Liu Guangbo, Lanham Clayton, Buchan J Ross, Kaplan Matthew E
Department of Molecular and Cellular Biology; University of Arizona, Tucson, Arizona, United States of America.
Functional Genomics Core facility, University of Arizona, Tucson, Arizona, United States of America.
PLoS One. 2017 Mar 20;12(3):e0174128. doi: 10.1371/journal.pone.0174128. eCollection 2017.
Saccharomyces cerevisiae (budding yeast) is a powerful eukaryotic model organism ideally suited to high-throughput genetic analyses, which time and again has yielded insights that further our understanding of cell biology processes conserved in humans. Lithium Acetate (LiAc) transformation of yeast with DNA for the purposes of exogenous protein expression (e.g., plasmids) or genome mutation (e.g., gene mutation, deletion, epitope tagging) is a useful and long established method. However, a reliable and optimized high throughput transformation protocol that runs almost no risk of human error has not been described in the literature. Here, we describe such a method that is broadly transferable to most liquid handling high-throughput robotic platforms, which are now commonplace in academic and industry settings. Using our optimized method, we are able to comfortably transform approximately 1200 individual strains per day, allowing complete transformation of typical genomic yeast libraries within 6 days. In addition, use of our protocol for gene knockout purposes also provides a potentially quicker, easier and more cost-effective approach to generating collections of double mutants than the popular and elegant synthetic genetic array methodology. In summary, our methodology will be of significant use to anyone interested in high throughput molecular and/or genetic analysis of yeast.
酿酒酵母（芽殖酵母）是一种强大的真核模式生物，非常适合进行高通量遗传分析，它一次又一次地带来了深刻见解，加深了我们对在人类中保守的细胞生物学过程的理解。为了外源蛋白表达（如质粒）或基因组突变（如基因突变、缺失、表位标签）的目的，用DNA对酵母进行醋酸锂（LiAc）转化是一种有用且长期确立的方法。然而，文献中尚未描述一种可靠且经过优化的高通量转化方案，该方案几乎不存在人为误差风险。在此，我们描述了一种可广泛应用于大多数液体处理高通量机器人平台的方法，这些平台如今在学术和工业环境中已很常见。使用我们优化后的方法，我们每天能够轻松转化约1200个单菌株，从而在6天内完成典型基因组酵母文库的完全转化。此外，将我们的方案用于基因敲除目的，与流行且精妙的合成遗传阵列方法相比，还为生成双突变体集合提供了一种可能更快、更简便且更具成本效益的方法。总之，我们的方法对于任何对酵母高通量分子和/或遗传分析感兴趣的人都将具有重要用途。
