2 nitrophenyl β d galactopyranoside

Gentamicin, agarose, chloroform and casamino acids were obtained from Fisher Scientific (Ottawa, ON, Canada). Erythromycin was purchased from Caledon Laboratories LTD (Georgetown, WA, Canada). Phenylalanine-Arginine-β-Naphthylamide, cholesterol and Triton X-100 were purchased from Sigma Aldrich (Oakville, ON, Canada). The compound 2-Nitrophenyl-β-d-galactopyranoside was obtained from Thermo Fisher Scientific (Ottawa, ON, Canada). DPPC (1,2-Dipalmitoyl-sn-glycero-3-phosphocholine) was obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Mueller–Hinton broth (MHB) and Lysogeny broth (LB) and agar were purchased from Becton Dickinson (Francklin Lakes, NJ, USA) and Becton Dickinson Microbiology Systems (Oakville, ON, Canada), respectively. ABt medium and Z buffer were prepared as described previously [47 (link),51 (link)].

To determine β‐galactosidase activity of yeast cells carrying the UPRE‐LacZ reporter, exponentially growing cells were lysed in Buffer Z (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, 50 mM 2‐mercaptoethanol) supplemented with 0.001% SDS and 2.5% chloroform. Lysates were incubated at 30°C for 15 minutes and the reaction was initiated with the addition of the substrate 2‐Nitrophenyl β‐D‐galactopyranoside (Thermo Scientific). When the mixture acquired a pale yellow colour the reaction was terminated by the addition of Na₂CO₃ and the absorbance at 420 nm and 550 nm was measured. Miller units were calculated according to the following formula: Miller Units = 1000 x (A420 ‐ (1.75 x A550)]/[Reaction time x Volume x OD600).