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: Biochemical Characterization of the IFRS/tRNA Pyl Pair 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