F500 plate reader

KaiA, KaiB, KaiC were prepared according to standard protocols^{43 (link)}, but the anion exchange chromatography step was omitted in the KaiC purification protocol. Fluorescently labeled KaiB (KaiB-IAF) was prepared by an overnight labeling reaction of KaiB-K25C with 6-iodoacetofluorescein^{43 (link)}. Protein concentrations were measured via densitometry on an SDS-PAGE gel against a BSA standard (Biorad). KaiABC reactions at different concentration were prepared by serial dilution of a master mix prepared at 2× standard concentration (7 μM KaiB and KaiC, 3 μM KaiA, 0.4 μM KaiB-IAF) in standard reaction buffer containing 2.5 mM ATP (20 mM Tris [pH 8], 150 mM NaCl, 5 mM MgCl2, 10% glycerol, 0.5 mM EDTA, 2.5 mM ATP). To monitor oscillations, reactions were transferred to a black 384-well plate (25 μL/well) and placed into a Tecan F500 plate reader pre-warmed to 30 °C^{43 (link)}. Parallel and perpendicular fluorescence signals (exc. 485 nm, 20 nm bandpass; emm. 535 nm, 25 nm bandpass; dichroic 510 nm.) were read out every 15 min. Wells containing buffer only were used as blanks. The G factor was adjusted to give 20 mP in the wells containing a solution of free fluorescein.

All laboratory evolution experiments were carried out by serial dilution in 24-well plates with breathable seals. Plates were grown on an orbital shaker at 600 rpm in 30 °C. Each evolving strain was grown on 1.2 ml of YP-xylulose medium until reaching stationary phase and then diluted daily by a factor of 1:64 into fresh media (6 generations per dilution). Growth of wild type and evolved populations was characterized using a Hamilton robotic system. Cultures were grown in 96-well plates with 600 rpm shaking in 30 °C. Mineral oil was added to each well to prevent evaporation. OD₆₀₀ measurements were taken during growth using a Tecan F500 plate reader.

eIF2B guanine nucleotide exchange activity was measured as described previously (Sekine et al., 2015 (link)), with minor modifications. Briefly, purified decameric eIF2B (final concentration 2.5 - 40 nM), phosphorylated or unphosphorylated eIF2 (final concentration 0 - 1 μM), ISRIB (a gift of Peter Fischer, U, Nottingham) dissolved in DMSO (final concentration 0.25 - 1 μM) or DMSO carrier control (final concentration < 5%^V/_V) were pre-assembled in assay buffer (20 mM HEPES-KOH pH 7.4, 150 mM KCl, 2 mM MgCl₂, 1 mM TCEP, 0.05 mg/mL bovine serum albumin, 0.01% Triton X-100, 1.5 mM GDP) and allowed to equilibrate for 10 minutes at room temperature in a low volume, “U” bottom, black 394 well plate (Corning, Cat #3667). At t = 0 purified eIF2(α^S51A), preloaded with BODIPY-FL-GDP (Invitrogen, G22360) (as previously described) (Sekine et al., 2015 (link)) was introduced at a final concentration of 125 nM and the fluorescence signal read kinetically in a Tecan F500 plate reader (Excitation wavelength: 485 nm, bandwidth 20 nm, Emission wavelength: 535 nm, bandwidth 25 nm). Where indicated, the data were fitted to a single-phase exponential decay function using GraphPad Prism V8: [Y = (Y0 - Plateau)^∗exp(-K^∗X) + Plateau], where Y0 is the Y value when X (time) is zero, Plateau is the Y value at infinite times, K is the rate constant expressed in reciprocal of the x axis time units.