Dahan Noa, Schuldiner Maya, Zalckvar Einat
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, 7610001, Israel.
Methods Mol Biol. 2017;1595:305-318. doi: 10.1007/978-1-4939-6937-1_28.
High-throughput methodologies have been extensively used in the budding yeast, Saccharomyces cerevisiae, to uncover fundamental principles of cell biology. Over the years, several collections of yeast strains (libraries) were built to enable systematic exploration of cellular functions. However, using these libraries experimentally is often labor intensive and restricted to laboratories that hold high throughput platforms. Utilizing the available full genome libraries we handpicked a subset of strains that represent all known and predicted peroxisomal proteins as well as proteins that have central roles in peroxisome biology. These smaller collections of strains, mini-libraries, can be rapidly and easily used for complicated screens by any lab. Since one of the libraries is built such that it can be easily modified in the tag, promoter and selection, we also discuss how these collections form the basis for creating a diversity of new peroxisomal libraries for future studies. Using manual tools, available in any yeast lab, coupled with few simple genetic approaches, we will show how these libraries can be "mixed and matched" to create tailor made libraries for screening. These yeast collections may now be exploited to study uncharted territories in the biology of peroxisomes by anyone, anywhere.
高通量方法已在芽殖酵母酿酒酵母中广泛应用,以揭示细胞生物学的基本原理。多年来,构建了几个酵母菌株文库,以便系统地探索细胞功能。然而,通过实验使用这些文库通常劳动强度大,并且仅限于拥有高通量平台的实验室。利用现有的全基因组文库,我们精心挑选了一组菌株,这些菌株代表了所有已知和预测的过氧化物酶体蛋白以及在过氧化物酶体生物学中起核心作用的蛋白。这些较小的菌株文库,即微型文库,可以被任何实验室快速且轻松地用于复杂的筛选。由于其中一个文库构建得使其在标签、启动子和选择方面易于修改,我们还将讨论这些文库如何为未来研究创建各种新的过氧化物酶体文库奠定基础。使用任何酵母实验室都具备的手动工具,再结合一些简单的遗传学方法,我们将展示如何将这些文库“混合搭配”以创建定制文库用于筛选。现在,任何人在任何地方都可以利用这些酵母文库来研究过氧化物酶体生物学中未知的领域。