Fig. 4. Equalizer-L combines NF and IFF circuitry to increase the range of gene dosage compensation.

a Equalizer-L produced lower cell-to-cell variability than either NF or IFF circuits alone. In this experiment and in simulations (d–g), doxycycline was used at the concentration producing the lowest cell-to-cell variability for each circuit (Equalizer-L, 1 ng/mL; NF, 10 ng/mL). **p < 0.01 (Tukey’s multiple comparison test). Square markers indicate n = 3 independent transfections. Simulation results closely matched the experimentally determined cell-to-cell output variability of the NF (b) and IFF (c) circuits. For b, the filled markers indicate the mean of n = 3 independent transfections per construct. For c, the square markers indicate independent transfections. The error bars are the SEM. Each simulation datapoint (open markers) was computed from 10,000 cells whose plasmid copy number was sampled from the estimated plasmid copy number distribution. See Supplementary Notes 1, 3, and 5 for simulation models and methods. d Deterministic simulations predicted that Equalizer-L has a wider compensation range than standalone NF and IFF circuits. The dashed gray curve (right axis) illustrates the estimated plasmid copy number distribution. Simulated overall expression rate (e), number of proteins translated per mRNA (f), and transcription rate per plasmid (g) for each topology. The dotted lines indicate the slopes corresponding to perfect dosage compensation. Source data are provided as a Source Data file.