Fig. 5. PERSIST dual function enables single-element positive feedback + repression motif.

a Schematic of positive feedback + repression motif. b Csy4 activates itself through its own PERSIST-ON motif and represses another element. Transfection efficiency of the EYFP reporter was tracked by a plasmid encoding constitutive tagBFP while Csy4-containing plasmid transfection efficiency was tracked by a plasmid encoding constitutive mKO2. Summary values were calculated for tagBFP-positive cells. n = 2 biologically independent samples, as shown by separate curves where each sample represents the evaluation of >1000 transfected cells (HEK293FT). The shaded region is the maximum interquartile range of all cells in each bin. c The positive feedback + repression motif can be used to improve the performance of the second stage of a three-stage repression cascade. An EYFP reporter tracks the output for subsequent additions of each stage. A larger percentage of stage-2 cells exhibit “ON" behavior with endoRNase positive feedback (right) compared to when repression alone is used (left), with a trade-off existing of an increase in the third stage response. Each bar represents an evaluation of cells within a large range of transfection levels of each stage (Supplementary Fig. 17). n = 2 biologically independent samples, where each sample represents the evaluation of >1000 transfected cells (HEK293FT). d The positive feedback + repression motif is used to make a genetic bistable switch. mKO2 and EYFP reporter expression was evaluated for iRFP720- and tagBFP-positive cells. Only bistable switch plasmids were used with no additional inputs, yet 97% of cells display expression in only a high-mKO2/low-EYFP (red dots) or high-EYFP/low-mKO2 (yellow dots) state after 48 hours. Few cells expressed high levels of both (blue dots) or none (gray dots). Percentages of cells in each state are shown in the inset and depicted in the bar. n = 1 representative sample (HEK293FT).