Fig. 6: Genetic stability of synthetic DNA sponge and its effect on circuit’s output gene expression noise.
From: Synthetic protein-binding DNA sponge as a tool to tune gene expression and mitigate protein toxicity
a Schematics showing the workflow of the stability assay of synthetic DNA sponges. The cells harboring the sponge plasmids were cultured in 1 mL of medium in a 96-deep well plate and were diluted every 12 h for 5 days (corresponding to approximately 100 generations in total). Every 24 h, the cells were harvested for plasmid extraction, restriction digestion and the plasmids were analyzed by electrophoresis (see Methods). b Image of a gel post electrophoresis showing the stability of synthetic single-layer DNA sponges of 320 × tetO, 40 × Ptet2, 80 × LBS, 40 × Pecf11 and dual-layer DNA sponges of 80 × LBS + 40 × tetO and 20 × LBS + 20 × Pecf11. The sponge plasmids extracted from the host cells after 1 or 100 generations were compared. The plasmids from 20th, 40th, 60th and 80th generations and another two biological replicates showing similar results are shown in Supplementary Fig. 13. The expected sizes of the selected DNA sponges are shown in Table 1. M, DNA marker. S, sponge. B, pSB3K3 backbone. The original image is provided as a Source Data file. c Schematics showing the study of noise effect of synthetic DNA sponge on synthetic circuit’s output gene expression. The output fluorescence was compared at single cell level between the host cells with and without the presence of sponge. The noise was measured on the basis of robust coefficient of variation (C.V.) of the circuit’s output gene expression. d Robust C.V. of the output gene expression (left) and dose-responses (right) of a two-layered AHL-responsive circuit (Fig. 3a) with or without tetO sponges from single cell assays. Values are mean ± s.d. indicated by shading (n = 3 biologically independent samples). The cells were induced with 0, 0.02, 0.10, 0.39, 1.56, 6.25, 25, 100, 400 and 1,600 nM of 3OC6HSL. e Robust C.V. of the output gene expression (left) and dose-responses (right) of a two-layered mercury-responsive circuit (Fig. 3e) with or without Pecf11 sponges from single cell assays. Values are mean ± s.d. indicated by shading (n = 3 biologically independent samples). The cells were induced with 0, 0.008, 0.016, 0.031, 0.063, 0.125, 0.25, 0.5, 1 and 2 μM of HgCl2. Full profile of dose-responses at single cell level and noise effect of other sponges are shown in Supplementary Figs. 5, 6, 9, 10g, 11d, 12i. Fluo., fluorescence. a.u., arbitrary units. Source data are provided as a Source Data file.
