Nislow Corey, Wong Lai Hong, Lee Amy Huei-Yi, Giaever Guri
Department of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Cold Spring Harb Protoc. 2016 Sep 1;2016(9):2016/9/pdb.prot088039. doi: 10.1101/pdb.prot088039.
The ability to measure and quantify the fitness of an entire organism requires considerably more complex approaches than simply using traditional "omic" methods that examine, for example, the abundance of RNA transcripts, proteins, or metabolites. The yeast deletion collections represent the only systematic, comprehensive set of null alleles for any organism in which such fitness measurements can be assayed. Generated by the Saccharomyces Genome Deletion Project, these collections allow the systematic and parallel analysis of gene functions using any measurable phenotype. The unique 20-bp molecular barcodes engineered into the genome of each deletion strain facilitate the massively parallel analysis of individual fitness. Here, we present functional genomic protocols for use with the yeast deletion collections. We describe how to maintain, propagate, and store the deletion collections and how to perform growth fitness assays on single and parallel screening platforms. Phenotypic fitness analyses of the yeast mutants, described in brief here, provide important insights into biological functions, mechanisms of drug action, and response to environmental stresses. It is important to bear in mind that the specific assays described in this protocol represent some of the many ways in which these collections can be assayed, and in this description particular attention is paid to maximizing throughput using growth as the phenotypic measure.
测量和量化整个生物体的适应性需要比单纯使用传统的“组学”方法更为复杂的方法，传统“组学”方法例如检测RNA转录本、蛋白质或代谢物的丰度。酵母缺失文库是唯一可用于检测此类适应性的、针对任何生物体的系统且全面的无效等位基因集合。由酿酒酵母基因组删除项目生成的这些文库，允许使用任何可测量的表型对基因功能进行系统和平行分析。每个缺失菌株基因组中设计的独特的20个碱基对的分子条形码便于对个体适应性进行大规模平行分析。在这里，我们介绍与酵母缺失文库一起使用的功能基因组学方案。我们描述了如何保存、繁殖和存储缺失文库，以及如何在单个和平行筛选平台上进行生长适应性测定。这里简要描述的酵母突变体的表型适应性分析为生物学功能、药物作用机制和对环境压力的反应提供了重要见解。需要牢记的是，本方案中描述的特定测定只是这些文库多种检测方式中的一部分，并且在本描述中特别关注以生长作为表型测量来最大化通量。
