Automated screening in environmental arrays allows analysis of quantitative phenotypic profiles in Saccharomyces cerevisiae

Abstract

A methodology for large-scale automated phenotypic profiling utilizing quantitative changes in yeast growth has been tested and applied to the analysis of some commonly used laboratory strains. This yeast-adjusted methodology is based on microcultivation in 350 µl liquid medium, where growth is frequently optically recorded, followed by automated extraction of relevant variables from obtained growth curves. We report that cultivation at this micro-scale displayed overall growth features and protein expression pattern highly similar to growth in well aerated medium-scale (10 ml) culture. However, differences were also encountered, mainly relating to the respiratory potential and the production of stress-induced proteins. Quantitative phenotypic profiles for the laboratory yeast strains W303, FY1679 and CEN-PK.2 were screened for in environmental arrays, including 98 different conditions composed of low, medium and high concentrations of 33 growth inhibitors. We introduce the concepts phenotypic index rate and phenotypic index stationary , which relate to changes in rate of growth and the stationary phase optical density increment, respectively, in a particular environment relative a reference strain. The laboratory strains presented selective phenotypic profiles in both phenotypic indexes and the two features appeared in many cases to be independent characteristics. We propose the utilization of this methodology in large-scale screening of the complete collection of yeast deletion mutants.

Sphingolipids contribute to acetic acid resistance in Zygosaccharomyces bailii

“…Measured cell density values were converted to equivalent OD 600 values using Equation (4). ${\mathrm{O}\mathrm{D}}_{600}=\frac{{\mathrm{normalO}\mathrm{normalD}}_{\mathrm{normalw}\mathrm{normali}\mathrm{normald}\mathrm{normale}\mathrm{normalb}\mathrm{normala}\mathrm{normaln}\mathrm{normald}450-580}}{\mathrm{normalP}\mathrm{normala}\mathrm{normalt}\mathrm{normalh}\mathrm{normall}\mathrm{normale}\mathrm{normaln}\mathrm{normalg}\mathrm{normalt}\mathrm{normalh}\left(\mathrm{c}\mathrm{m}\right)\times 1.32}$The nonlinear correlation between optical density and cell density at high cell concentrations was corrected for using Equation (5) (Warringer and Blomberg, 2003). ${\mathrm{O}\mathrm{D}}_{\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{d}}={\mathrm{O}\mathrm{D}}_{\mathrm{o}\mathrm{b}\mathrm{s}\mathrm{e}\mathrm{r}\mathrm{v}\mathrm{e}\mathrm{d}}+{\mathrm{O}\mathrm{D}}_{\mathrm{o}\mathrm{b}\mathrm{s}\mathrm{e}\mathrm{r}\mathrm{v}\mathrm{e}\mathrm{d}}^2\times 0.449+{\mathrm{O}\mathrm{D}}_{\mathrm{o}\mathrm{b}\mathrm{s}\mathrm{e}\mathrm{r}\mathrm{v}\mathrm{e}\mathrm{d}}^3\times 0.191$ …”

Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae

“…14, in synthetic defined medium containing 0.7 M NaCl, 650 g͞ml caffeine, or 300 g͞ml paraquat. Two consecutive precultures were run, each for 48 h. For experimental runs, strains were cultivated for 47 h with duplicates on separate 100-well plates (14). The rate of growth was calculated as reported in ref.…”

“…Using the clone collection, we next sought to identify membrane proteins that, when overexpressed, specifically inhibit or promote cell growth under various conditions. To this end, we screened for effects on cell growth caused by the overexpression of individual HA͞Suc2͞His4C fusions by microcultivation and automated recording of individual growth curves (14) in synthetic defined medium and in three different stress conditions (NaCl, caffeine, and paraquat). Typical growth curves in synthetic defined medium and paraquat for the STY50 strain (transformed with empty vector) and for strains overexpressing Erp1p and Ycl045cp (discussed below) are shown in Fig.…”

“…As described in Experimental Procedures, logarithmic strain coefficients (LSCs) were calculated by normalizing the growth rate for each strain to the average growth rate of four reference cultures of the STY50 strain included in each run. For stress conditions, LSC values were normalized further to the LSC values obtained in synthetic defined medium, providing a logarithmic phenotypic index (LPI) that gives a quantitative measure of the gene-by-environment interactions normalized for general growth defects (14).…”

An Integrated Laboratory Robotic System for Autonomous Discovery of Gene Function

“…We apply an empirical approach based on cubic spline polynomials. This incorporates techniques for handling missing data points and noise and comparisons indicate that on our data it provides improved results when compared with other empirical methods (e.g., see reference 21 ) or classical sigmoidal modeling approaches (e.g., see reference 22 ). The growth parameters extracted include maximum growth rate (and hence doubling time), lag time, start and end time points of the period of maximum exponential growth, OD at the end point of the period of maximum exponential growth, global maximum OD reached, and also a signal-to-noise ratio.…”