# Dataset for "An Hfq-dependent post-transcriptional mechanism fine tunes RecB expression in Escherichia coli" 
doi: https://doi.org/10.7554/eLife.94918.2

# Authors: 
Irina Kalita1,2,3,*; Ira Alexandra Iosub1,4,5; Lorna McLaren1,2; Louise Goossens1,2; Sander Granneman1,4; and Meriem El Karoui1,2,*

1 Centre for Engineering Biology, University of Edinburgh, UK
2 Institute of Cell Biology, University of Edinburgh, UK
3 Max Planck Institute for Terrestrial Microbiology & Center for Synthetic Microbiology (SYNMIKRO), Marburg, Germany
4 Institute of Quantitative Biology, Biochemistry and Biotechnology, University of Edinburgh, UK
5 The Francis Crick Institute, London, UK
*For correspondence: irykalita@gmail.com, meriem.elkaroui@ed.ac.uk
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## GENERAL INFORMATION 

1. Abstract 

All living organisms have developed strategies to respond to chromosomal damage and preserve genome integrity. One such response is the repair of DNA double-strand breaks (DSBs), one of the most toxic forms of DNA lesions. 
In Escherichia coli, DSBs are repaired via RecBCD-dependent homologous recombination. RecBCD is essential for accurate chromosome maintenance, but its over-expression can lead to reduced DNA repair ability. 
This apparent paradox suggests that RecBCD copy numbers may need to be tightly controlled within an optimal range. Using single-molecule fluorescence microscopy, we have established that RecB is present in very low abundance at mRNA and protein levels. 
RecB transcription shows high fluctuations, yet cell-to-cell protein variability remains remarkably low. Here, we show that the post-transcriptional regulator Hfq binds to recB mRNA and down-regulates RecB protein translation in vivo. 
Furthermore, specific disruption of the Hfq-binding site leads to more efficient translation of recB mRNAs. In addition, we observe a less effective reduction of RecB protein fluctuations in the absence of Hfq. 
This fine-tuning Hfq-mediated mechanism might have the underlying physiological function of maintaining RecB protein levels within an optimal range.

2. Dataset description 

This dataset contains the dataset presented in the aforementioned publication:   

- Fluorescence microscopy data obtained from single-molecule fluorescent in situ hybridization (FISH) and Halo-labelling (HaloTag) experiments: raw images (.TIF), examples of masks obtained from cell segmentation (.mat), and numerical data for quantification of foci in Escherichia coli cells (.CSV).  
- Fluorescence microscopy software used for cell segmentation and spot detection in FISH and HaloTag experiments (.mat).
- Cell growth and viability assays data: optical density measurements at 600nm (OD600) and survival factor calculated using colony forming unit assays (.CSV files).
- RT-qPCR data: reverse transcription quantitative real-time PCR data (.CSV).

3. Data reuse

The fluorescence microscopy image dataset might be of interest as a control dataset for those testing different algorithms for detection of flurescent spots in crowded environments, as well as testing algorithms for automated cell segmentation. 
This dataset might also be useful for those who would like to learn how to use the methods for spot detection in FISH and HaloTag experiments.


## DATASET FOLDER/FILE OVERVIEW  

The dataset is divided in main folders by data type:
-"Fluorescence_microscopy_data", 
-"Fluorescence_microscopy_SRC" (SRC=source code)
-"Growth-and-viability_data"
-"RT-qPCR_data"

Within these data type folders, the data is divided in a "folder by figure" structure, meaning the data is separated according to the order that they appear in the publication. 
Specific README files for each data type containing more detailed information can be found within each main data type folder:
See README-FM, README-GV, README-PCR and README-SRC for fluorescence microscopy, growth and viability data, RT-qPCR and analysis code for microscopy images, respectively.  

Folder overview:  


|-- Fluorescence_microscopy_SRC
|   |-- cell_segmentation
|   |   `-- License.txt
|   |-- spot_quantification
|   |   |-- License.txt
|   |   `-- spatzcells_documentation.pdf
|   `-- README-SRC.txt
|-- Fluorescence_microscopy_data
|   |-- Fig1-RecB_mRNAs_abundance
|   |   |-- Example_masks
|   |   |   |-- delta_recB_FISH_S2_masks
|   |   |   `-- wild_type_FISH_S2_masks
|   |   |-- Images_raw
|   |   |   |-- delta_recB_FISH_S1
|   |   |   |-- delta_recB_FISH_S2
|   |   |   |-- delta_recB_FISH_S3
|   |   |   |-- wild_type-degr_rifamp_FISH_S1
|   |   |   |-- wild_type-degr_rifamp_FISH_S2
|   |   |   |-- wild_type_FISH_S1
|   |   |   |-- wild_type_FISH_S2
|   |   |   `-- wild_type_FISH_S3
|   |   `-- Quantification_results
|   |-- Fig1-S2-recB_overexpr_arabinose
|   |   |-- Images_raw
|   |   |   |-- delta_recB_pBAD33_FISH
|   |   |   |-- delta_recB_pIK02_arab_FISH
|   |   |   `-- wild_type_pBAD33_FISH
|   |   `-- Quantification_results
|   |-- Fig2-RecB_protein_abundance
|   |   |-- Example_masks
|   |   |   |-- wild_type_HaloTag_S1_masks
|   |   |   `-- wild_type_no_HaloTag_neg_ctrl_masks
|   |   |-- Images_raw
|   |   |   |-- recB_removal_HaloTag_S1
|   |   |   |-- recB_removal_HaloTag_S2
|   |   |   |-- wild_type_HaloTag_S1
|   |   |   |-- wild_type_HaloTag_S2
|   |   |   |-- wild_type_HaloTag_S3
|   |   |   `-- wild_type_no_HaloTag_neg_ctrl
|   |   `-- Quantification_results
|   |-- Fig3-RecB_DSB_ciprofloxacin
|   |   |-- Images_raw
|   |   |   |-- off_cipro_FISH_S1
|   |   |   |-- off_cipro_FISH_S2
|   |   |   |-- off_cipro_FISH_S3
|   |   |   |-- off_cipro_HaloTag_S1
|   |   |   |-- off_cipro_HaloTag_S2
|   |   |   |-- off_cipro_HaloTag_S3
|   |   |   |-- on_cipro_FISH_S1
|   |   |   |-- on_cipro_FISH_S2
|   |   |   |-- on_cipro_FISH_S3
|   |   |   |-- on_cipro_HaloTag_S1
|   |   |   |-- on_cipro_HaloTag_S2
|   |   |   `-- on_cipro_HaloTag_S3
|   |   `-- Quantification_results
|   |-- Fig4-RecB_delta_hfq
|   |   |-- Images_raw
|   |   |   |-- delta_hfq_FISH_S1
|   |   |   |-- delta_hfq_FISH_S2
|   |   |   |-- delta_hfq_FISH_S3
|   |   |   |-- delta_hfq_HaloTag_S1
|   |   |   |-- delta_hfq_HaloTag_S2
|   |   |   |-- delta_hfq_HaloTag_S3
|   |   |   |-- delta_hfq_HaloTag_S4
|   |   |   `-- delta_hfq_HaloTag_S5
|   |   `-- Quantification_results
|   |-- Fig5-RecB_post_transc_regulation
|   |   |-- Images_raw
|   |   |   |-- RecB-5UTR_deletion_FISH_S1
|   |   |   |-- RecB-5UTR_deletion_FISH_S2
|   |   |   |-- RecB-5UTR_deletion_HaloTag_S1
|   |   |   |-- RecB-5UTR_deletion_HaloTag_S2
|   |   |   |-- RecB-5UTR_deletion_HaloTag_S3
|   |   |   |-- delta_hfq_pQE-Hfq_HaloTag_S1
|   |   |   |-- delta_hfq_pQE-Hfq_HaloTag_S2
|   |   |   |-- wild_type_pZA21-ChiX_HaloTag_S1
|   |   |   `-- wild_type_pZA21-ChiX_HaloTag_S2
|   |   `-- Quantification_results
|   |-- Fig5-S1-RecB_Hfq_complem_controls
|   |   |-- Images_raw
|   |   |   |-- delta_hfq_pQE80L_HaloTag_S1
|   |   |   `-- delta_hfq_pQE80L_HaloTag_S2
|   |   `-- Quantification_results
|   |-- Fig5-S2-RecB_ChiX_overexpr_controls
|   |   |-- Images_raw
|   |   |   |-- wild_type_pZA21-CyaR_HaloTag_S1
|   |   |   |-- wild_type_pZA21-CyaR_HaloTag_S2
|   |   |   |-- wild_type_pZA21_HaloTag_S1
|   |   |   `-- wild_type_pZA21_HaloTag_S2
|   |   `-- Quantification_results
|   |-- Fig5-S3-RecB_delta_chiX
|   |   |-- Images_raw
|   |   |   |-- delta_chiX_HaloTag_S1
|   |   |   `-- delta_chiX_HaloTag_S2
|   |   `-- Quantification_results
|   `-- README-FM.txt
|-- Growth-and-viability_data
|   `-- README-GV.txt
|-- RT-qPCR_data
|   `-- README-PCR.txt
|-- License.txt
`-- README.txt

Note: For clarity, the data files were removed from the directory tree.