Phenomic Assessment of Genetic Buffering by Kinetic Analysis of Cell Arrays

Abstract

Summary Quantitative high throughput cell array phenotyping (Q-HTCP) is applied to the genomic collection of yeast gene deletion mutants for systematic, comprehensive assessment of the contribution of genes and gene combinations to any phenotype of interest (phenomic analysis). Interacting gene networks influence every phenotype. Genetic buffering refers to how gene interaction networks stabilize or destabilize a phenotype. Like genomics, phenomics varies in its resolution with there being a tradeoff allocating a greater number of measurements per sample to enhance quantification of the phenotype vs. increasing the number of different samples by obtaining fewer measurement per sample. The Q-HTCP protocol we describe assesses 50,000–70,000 cultures per experiment by obtaining kinetic growth curves from time series imaging of agar cell arrays. This approach was developed for the yeast gene deletion strains, but it could be applied as well to other microbial mutant arrays grown on solid agar media. The methods we describe are for creation and maintenance of frozen stocks, liquid source array preparation, agar destination plate printing, image scanning, image analysis, curve fitting and evaluation of gene interaction.

Gene-nutrient interaction markedly influences yeast chronological lifespan

“…To clarify the potential for media and genetic factors to influence CLS, we investigated the different experimental conditions employed by the three previous genome-wide CLS screens using a single platform for measuring CLS (Louie and others 2012; Rodgers and others 2014; Shah and others 2007). Quantitative high-throughput cell array phenotyping ( Q-HTCP ) was used to measure CLS in six different media conditions and three strains with differing ploidy and auxotrophic requirements.…”

“…Q-HTCP was performed as previously described (Hartman IV and Tippery 2004; Rodgers and others 2014; Shah and others 2007). Briefly, a 384-well happy array containing the 3 reference strains was grown in YPD for 2 days and used to inoculate the six different media (see Fig.…”

Long‐range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels

“…Cell arrays were created robotically with the Sciclone ALH3000 liquid handling system (Caliper Life Sciences/Perkin Elmer, Hopkinton, MA, USA), manipulating a 384-pin tool with FP6-style pins (V&P Scientific, San Diego, CA, USA) for transferring cells. Plates were incubated at 30°C and scanned manually (Yor1/oligomycin experiments) or robotically as previously described (15,(27)(28)(29). Pixel intensities were derived from time series images for each culture spot and fitted to a logistic growth equation, which was used to obtain the growth parameters (29).…”

A humanized yeast phenomic model of deoxycytidine kinase to predict genetic buffering of nucleoside analog cytotoxicity

“…Q-HTCP, an automated method of collecting growth curve phenotypes for the YKO/KD library arrayed onto agar media, was used to obtain phenomic data [38]. A Caliper Sciclone 3000 liquid handling robot was used for cell array printing, integrated with a custom imaging robot (Hartman laboratory) and Cytomat 6001 (Thermo Fisher Scientific, Asheville, NC, USA) incubator.…”

“…A Caliper Sciclone 3000 liquid handling robot was used for cell array printing, integrated with a custom imaging robot (Hartman laboratory) and Cytomat 6001 (Thermo Fisher Scientific, Asheville, NC, USA) incubator. Images of the 384-culture arrays were obtained approximately every 2-3 hours and analyzed as previously described [9,38]. To obtain CPPs, image analysis was performed in Matlab and data were fit to the logistic equation, G(t) = K/(1 + e −r(t−l) ), assuming G(0) < K, where G(t) is the image intensity of a spotted culture vs. time, K is the carrying capacity, r is the maximum specific growth rate, and l is the moment of maximal absolute growth rate, occurring when G(t) = K/2 (the time to reach half of carrying capacity) [7].…”