A prototrophic deletion mutant collection for yeast metabolomics and systems biology

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1 Supplementary Material for A prototrophic deletion mutant collection for yeast metabolomics and systems biology Michael Mülleder 1+, Floriana Capuano 1+, Pınar Pir 1, Stefan Christen 2, Uwe Sauer 2, Stephen G Oliver 1 and Markus Ralser 1 1 Cambridge Systems Biology Centre and Department of Biochemistry, University of Cambridge, 80 Tennis Court Rd, Cambridge CB21GA, United Kingdom 2 Institute for Molecular Systems Biology, ETH Zurich, Switzerland Supplementary Figure 1 phlum (Addgene ID 40276), a singly copy centromeric vector (minichromosome) for restoring prototropy in the BY version of the S288c yeast gene deletion library 2. MET15, URA3 and LEU2 genes including their native promoter and terminator sequences were amplified by PCR from the prs plasmid series 3 and cloned in alternating orientations into the prs313 backbone containing the native HIS3 gene. Unique restriction sites were introduced between each marker and facilitate selective excision of the genes. The final plasmid was verified by re-sequencing. Transformation of phlum into BY4741 based strain restores prototrophy maintaining i) full compatibility with existing genome resources and ii) possibility for counter selection using URA3 or HIS3 counterselecting agents. Muelleder et al, Nature Biotechnology: doi: /nbt

2 Supplementary Figure 2: phlum fully restores prototrophy in BY4741. Growth of prototrophic BY4741 phlum compared to isogenic strains (also carrying phlum) in which either HIS3, LEU2, URA3 or MET15 endogenous loci have been repaired (The strains are thus 2n for the respective genes), in synthetic minimal media.(n=9, error bars +/- SD). Supplementary Figure 3. ade12δ as plate orientation/identity identifier and repetitive marker. Yeast lacking the adenylosuccinate synthase Ade12p acquire an intense red colour 4. An ade12δ strain was included as repetitive control for metabolomics experiments, and to facilitate visual plate orientation (position H2) and identification (position indicating the plate number, shown is plate 34). If necessary, the strain can be negatively selected using its natural adenine auxotrophy. Muelleder et al, Nature Biotechnology: doi: /nbt

3 Sample µmax (h - 1 ) SC media s.d. µmax (h - 1 ) SM media s.d. leu2δ ura3δ met15δ his3δ leu2δ met15δ his3δ ura3δ met15δ his3δ leu2δ ura3δ met15δ leu2δ ura3δ his3δ met15δ his3δ leu2δ met15δ ura3δ met15δ leu2δ his3δ ura3δ his3δ leu2δ ura3δ met15δ his3δ leu2δ ura3δ prototroph Supplementary Table 1. Maximum specific growth rates of 16 strains harboring the possible combinations of auxotrophic markers used in the MATa version of the auxotrophic S288c gene deletion collection 2. The different strains were grown in either synthetic complete (SC) or synthetic minimal (SM) media. Growth was recorded in 96 well plates (n=5), Vol 200µl, by measuring OD 595 using a FLUOstar OPTIMA (BMG Labtech) plate reader. Maximum growth rate was determined using a model free spline fit within the R package grofit 5, values are given per hour. Muelleder et al, Nature Biotechnology: doi: /nbt

4 Gene NOP10 PCF11 ERG7 NCP1 TAF1 TAF7 PRE5 RLI1 PRE4 PRO3 ERG1 SSY1 SAH1 Confirmation of previous results Confirmation of previous results Supplementary Table 2: Lethal phenotypes rescued by prototrophy. 370 strains of the titratable-promoter (Tet-OFF) essential collection 6 exhibit a lethal phenotype after being replicated to SC media containing 40µg/ml doxycycline (DOX). After restoring prototrophy by transforming the library with phlum, the genes listed in the table lost the lethal phenotype (all candidates were verified with two independent transformations, see also Fig 1f). The screen confirmed phenotypes for SSY1 7,8 and SAH1 9, both previously reported to be synthetic lethal with auxotrophic markers. Muelleder et al, Nature Biotechnology: doi: /nbt

5 Supplementary Methods Saccharomyces cerevisiae growth media recipes 1. Yeast extract peptone dextrose (YPD) 20 g / l peptone BD Bacto g / l yeast extract BD Bacto g / l glucose MERCK Synthetic minimal media (SM)* 6.7 g / l yeast nitrogen base (YNB) 10 Sigma Y g / l glucose MERCK mg / l uracil, in 200µM NaOH Sigma U mg / l methionine * Sigma M mg / l leucine Sigma L mg / l histidine Sigma H Synthetic complete media (SC) 6.7 g / l yeast nitrogen base (YNB) 10 Sigma Y g / l glucose MERCK mg / l uracil in 200µM NaOH Sigma U mg / l adenine hemisulfate, in 100µM NaOH Sigma A mg / l leucine Sigma L mg / l histidine Sigma H mg / l tryptophan Sigma T complete supplement mixture (CSM) (-Ade-His-Leu-Trp-Ura) containing: MPBio mg/l L-arginine HCl 80 mg/l L-aspartic acid 50 mg/l L-isoleucine 50 mg/l L-lysine HCl 20 mg/l L-methionine 50 mg/l L-phenylalanine 100 mg/l L-threonine 50 mg/l L-tyrosine 140 mg/l L-valine *Figure 1c: For the 1X/10X comparison, the following supplement concentrations were used: 20mg/l (1x media); 200mg/l (10x media) uracil, 20mg/l; 200mg/l methionine, 60mg/l; 600mg/l leucine Muelleder et al, Nature Biotechnology: doi: /nbt

6 and 20mg/l; 200mg/l histidine. Muelleder et al, Nature Biotechnology: doi: /nbt

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