Superloser: A Plasmid Shuffling Vector for Saccharomyces cerevisiae with Exceedingly Low Background

Here we report a new plasmid shuffle vector for forcing budding yeast (Saccharomyces cerevisiae) to incorporate a new genetic pathway in place of a native pathway - even an essential one - while maintaining low false positive rates (less than 1 in 10(8) per cell). This plasmid, dubbed "Superloser," was designed with reduced sequence similarity to commonly used yeast plasmids (i.e., pRS400 series) to limit recombination, a process that in our experience leads to retention of the yeast gene(s) instead of the desired gene(s). In addition, Superloser utilizes two orthogonal copies of the counter-selectable marker to reduce spontaneous 5-fluoroorotic acid resistance. Finally, the CEN/ARS sequence is fused to the -10 promoter, which disrupts plasmid segregation in the presence of the sugar galactose, causing Superloser to rapidly be removed from a population of cells. We show one proof-of-concept shuffling experiment: swapping yeast's core histones out for their human counterparts. Superloser is especially useful for forcing yeast to use highly unfavorable genes, such as human histones, as it enables plating a large number of cells (1.4x10(9)) on a single 10 cm petri dish while maintaining a very low background. Therefore, Superloser is a useful tool for yeast geneticists to effectively shuffle low viability genes and/or pathways in yeast that may arise in as few as 1 in 10(8) cells.

在此我们报道了一种新的质粒洗牌载体，它可迫使芽殖酵母（酿酒酵母）整合一个新的遗传途径来替代一个天然途径——即使是一个必需途径——同时保持较低的假阳性率（每个细胞低于10⁻⁸）。这种被称为“超级失败者”的质粒，其设计使其与常用的酵母质粒（即pRS400系列）具有较低的序列相似性，以限制重组，根据我们的经验，重组过程会导致保留酵母基因而非所需基因。此外，“超级失败者”利用了两个正交的反向选择标记拷贝来降低自发的5 - 氟乳清酸抗性。最后，着丝粒/自主复制序列（CEN/ARS）与 - 10启动子融合，这会在半乳糖存在的情况下破坏质粒分离，导致“超级失败者”从细胞群体中迅速被去除。我们展示了一个概念验证的洗牌实验：将酵母的核心组蛋白替换为人类的相应组蛋白。“超级失败者”对于迫使酵母使用极不利的基因（如人类组蛋白）特别有用，因为它能够在一个10厘米的培养皿中培养大量细胞（1.4×10⁹个），同时保持非常低的背景。因此，“超级失败者”是酵母遗传学家有效洗牌酵母中低活力基因和/或途径的有用工具，这些基因和/或途径可能仅在10⁻⁸的细胞中出现。