Figure 3

Detection of unnatural amino acid incorporation by Western blot and fluorescence imaging.

(A) Schematic representation of mutation sites in Rlc1p-GFP and Glutathion-S-transferase (GST). (B) Top: S. pombe cells expressing E. coli TyrRS and Rlc1p-TAG-GFP from a plasmid (MBY 8965) were grown at 30 °C in the presence or absence of 1 mM azido-phenylalanine to exponential growth phase and live cells were imaged using Andor Spining Disk. 5 μm. Bottom: S. pombe cells expressing E. coli AzFRS and Rlc1p-TAG-GFP from a plasmid (MBY 8966) were grown at 30 °C in the presence or absence of 1 Mm azido-phenylalanine to exponential growth phase. Cell were imaged using Andor Spining Disk. 5 μm. Please refer to Supplemental Table S1 for the strain numbers. (C) AzF has no adverse effect on cell growth and cell cycle progression. The E. coli TyrRS cells expressing Rlc1p-TAG-GFP in the absence or presence of AzF were quantified. The relative proportions of mononucleated cells (shown as blue bars), mononucleated cells with actomyosin ring (shown as red bars), binucleated cells (shown as purple bars) and binucleated cells with actomyosin ring (shown as green bars) were quantitated. The mean relative proportion of each category from three independent experiments is shown (N > 60 for each strain in each experiment). (D) The cell extract were prepared from E. coli TyrRS strain expressing GST (G6stop)-6xHIS (MBY 10031) in the AzF+ and AzF− medium, respectively. The same as E. coli AzFRS strain expressing GST (G6stop)-6xHIS (MBY 10026). In total, all the four samples were purified with Ni-NTA, respectively and detected with α-GST antibodies. Please refer to Supplemental Table S1 for the strain number.