Erlenmeyer flasks

The saltwater medium A+ used in batch experiments contained, per liter, 18 g NaCl, 5 g MgSO₄⋅7H₂O, 1 g NaNO₃, 0.6 g KCl, 0.05 g KH₂PO₄, 0.03 g Na₂-EDTA, 0.27 g CaCl₂, 1 g Trizma base (Tris), 1 mL L⁻¹ of 3.89 g L⁻¹ FeCl₃⋅6H₂O stock in 0.1 N HCl, and 1 mL L⁻¹ of P1 metals micronutrient solution. The P1 stock solution contained, per liter, 34.26 g H₃BO₃, 4.32 g MnCl₂⋅4H₂O, 0.315 g ZnCl, 0.03 g MoO₃ (85%), 12.15 mg CoCl₂⋅6H₂O, and 3 mg CuSO₄⋅5H₂O. For A+ medium without nitrogen (−N), NaNO₃ was replaced by an equimolar amount of NaCl.
Liquid cell cultures were grown using a rotary shaker under constant illumination in an atmosphere of 1% CO₂, 34°C, and 160 μmol photons m⁻² s⁻¹ (μmol m⁻² s⁻¹ hereafter) photosynthetically active radiation (PAR) in 250-mL Erlenmeyer flasks with soft caps to facilitate gas exchange (VWR, Radnor, PA, USA). Batch flask cultures were grown in quadruplicate and standardized to 2.5 mg L⁻¹ chlorophyll a at the beginning of each experiment. Pre-cultures were similarly normalized and grown to mid-linear phase (15–25 μg mL⁻¹ chlorophyll a), whereupon cells were concentrated by centrifugation and resuspended in fresh medium for experimental replicates, which were sampled over a time course.

RBDs for SARS-CoV-2 and the seasonal coronaviruses were expressed in the Expi293F human expression system (Gibco, Maryland, USA) as secreted proteins. In brief, the gene encoding each RBD with the signal peptide of human tissue plasminogen activator and expression tags in the following configuration: (MDAMKRGLCCVLLLCGAVFVSP)-RBD protein-(GGGGS)₃-HaloTag-3xFLAG, was synthesized into the pcDNA3.4 expression vector. Plasmid DNA was prepped with NucleoBond Xtra Midi kit for transfection-grade plasmid DNA (Macherey-Nagel, PA, USA). Expi293F cells (Gibco) were grown in Opti-MEM Reduced Serum Medium (Gibco) and transfected using the ExpiFectamine293 Transfection Kit (Gibco). The RBD proteins were expressed in Erlenmeyer Flasks (VWR International, Pennsylvania, USA) at 37°C with 8% CO₂ on an orbital shaker (Laboratory Supply Network, USA). At 4.5 days post-transfection, the cells were harvested and centrifuged 20 min at 4,000 × g at 4°C. The supernatant was collected at 3,000 × g for 25 min at 4°C, filtered using a 0.22-µm Stericup filter, and stored at −80°C until usage.

Seed cultures were grown under 30 μmol photons m⁻² s⁻¹ at 30 °C in BG11 with appropriate antibiotic(s) in 100 mL Erlenmeyer flasks (VWR) until OD₇₅₀ = 1.5–2.0. The seed cultures were then used to inoculate 25 mL experimental cultures to OD₇₅₀ = 0.1 in BioLite 25 cm² plug-sealed tissue culture flasks (Thermo Fisher Scientific). The medium used for experimental cultures was BG11 with addition of 50 mM NaHCO₃ (Sigma-Aldrich) and appropriate antibiotic(s) (final concentrations: chloramphenicol, 10 μg mL⁻¹; spectinomycin, 25 μg mL⁻¹; erythromycin, 25 μg mL⁻¹; and kanamycin, 25 μg mL⁻¹). All experimental cultures were prepared in quadruplicates. The flasks were shaken horizontally at 120 rpm, under 50 μmol photons m⁻² s⁻¹ at 30 °C. Two milliliters of culture were sampled from each flask every second day for measurements and 2 mL of fresh BG11 medium with addition of 500 mM NaHCO₃ (Sigma-Aldrich) and appropriate antibiotic(s) were added back. The pH of experimental cultures was measured with MColorpHast™ pH-indicator strips (pH 6.5–10) (Merck) and the cultures pH were adjusted to the range between 7–8 using 37% HCl (Sigma-Aldrich).