Fig. 5.

Mitigating competition for shared limited resources with antithetic integral and PI feedback. (A) A genetic implementation of an antithetic integral and PI feedback circuit for mitigating the effects of limited shared resources. The antithetic integral and PI feedback circuit characterized in Figs. 2 and 3 are repurposed to mitigate the coupling of gene expression induced by shared pools of finite resources. Varying the amounts of an additional disturbance plasmid that constitutively expresses the fluorescent protein miRFP670 introduces a disturbance to the amount of available resources, which indirectly affects the expression levels of tTA-mCitrine-SMASh. (B) Steady-state rejection of disturbances to available limited shared resources. The activator plasmid and antisense plasmid for all conditions were transiently transfected at a setpoint ratio of 1/2 together with disturbance strengths varying from 0.6 to 3.5. The Top and Bottom rows, respectively, show the fluorescence of the miRFP670 disturbance and mCitrine output normalized to the lowest disturbance strength. The disturbance strength describes the amount of disturbance plasmid relative to the activator plasmid. Unpaired two-sided T test with regard to lowest disturbance strength (0.6) for all controllers, for open and closed loop. P value: **** < 0.0001, *** < 0.0005, ** < 0.005, * < 0.05. (C) Reduction in cell-to-cell variability as a result of PI feedback control. The coefficient of variation squared was computed for the first two disturbance strengths and normalized two the I-Control condition. Unpaired two-sided T test with regard to I-Control for both setpoints shown. P value: **** < 0.0001, *** < 0.0005, ** < 0.005, * < 0.05. The data are shown as the mean ± SE for n = 3 technical replicates per condition. The unnormalized data are shown in SI Appendix, Fig. S13 and provided in a separate file.