Superloser: A plasmid shuffling vector for Saccharomyces cerevisiae with exceedingly low background

Max A.B. Haase, David M. Truong, Jef D. Boeke

    Research output: Contribution to journalArticlepeer-review

    Abstract

    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 108 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 URA3 to reduce spontaneous 5-fluoroorotic acid resistance. Finally, the CEN/ARS sequence is fused to the GAL1-10 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 (1.4×109) 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 108 cells.

    Original languageEnglish (US)
    Pages (from-to)2699-2707
    Number of pages9
    JournalG3: Genes, Genomes, Genetics
    Volume9
    Issue number8
    DOIs
    StatePublished - Aug 1 2019

    Keywords

    • 5-FOA
    • Counterselection
    • Humanization
    • Plasmid shuffling
    • S. cerevisiae

    ASJC Scopus subject areas

    • Molecular Biology
    • Genetics
    • Genetics(clinical)

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