Hypersensitivity of the ABC16-monster to drugs. (a) Pie chart showing the number of drugs to which the ABC16-monster or the previously-described drug-hypersensitive AD strain is more sensitive. (b) A comparison of ABC16-monster and single-mutant drug sensitivity. The IC₅₀ for each strain/drug combination relative to that of the corresponding non-mutant/drug combination was calculated. Area of each shape is proportional to this relative IC₅₀ value. *: The minimum value among the relative IC₅₀ values for single mutants is indicated for comparison with the relative IC₅₀ of the ABC16-monster (c) Exponential growth rates of the ABC16-monster (green), non-mutant (blue), and single-deletion strains (grey) as a function of concentration of tamoxifen, fluconazole, and valinomycin.

Design of the Green Monster process. (a) Schematic overview of the process In yeast, crossing different haploid single-mutants generates 0-deletion (off-white), 1-deletion (light green), and 2-deletion (dark green) cells. From this mixture, flow cytometry enriches for 2-deletion cells. Higher-order multi-mutants are assembled via repeated rounds of sexual assortment and enrichment. (b) Universal GFP deletion cassette replaces KanMX4 in 'your favorite gene' (YFG) via recombination within TEF subsequences, while maintaining barcodes (stripes) intact. The inducible tetO₂ promoter allows titration of GFP expression. Transcriptional terminators (brown) and the TEF promoter (light blue) and terminator (dark blue) are shown. Selectable marker: URA3.. (c) GMToolkits, inserted at the CAN1 locus, contain rtTA21, either KanMX4 and STE2pr-Sp-his5 (GMToolkit-a) or NatMX4 20 and STE3pr-LEU2 19 (GMToolkit-α).

Demonstration of the Green Monster process. (a) Simulations showing that >99% of a cell population accumulate all 24 deletions in eight (top), 12 (middle), or 19 rounds (bottom), with greater efficiency for lower coefficient of variation (CV) of GFP intensity (achievable using internal standard to control for noise (b) Simulation showing that 24 linked deletions with the meiotic crossover probability between adjacent loci (P(R)) of 5% can assemble in 16 rounds when the GFP CV is 50%. (c) Cell sorting strategy. Histograms (y-axis: the percentage of maximum cell number) are shown for GFP intensity in no-GFP cells (0Δ), single-GFP cells (1Δ), and a haploid ‘meiotic mix’ resulting from a cross of two single-GFP strains, with an expected 1:2:1 ratio of no-GFP, one-GFP, and two-GFP cells. The brightest 1% of the cells in the meiotic mix were collected (red filled area). (d) GFP intensity of multi-mutants. Histograms are shown for no-GFP, 1-GFP, 2-GFP, 4-GFP, 8-GFP and 16-GFP ‘ABC16-monster’ cells (isogenic populations). (e) Fluorescence micrographs showing non-mutant (top left), double-mutant (top right), the ABC16-monster (bottom left), and a mixture of double mutant and ABC16-monster (bottom right) cells. Identical exposure, brightness, and contrast settings were used for images. Scale bar indicates 10 µm. (f) The en masse Green Monster process. Average deletion numbers for each round from three independent processes are plotted (red, blue, and yellow). Error bars indicate standard deviations (n = 21 – 34).