A library of reporters of the global regulators of gene expression of Escherichia coli
Abstract
The topology of the transcription factor network (TFN) of E. coli is far from uniform, with 22 global regulator (GR) proteins controlling one-third of all genes. So far, their production rates cannot be tracked by comparable fluorescent proteins. We developed a library of fluorescent reporters for 16 GRs. Each consists of a single-copy plasmid coding for GFP fused to the full-length copy of the native promoter. We tracked their activity in exponential and stationary growth, and under weak and strong stresses. We show that the reporters have high sensitivity and specificity to all stresses tested and detect single-cell variability in transcription rates. Given the influence of GRs on the TFN, we expect that the new library will contribute to dissecting global transcriptional stress-response programs of E. coli. Moreover, it can be invaluable in bio-industrial applications that tune those programs to, instead of cell growth, favor productivity while reducing energy consumption.
README: A library of reporters of the global regulators of gene expression of Escherichia coli
Recommended citation for this dataset: S. Dash, R. Jagadeesan, I.S.C. Baptista, V. Chauhan, V. Kandavalli, S.M.D. Oliveira and A.S. Ribeiro. (2024). A library of reporters of the global gene expression regulators of Escherichia coli; Dryad (dataset)
DATA & FILE OVERVIEW
Flow_Cytometry
- This folder contains all flow cytometry data used in the project. The folder structure is organized as a function of the figures in the paper.
- 01_Optimal_Conditions: Related to Figure 1C and 4A, 4B of main manuscript
- 02_Strong_Stress: Related to Figure 6(A-D) and of main manuscript and Figure S11 of Supplementary information
- Note: All flow cytometry data was acquired using an ACEA NovoCyte Flow Cytometer (ACEA Biosciences Inc., San Diego, USA). All flow cytometry raw data is provided as *.csv files. The units are in arbitrary units (a.u.). Each raw file contains 26 columns, out of which we used the following: ‘FITC-H’ and ‘Width’. Specifically, the parameter FITC-H corresponds to the fluorescein isothiocyanate detection channel with a 530/30 nm emission filter, with a core diameter of 7.7 μM and a PMT voltage of 600. This channel informs on the expression level of GFP proteins. The parameter 'Width' measures the duration of the signal, not impacted by the PMT voltage, which correlates with cell size. Finally, each file contains 50000 rows (excluding the heading row). Each row corresponds to a single-cell measurement.
Microscopy
- This folder contains all microscopy data used in the project. The folder structure is organized as a function of the figures in the paper.
- 01_Phase_Contrast_Images: Related to Figures S2A of Supplementary information
Plasmid_Sequences
- This folder contains GR reporter plasmid maps (generated by Snapgene) and FASTA files of their DNA sequences.
- 01_FASTA files: Related to Figures 2A of main manuscript
- Note: Plasmid maps are given in the file named "Plasmid_Maps".
RNAseq_TPM
- This folder contains TPM-normalized mRNA counts of GR genes.
- 01_RNAseq_TPM: Related to Figure 2B of the main manuscript
- Note: Raw RNA-seq data was published in (DOI: 10.1093/nar/gkac540) and can be accessed using the NCBI GEO accession code GSE178281.
RT_PCR
- This folder contains RT-PCR data to study the relative mRNA production rates of the GRs.
- 01_GR_Data: Related to Figures 2A of main manuscript
- 02_GFP_Data: Related to Figures S10 of Supplementary information
Spectrophotometry
- This folder contains the OD and Fluorescence data for all the GR plasmids. The units are in arbitrary units (a.u.). Related to Figures 3A, 3B, 5(A-D) and Figs. S4, S7, S9 of the Supplementary information;
- Subfolder name: Fluorescence
- 01_Growth_Transition: Related to Figures 3A of the main manuscript
- 02_RNA_Seq_Correlation: Related to Figures 2E of the main manuscript
- 03_Weak_Stresses: Related to Figures 5 (A-D) of the main manuscript
- Subfolder name: OD
- 01_LB_Growth_curves: Related to Figure 1B of the main manuscript
- 02_M9_Glucose_Growth_curves: Related to Figure 1B of the main manuscript
- 03_Stress_growth_curves: Related to S7 of the Supplementary information
DATA-SPECIFIC INFORMATION FOR:
Flow_Cytometry\01_Optimal_Conditions:
To evaluate the relationship between the squared coefficient of variation (CV2) and mean protein numbers of all GRs and compare their single-cell variability in RNA production dynamics. Additionally, we also measured the squared coefficient of variation (CV2) of YFP-tagged GR strains from the YFP fusion library.
Flow_Cytometry\02_Strong_Stress:
To evaluate the relationship between single-cell mean GFP levels of all GRs and their pulse width (as a proxy for cell size) when subjected to several strong stress conditions.
Microscopy\01_Phase_Contrast_Images:
To compare the average cell size of 7 GR reporter plasmid-containing cells with the WT (MG1655) in optimal growth conditions.
Cases:
- 3.1: Cra: Phase contrast microscopy data of 5 panels.
- 3.2: CRP: Phase contrast microscopy data of 4 panels.
- 3.3: Fur: Phase contrast microscopy data of 5 panels.
- 3.4: HNS: Phase contrast microscopy data of 5 panels.
- 3.5: LexA: Phase contrast microscopy data of 5 panels.
- 3.6: MarA: Phase contrast microscopy data of 5 panels.
- 3.7: MG1655: Phase contrast microscopy data of 5 panels.
- 3.8: SoxS: Phase contrast microscopy data of 5 panels.
Spectrophotometry\01_Fluorescence:
Cases:
- 4.1: Growth_Transition: To compare the transcription dynamics of 16 GRs using reporter plasmids in optimal growth conditions (Growth phase transition.xlsx).
- 4.2: RNA_Seq_Correlation: To compare the relative chromosomal GR gene RNA production levels of 18 GRs (measured by RNA seq) with the relative GFP levels (measured by Spectrophotometry using reporter plasmids in optimal growth conditions (LB_measurements.xlsx)).
- 4.1: Weak_Stresses: To compare different GR transcription dynamics using reporter plasmids when subjected to excess Iron, Tetracycline, Kanamycin, and Oxidative stress (Weak stresses.xlsx). Note that the strength of the stress was tuned to not disturb the growth rates as compared to optimal conditions.
Spectrophotometry\02_OD:
Cases:
- 4.1: Growth_curves: To compare the growth rates of cells containing 18 GR reporter plasmids with the WT strain in optimal growth conditions (Growth_curves.xlsx).
- 4.2: Stress_growth_curves: To compare the growth rates of cells containing 4 GR reporter plasmids with the WT strain when subjected to weak as well as strong stress conditions (Stress_growth_curves.xlsx).