Figure Lengend Snippet: Tandem mass spectrometric analysis of the LSTQS208VLXK fragment of sfGFP purified from E. coli expressing tRNAPylACU. The notations b0 and y0 denote loss of water from the b and y fragments, respectively. X denotes 3-I-Phe incorporated at the targeted Ser208AGU position. This peptide contains 3 different serine codons, of which only 1 was encoded by AGU (bolded), decoded by tRNAPylACU, and reassigned to 3-I-Phe.

Article Snippet: The aminoacylation assays were carried out for 60 min at 37 °C in aminoacylation buffer containing 100 mM Tris-HCl (pH 7.0), 10 mM MgCl 2 , 50 mM potassium chloride, and 5 mM DTT, with 1 mM 3-I-Phe (ChemImpex), 2 mM ATP, 500 nM IFRS, and 5 µM 32 P 3′-end-labeled transcript.

Techniques: Purification, Expressing

Journal: ACS synthetic biology

Article Title: Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli

doi: 10.1021/acssynbio.5b00197

Figure Lengend Snippet: Extracted ion chromatograms from SRM analyses to quantify 3-I-Phe incorporation at Ser208AGU of sfGFP. The extent of 3-I-Phe incorporation was measured by quantitative mass spectrometric analyses (SRM) to be 65 ± 17% at position Ser208AGU. Signals from known amounts of the isotopically labeled Ser (blue, in foreground; 100 fmol) and 3-I-Phe (pink, in foreground; 500 fmol) peptides are superposed on signals from the digested sample, which contained a heterogeneous mixture of Ser (blue, at back; 432.6 fmol), 3-I-Phe (pink, at back; 776.5 fmol), Thr, and Phe peptides.

Article Snippet: The aminoacylation assays were carried out for 60 min at 37 °C in aminoacylation buffer containing 100 mM Tris-HCl (pH 7.0), 10 mM MgCl 2 , 50 mM potassium chloride, and 5 mM DTT, with 1 mM 3-I-Phe (ChemImpex), 2 mM ATP, 500 nM IFRS, and 5 µM 32 P 3′-end-labeled transcript.

Techniques: Labeling