Expanding tunable selection in yeast using auxotrophic markers URA3 and TRP1

Auxotrophic markers are fundamental tools in yeast genetics, but their use is traditionally binary (growth or no growth), which limits quantitative analysis. Here we repurpose the classic auxotrophic markers URA3 and TRP1 as tunable selection tools that quantitatively link gene-expression levels to growth outcomes. In uracil-deficient medium (SC–Ura), URA3 yields a positive, semi-quantitative growth response proportional to its expression level. Conversely, under selection with 5-fluoroanthranilic acid (5-FAA), TRP1 shows an inverse relationship between expression and survival: higher TRP1 expression triggers greater 5-FAA toxicity and reduced growth. Using integrative plasmids to vary gene copy number, we tuned expression levels and found that even ∼2–4-fold differences produce distinct growth phenotypes. We also obtained dose–response results correlating marker expression with growth rates. Finally, applying this system to a synthetic mitochondrial mRNA localization scheme, we detected the resulting increase in protein expression via the growth-based readout. By converting traditional binary markers into tunable, quantitative selectors, this platform expands the yeast toolkit for evaluating gene expression differences and synthetic circuit function with simple growth assays.