(A) Monoculture colonies on MSgg agar (basal knockdown) exhibited more variation in size than monoculture colonies on LB agar after 16 hr of growth. With full knockdown, there were many monoculture colonies with small size relative to controls on LB and on MSgg after 16 hr. The CRISPRi library is within the white boxes, and colonies outside of the boxes are parent-GFP controls. The distance between the centers of neighboring colonies is 9 mm. Data for colony areas is in Supplementary file 2. (B) A few knockdowns exhibited high competitive fitness in co-culture despite having reduced colony sizes in monoculture in basal knockdown conditions (orange), and one non-essential gene knockdown in the library had reduced competitive fitness (blue). The rest of the strains are in gray, and the controls are in black. (C) A reaction-diffusion model of colony growth (Materials and methods) recapitulates competitive fitness when DC = 0.003 mm2 hr−1; competitive fitness is not strongly perturbed by 10-fold changes in K/n0 and Dn/DC. Parameters M1 = 0.0175 min−1, M1/M2 = 0.8, r0 = 1 mm, and C0/(n0/b)=0.001 are estimates from experimental data. The black line shows the simulated competitive fitness corresponding to the colony in the inset of Figure 2E in which both competitive fitness and colony size data were recapitulated with K/n0 = 0.05 and Dn/DC = 100. Competitive fitness was largely unchanged if Dn/DC = 1000 (dark pink, partly beneath black line) or Dn/DC = 10 (light pink, partly beneath black line), or if K/n0 = 1.0 (dark blue) or K/n0 = 0.005 (light blue, partly beneath dark pink line). (D) Strains that competed well at full knockdown included genes related to metabolism, gene regulation, prophage, and cell envelope (divIB), along with yloU, a gene of unknown function. Merged images show the parent-RFP false-colored in magenta and the knockdown strain false-colored in green. The 100% parent-RFP, 50% parent-RFP+50% parent-GFP, and 100% parent-GFP controls are shown to the right of the knockdowns. Scale bar: 5 mm. (E) Competitive fitnesses of the knockdowns with the highest fitness values at 16 hr were mostly stable over time with full CRISPRi induction. Curves are means, and shaded regions represent one standard error of the mean (n = 3 biological replicates).