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

Area of Science:

• Molecular Biology
• Yeast Genetics
• Synthetic Biology

Background:

• Genetic pathway replacement is crucial for understanding gene function and engineering cellular processes.
• Existing methods for replacing essential genes in yeast often suffer from high false positive rates and inefficiency.
• Recombination between similar DNA sequences can lead to the retention of native genes, hindering successful pathway shuffling.

Purpose of the Study:

• To develop a highly efficient and accurate plasmid shuffle vector for budding yeast (Saccharomyces cerevisiae).
• To enable the replacement of essential native genetic pathways with new ones, including unfavorable genes.
• To minimize false positive rates in genetic engineering experiments.

Main Methods:

• Designed the "Superloser" plasmid vector with reduced sequence similarity to common yeast plasmids to limit recombination.
• Incorporated two orthogonal copies of the counter-selectable marker URA3 to decrease spontaneous 5-fluoroorotic acid resistance.
• Fused the CEN/ARS sequence to the GAL1-10 promoter to ensure rapid plasmid removal in the presence of galactose.

Main Results:

• Demonstrated successful proof-of-concept by swapping yeast core histones for human counterparts.
• Achieved very low false positive rates (less than 1 in 10^8 cells).
• Enabled plating of a large number of cells (1.4x10^9) on a single petri dish with a low background.

Conclusions:

• Superloser is a powerful and reliable tool for yeast geneticists.
• Facilitates the effective shuffling of low viability genes and pathways in Saccharomyces cerevisiae.
• Significantly improves the efficiency and accuracy of genetic engineering in yeast.