Imaging Tae1 Expression and Protein Synthesis

Chemically competent cells were transformed with pBAD24 constructs: (pBAD24::tae1^WT, pBAD24::tae1^C30A,or pBAD24) and selected with Carb overnight in liquid LB. Cultures were diluted by 1:100 and grown in LBNS+ Carb+ 100μM IPTG at 37°C with shaking. At early log phase (~80 minutes) 0.125% arabinose was added to induce Tae1 expression. After 35 minutes, 13μM O-propargyl-puromycin (OPP) was added to cultures to label new peptide synthesis before harvesting (Click-iT Plus OPP Alexa Fluor 488 Protein Synthesis Assay Kit, Invitrogen)[88 (link)]. After labelling, cells were pelleted and fixed in 3.7% formaldehyde in PBS. Cells were permeabilized with 0.3% Triton X-100 in PBS for 15 min, then labelled for imaging with Click-iT reaction cocktail for 20 min in the dark, washed then resuspended in PBS. Fluorescence imaging was performed on a Nikon Eclipse Ti2-E inverted microscope equipped with a 100x/1.40 oil-immersion objective and an EMCCD camera (Prime 95B). The 488-nm laser illumination fluorescence and phase-contrast images were captured using NIS-Elements AR Viewer 5.20 and analyzed using MicrobeJ software for Fiji [84 (link),87 (link)].