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

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 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 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 (1.4x10 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.

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“…Histone-humanized yeasts are generated by using a low background dual-plasmid histone shuffle assay followed by counterselection of the yeast histone genes, which are linked to the URA3 marker, with 5-FOA (Figure 1A, S1A; (Haase et al, 2019; Truong and Boeke, 2017). After approximately 2 - 4 weeks of growth, we observed candidate humanized colonies as indicated by the appearance of small colonies, whereas larger colonies retained yeast histone genes (Figure S1B).…”

“…We humanized yeast’s core histone genes as previously reported (Haase et al, 2019; Truong and Boeke, 2017). Briefly, a shuffle strain – which has the four histone gene clusters chromosomally deleted and has a single set of yeast histone genes on a URA3 plasmid – is transformed with a TRP1 plasmid encoding a single set of human histone genes.…”

“…Once the culture has reached saturation, a range of volumes (typically 1uL for a robust humanizing strain or up to 1mL for a weak humanizing strain) are plated to SC-Trp agar plates containing 5-FOA (1 mg mL-1), to counter select the URA3 marker (Haase et al, 2019). Plates are sealed in a Tupperware box with a damp paper towel and growth at 30 • C is monitored for up to 4 weeks, but longer in some cases.…”

“…Colonies which appeared after 7 days of growth on plates with 5-FOA were PCR genotyped to confirm the loss the yeast histone genes (Haase et al, 2019). Confirmed clones were patched to rich media agar plates (YPD) and stored in glycerol at -80 • C. A portion of each patch was then cultured in SC-Trp and grown to saturation (generation 0).…”

“…We have previously shown that the budding yeast, Saccharomyces cerevisiae , can subsist with human histone comprised chromatin (Truong and Boeke, 2017). However, a large bottleneck limits how readily this occurs, with just ~1 in 10 9 cells surviving the initial histone swap (Haase et al, 2019). The histone-humanized cells are initially extremely unfit, with a generation time ranging from 8-12 hours, but they quickly adapt and fitness improves.…”