Abstract
Systematic analysis of gene overexpression phenotypes provides insight into gene function, enzyme targets, and biological pathways. Here, we describe a novel functional genomics platform that enables a highly parallel and systematic assessment of overexpression phenotypes in pooled cultures. First, we constructed a genome-level collection of ~5100 yeast Barcoder strains, each of which carries a unique barcode, enabling pooled fitness assays with a barcode microarray or sequencing readout. Second, we constructed a yeast ORF galactose-induced overexpression array by generating a genome-wide set of yeast transformants, each of which carries an individual plasmid-born and sequence-verified ORF, derived from the S. cerevisiae FLEX collection. We combined these collections genetically using Synthetic Genetic Array (SGA) methodology, generating ~5100 strains, each of which is barcoded and overexpresses a specific ORF, a set we termed “barFLEX”. Additional SGA allows the barFLEX collection to be moved into different genetic backgrounds. As a proof-of-principle, we describe the properties of the barFLEX overexpression collection and its application in synthetic dosage lethality studies under different environmental conditions.