Figure 1
From: Site Specific Genetic Incorporation of Azidophenylalanine in Schizosaccharomyces pombe
Suppression by orthogonal tRNA/aaRS pairs in S. pombe.
(A) Schematic representation of the DNA constructs. Construct 1: expressing S. pombe methionine tRNA (Sp tRNAMet) and S. pombe serine tRNA (Sp tRNASer). Construct 2: expressing S. pombe methionine tRNA (Sp tRNAMet) and sup3–5 (an anticodon mutant of S. pombe tRNAser). Construct 3: expressing B. stearothermophilus tyrosine tRNA carrying CUA in the anti-codon loop (Bst tRNATyr) and sup3–5. Construct 4: expressing E. coli tyrosine tRNA carrying CUA in the anti-codon loop (E. coli tRNATyr) and sup3–5. Constructs 2–4 carry a selectable marker, His5p, which carries an amber stop codon at position 63. (B) Strain construction. Strain 1: E. coli TyrRS was integrated at the leu1 locus and expressed under act1 promoter. Strain 2: E. coli AzFRS was integrated at the leu1 locus and expressed under act1 promoter. (C) Spot assay of amber codon suppression in S. pombe. The numbers above the cell spot assay images represents the DNA constructs (schematized in Fig. 1A) that were introduced into the ade6–704 his5Δ strain. (D) Spot assay for the AzF incorporation into His5p. In each blot, the left colony is formed by the S. pombe strain expressing E. coli TyrRS and E. coli tRNATyr. The right colony is formed by the S. pombe strain expressing E. coli AzFRS and E. coli tRNATyr. Both strains are plated in the minimal medium lacking adenine (top), histidine (middle) and histidine supplemented with 1 mM AzF (bottom), respectively.
