Bacteria evolved genes whose single-cell distributions of expression levels are broad, or even bimodal. Evidence suggests that they might enhance phenotypic diversity for coping with fluctuating environments. We identified seven genes in E. coli with bimodal (low and high) single-cell expression levels under standard growth conditions and studied how their dynamics are modified by environmental and antibiotic stresses known to target gene expression.  We found that all genes lose bimodality under some, but not under all, stresses. Also, bimodality can reemerge upon cells returning to standard conditions, which suggests that the genes can switch often between high and low expression rates. As such, these genes could become valuable components of future multi-stable synthetic circuits. Next, we proposed models of bimodal transcription dynamics with realistic parameter values, able to mimic the outcome of the perturbations studied. We explored several models’ tunability and boundaries of parameter values, beyond which it shifts to unimodal dynamics. From the model results, we predict that bimodality is robust, and yet tunable, not only by RNA and protein degradation rates, but also by the fraction of time that promoters remain unavailable for new transcription events. Finally, we show evidence that, although the empirical expression levels are influenced by many factors, the bimodality emerges during transcription initiation, at the promoter regions and, thus, may be evolvable and adaptable. # Bimodality in E. coli gene expression: sources and robustness to genome-wide stresses [https://doi.org/10.5061/dryad.dz08kps5n](https://doi.org/10.5061/dryad.dz08kps5n) This dataset contains flow cytometry, microscopy and spectrophotometry data. It also contains the manual classification of bimodality. ## Description of the data and file structure #### - 01 Flow cytometry: This folder contains all flow cytometry data used in the project. **Note 1**: Unless stated otherwise, in the subfolders, there are 3 CSV files labelled "Sample 1", "Sample 2", and "Sample 3" that represent the measurements of 3 biological replicates in a given condition. **Note 2**: All CSV files in this folder contain fluorescence data in arbitrary units (AU) for the various flow cytometry detection channels. * 01 WT: Contains data measured by flow cytometry of MG1655 strain without fluorescent tags. * 01 Standard growth: Contains data measured in standard growth condition. * 02 Stationary growth: Contains data measured in stationary growth condition. * 03 Novobiocin: Contains data measured when cells are subjected to novobiocin. * 04 Cold shock: Contains data measured when cells are subjected to cold shock. * 05 Rifampicin: Contains data measured when cells are subjected to rifampicin. * 06 Streptomycin: Contains data measured when cells are subjected to streptomycin. * 02 YFP strains: Contains data measured by flow cytometry of YFP-tagged strains. * 01 Unimodal genes: Contains data regarding 49 strains classified as having unimodal gene expression distributions. * 01 Standard growth: Data measured in standard growth condition. Contains 49 folders, each one named after the gene tagged with YFP. * 02 Bimodal genes: Contains data regarding 7 strains classified as having bimodal gene expression distributions. * 01 Standard growth: Data measured in standard growth condition. Contains 7 folders, each one named after the gene tagged with YFP. * 02 Stationary growth: Data measured in stationary growth condition. Contains 7 folders, each one named after the gene tagged with YFP. * 03 Novobiocin: Data measured when cells are subjected to novobiocin. Contains 7 folders, each one named after the gene tagged with YFP. * 04 Cold shock: Data measured when cells are subjected to cold shock. Contains 7 folders, each one named after the gene tagged with YFP. * 05 Rifampicin: Data measured when cells are subjected to rifampicin. Contains 7 folders, each one named after the gene tagged with YFP. * 06 Streptomycin: Data measured when cells are subjected to streptomycin. Contains 7 folders, each one named after the gene tagged with YFP. * 07 Growth phase shifts: Data measured when cells are subjected to growth phase shifts for 3 generations.  * 01 Generation 1: Data pertaining to the measurements of the first generation cells. * 01 Early exponential: Measurements made at the early exponential growth phase conditions. Contains 7 folders, each one named after the gene tagged with YFP. * 02 Exponential: Measurements made at the early exponential growth phase conditions. Contains 7 folders, each one named after the gene tagged with YFP. * 03 Early stationary: Measurements made at the early exponential growth phase conditions. Contains 7 folders, each one named after the gene tagged with YFP. * 04 Stationary: Measurements made at the early exponential growth phase conditions. Contains 7 folders, each one named after the gene tagged with YFP. * 02 Generation 2: Data pertaining to the measurements of the second generation cells. * 01 Early exponential: Measurements made at the early exponential growth phase conditions. Contains 7 folders, each one named after the gene tagged with YFP. * 02 Exponential: Measurements made at the early exponential growth phase conditions. Contains 7 folders, each one named after the gene tagged with YFP. * 03 Early stationary: Measurements made at the early exponential growth phase conditions. Contains 7 folders, each one named after the gene tagged with YFP. * 04 Stationary: Measurements made at the early exponential growth phase conditions. Contains 7 folders, each one named after the gene tagged with YFP. * 03 Generation 3: Data pertaining to the measurements of the third generation cells. * 01 Early exponential: Measurements made at the early exponential growth phase conditions. Contains 7 folders, each one named after the gene tagged with YFP. * 02 Exponential: Measurements made at the early exponential growth phase conditions. Contains 7 folders, each one named after the gene tagged with YFP. * 03 Early stationary: Measurements made at the early exponential growth phase conditions. Contains 7 folders, each one named after the gene tagged with YFP. * 04 Stationary: Measurements made at the early exponential growth phase conditions. Contains 7 folders, each one named after the gene tagged with YFP. * 03 GFP promoter fusions: Contains data measured by flow cytometry of cells with a low-copy plasmid with transcriptional fusions of GFP of promoters.  * 01 Bimodal genes: Contains data regarding 6 strains classified as having bimodal gene expression distributions. * 01 Standard growth: Data measured in standard growth condition. Contains 6 folders, each one named after the gene whose promoter is tagged with GFP. #### - 02 Spectrophotometry: This folder contains all spectrophotometry data used in the project. * 01 Growth: This folder contains an Excel file (named "OD600 data") with optical density (OD600), a unitless absorbance measurement, for MG1655 under standard growth conditions. Growth is tracked over time, with time recorded in minutes (min). #### - 03 Microscopy: This folder contains all microscopy data used in the project. * 01 YFP strains: Contains data measured by flow cytometry of YFP-tagged strains. * 01 Unimodal genes: Contains data regarding 2 strains classified as having unimodal gene expression distributions. Contains 2 folders, each one named after the gene tagged with YFP. * 02 Bimodal genes: Contains data regarding the 7 strains classified as having bimodal gene expression distributions. Contains 7 folders, each one named after the gene tagged with YFP. **Note**: For both folders: In each gene folder, there are various folders, each for a panel. In each panel folder, there are confocal images of YFP tagged strain along with the correspondent phase contrast image. Confocal images have the prefix "C2" and the phase contrast images have the prefix "C1". #### - 04 Manual Classification: This folder contains an excel file (named "Manual Classification Analysis") with the results of bimodality classification of genes in standard growth conditions (made by 15 individuals).

2025