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).