Fig. 2: Burden imposed by genetic circuits in mammalian cells.

a Left: As the total plasmid amount increases, the total expression plateaus. Right: Titration of two plasmids expressing the fluorescent proteins mCitrine and mRuby3 from EF1α promoters in ratios from 1:4 to 4:1 (total of 50 ng, top right; or 500 ng of DNA, bottom right). N = 3 biological replicates. Source data are provided as a Source Data file. b Two plasmids were co-transfected, one constitutively expressing capacity monitor and tTA from a strong constitutive promoter and the other expressing X-tra from a tTA responsive promoter. Capacity monitor levels counterbalance the increase in X-tra expression. Flow cytometry data are normalized to the expression at maximal Dox. N = 3 biological replicates. Source data are provided as a Source Data file. c mRNA quantification of X-tra and a capacity monitor expressed at different molar ratios. As the X-tra increases, the mRNA levels of the capacity monitor decreases. N = 4 biological replicates. qPCR analysis was performed 48 h post-transfection and data show fold change ± SE. Source data are provided as a Source Data file. d Cells transfected with a plasmid expressing two fluorescent proteins from a bidirectional promoter were sorted according to high, intermediate, or no fluorescence (Supplementary Fig. 4) for mRNA extraction. mRNA levels expressed from endogenous genes decrease in cells with intermediate and high fluorescence. N = 3 biological replicates. Data show fold change ± SE. Individual values are plotted in Supplementary Fig. 28. Source data are provided as a Source Data file. e Capacity monitor levels are higher with an HDV ribozyme rapidly degrading the capacity monitor mRNA than with an inactive mutant, suggesting a sequestration of transcriptional resources. N = 3 biological replicates (N = 2 for HDV−, 1.6 ng/μL DOX). Source data are provided as a Source Data file. f The synthetic intron shows higher X-tra levels compared to a control and leads to reduced capacity monitor levels. N = 4 biological replicates. Source data are provided as a Source Data file. g Repressed X-tra expression leads to increased capacity monitor levels. N = 2 biological replicates for L7Ae and N = 4 for Ms2-cNOT7. Source data are provided as a Source Data file. h When X-tra is downregulated by miR-221 endogenously expressed in HEK293T cells, the capacity monitor levels increase. All flow cytometry data were acquired 48 h post-transfection and are plotted as mean ± SE. SE standard error, r.u. relative units. N = 2 biological replicates. Source data are provided as a Source Data file. Unpaired two-sided T-test. P value: ****<0.0001, ***<0.0005, **<0.005, *<0.05.