Fig. 4: Negative and positive cascaded gene expression reactions on a single DNA molecule.

a Schematic of a DNA molecule encoding the cI repressor gene for repression of the HT reporter protein on the same DNA. b Fluorescent images of protein synthesis on DNA molecules encoding only the HT protein fused to a 730 nt extension (n = 4 independent experiments), with cI gene on the same DNA (n = 3 independent experiments), and with cI gene encoded on a second DNA molecule in the same flow channel (n = 4 independent experiments). Scale bar, 5 µm. Number of expression spots with the three different experimental setups. Experiments were replicated (small circles) to obtain average values (gray bars). The error bars are given as SD. c Schematic of a DNA molecule producing a T7 RNAP (T7pol) to drive a downstream gfp gene under the control of a T7 promoter (P_T7) on the same DNA. d Exemplary fluorescent images of protein synthesis on DNA encoding the gene for the T7 RNAP without gfp gene (n = 5 independent experiments), with encoded T7 RNAP and gfp gene (n = 4 independent experiments), and the genes for T7 RNAP and GFP split between two DNA molecules (n = 3 independent experiments) immobilized in the same flow channel. Scale bar, 5 µm. DNA molecules with protein build-up are indicated by red arrow heads. The signal-to-background ratio (SBR) of single dye-bound HT proteins surface-immobilized with antibodies and manually extracted gene expression spots with the three different experimental setups (from left to right, n = 21,26,18,13 expression spots over 2 independent experiments). Boxplots indicate median (red line), first and third quartile (black box), and 1.5 times within the inter-quartile range (whiskers) with individual data points shown as circles. e Fluorescence image of a DNA molecule and time series of HT-T7 RNAP accumulating on the DNA. Similar traces were observed for many DNA molecules and n = 4 independent experiments. f Fluorescence images of a very dilute single DNA molecule co-localized to HT-T7 RNAP signal with high SBR. Data was replicated with two independent expression experiments. Source data are provided as a Source Data file.