High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots.
High-throughput transformation of Saccharomyces cerevisiae using liquid handling robots.
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DOI：
10.1371/journal.pone.0174128
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Kaplan ME
中科院分区：
文献类型：
--
作者：
Liu G;Lanham C;Buchan JR;Kaplan ME
酿酒酵母（芽殖酵母）是一种强大的真核模型生物，非常适合高通量遗传分析，一次又一次地产生见解，进一步加深我们对人类保守的细胞生物学过程的理解。为了外源蛋白表达（例如，质粒）或基因组突变（例如，基因突变、缺失、表位标记），用 DNA 进行酵母的醋酸锂 (LiAc) 转化是一种有用且长期建立的方法。然而，文献中尚未描述一种几乎不存在人为错误风险的可靠且优化的高通量转化方案。在这里，我们描述了一种可以广泛转移到大多数液体处理高通量机器人平台的方法，这些平台现在在学术和工业环境中很常见。使用我们的优化方法，我们每天能够轻松转化大约 1200 个菌株，从而在 6 天内完成典型基因组酵母文库的完全转化。此外，与流行且优雅的合成基因阵列方法相比，使用我们的方案进行基因敲除还提供了一种可能更快、更容易且更具成本效益的方法来生成双突变体集合。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具有重要意义。
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.