DOI: 10.5061/dryad.b2rbnzsm8
title: A library of reporters of the global regulators of gene expression of Escherichia coli

description: 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.
description: # 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).