NextSeq 500 paired end sequencing; Quantification of global gene expression control by growth rate and metabolic cues
Quantification of global gene expression control by growth rate and metabolic cues
The dead volume was collected with a syringe and discarded. For transcriptome sampling, biomass was collected from the reactor with a syringe and injected into chilled 50 ml Falcon tubes filled with 35 mL crushed ice. Samples were centrifuged for 4 min at 3,000 x g at 4oC; cell pellets were washed once with 1 mL of chilled water, transferred into Eppendorf tubes, flash frozen in liquid nitrogen, and stored at -80oC until analysis. The yeast Saccharomyces cerevisiae CEN.PK113-7D (MATa, MAL2-8c, SUC2) was used for all experiments. Cells were stored in aliquoted glycerol stocks at -80 oC. Chemostat experiments were carried out in DASGIP 1L bioreactors (Jülich, Germany) equipped with off-gas analysis, pH, temperature and dissolved oxygen sensors. Chemostat experiments were carried out at 30 oC, pH 5, working volume 0.5 L, aeration 1 vvm, pO2 >30%, agitation speed 800 rpm. For GR subset (Fig 1B) chemostats, media contained 0.5 g L-1 (NH4)2SO4, as well as: glucose⋅H2O, 7.5 g L-1; KH2PO4, 3 g L-1; MgSO4·7H2O, 0.5 g L-1; trace metals solution, 1 ml L-1; vitamin solution, 1 ml L-1; antifoam, 0.1 ml L-1. For AA subset, nitrogen-limited chemostats, media contained either 1.24 g L-1  Phe;  0.98 g L-1 Ile; 0.5 g L-1  (NH4)2SO4; or 0.55 g L-1  Gln, as well as: glucose⋅H2O, 7.5 g L-1; KH2PO4, 3 g L-1; MgSO4·7H2O, 0.5 g L-1; trace metals solution, 1 ml L-1; vitamin solution, 1 ml L-1; antifoam, 0.1 ml L-1. For AA subset, carbon-limited chemostats on Phe, Ile, and ammonium, media contained either 12.35 g L-1  Phe; 9.81 g L-1  Ile; or 5 g L-1  (NH4)2SO4, as well as: glucose⋅H2O, 7.5 g L-1; KH2PO4, 3 g L-1; MgSO4·7H2O, 0.5 g L-1; trace metals solution, 1 ml L-1; vitamin solution, 1 ml L-1; antifoam, 0.1 ml L-1. For AA subset, carbon-limited chemostats on Gln, media contained 5.54 g L-1  Gln with either 7.5 g L-1 glucose⋅H2O (labeled “Q” in Fig 1B) or 1.21 g L-1 glucose⋅H2O (labeled “Q*” in Fig 1B), as well as: KH2PO4, 3 g L-1; MgSO4·7H2O, 0.5 g L-1; trace metals solution, 1 ml L-1; vitamin solution, 1 ml L-1; antifoam, 0.1 ml L-1. The trace metals solution contained: EDTA (sodium salt), 15.0 g L-1; ZnSO4⋅7H2O, 4.5 g L-1; MnCl2⋅2H2O, 0.84 g L-1; CoCl2⋅6H2O, 0.3 g L-1; CuSO4⋅5H2O, 0.3 g L-1; Na2MoO4⋅2H2O, 0.4 g L-1; CaCl2⋅2H2O, 4.5 g L-1; FeSO4⋅7H2O, 3.0 g L-1; H3BO3, 1.0 g L-1; and KI, 0.10 g L-1. The vitamin solution contained: biotin, 0.05 g L-1; p-amino benzoic acid, 0.2 g L-1; nicotinic acid, 1 g L-1; Ca-pantothenate, 1 g L-1; pyridoxine-HCl, 1 g L-1; thiamine-HCl, 1 g L-1 and myo-inositol, 25 g L-1. RNA was extracted using Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany). The Illumina High-Output Kit (2×75 bp) was used to prepare mRNA samples for sequencing.