Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments
December 21st, 2017
This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.
The overall goal of this protocol is to label individual transcripts in E.Coli for fluorescently labeled DNA probes for visualization in single-molecule FiSH experiments. This method can help answer key questions in the systems biology field, such as characterizing transcripts cell to cell ability of field of interest. As well as the transcripts of cell wall localization.
The main advantage of this technique is that it provides quantity of information about the statistics of transcriptional processes. To begin the protocol, grow E.Coli MG1655 in LB medium overnight in an orbital shaker, at 260 RPM and 37 degrees Celsius. The next day, dilute the overnight culture at one to 100 in fresh medium and measure its optical density in a spectrophotometer, up to a value of 0.2 to 0.4.
Next, pellet the cells by centrifugation for five minutes at 4, 500 G at four degrees Celsius. Remove the supernatant from all tubes. Add one milliliter of 1XPBS to each tube and re-suspend the pellet to minimize cell to cell aggregation.
Then add four milliliters of fixation buffer and gently vortex the tube. Incubate the samples in an orbital shaker. Re-suspend the pellet in order to minimize cell to cell aggregation, which may interfere with cell imaging.
After incubation, pellet the cells, remove the supernatant, and add one milliliter of 1XPBS. Transfer suspensions to 1.8 milliliter conical bottom microcentrifuge tubes. Pellet the cells by centrifugation.
Begin bacteria permeabilization by carefully removing the supernatant. Use small volume pipettes to remove all of the liquid outside the pellet. Next, re-suspend the pellet in 300 microliters DEPC treated water.
Add 350 microliters of high grade absolute ethanol and mix the solution gently by inverting the tube. Add another 350 microliters of ethanol and mix again. Then rotate the sample with the tube rotator at 20 RPM for one hour at room temperature.
Pellet the cells from the permeabilization. After centrifugation, remove the supernatant. Add one milliliter of 20%wash buffer to the samples.
Leave them standing for several minutes. Next, pellet cells by centrifugation for seven minutes at 750 G and four degrees Celsius. Remove the supernatant very gently and use small volume pipettes to remove all the liquid outside the pellet.
Then add the oligonucleotide probe set stock solution to the hybridization buffer aliquot so that the final oligonucleotide concentration is 250 nanomolar and vortex the tube vigorously. Suspend the samples in 50 microliters of hybridization buffer that contains the oligonucleotide probes, while avoiding the generation of bubbles. Leave the samples overnight, in the dark, on a heat block at 30 degrees Celsius.
To prepare washing, obtain one new micro-centrifuge tube for each sample to image. Add one milliliter wash buffer to each tube. Add 10 to 15 microliters from the hybridized samples to the new tube that contains the wash buffer.
Pellet the cells by centrifugation. Then, remove the supernatant. Wash the samples two more times.
Re-suspend the pellet in 25 microliters wash buffer. Prepare an agarose gel by adding 150 milligrams of agarose to 10 milliliters of 2X SSC buffer. Microwave the solution for 10 to 15 seconds each time, until agarose fully dissolves.
Then set the tube aside to cool for a few minutes. Next, place a separating, silicone frame on the slide so that the slide's center remains exposed. Place a 10 millimeter wide, one to two millimeter thick rigid ring in the center of the slide.
Deposit a small amount of gel into the center for sealing and wait a few minutes for the gel to harden. Then add more gel, until a high dome shape is formed. Wait about 10 minutes for the gel to harden and remove the ring.
Deposit 10 microliters of wash bacterial cells onto the gel and seal it with a number zero cover slip slide. Keep the sample in the dark to prevent photo-bleaching. smFiSH measurements of E.coli cells show the level of galK induction in response to variable extra-cellular D-fucose concentrations.
The background signal is shown by a galK deleted strain, or delta galK JW0740 as a negative control. Numerous spots are seen in most cells. Corresponding to single or few galK transcripts when two millimolar D-fucose is added to the bacterial culture.
Whereas, a few spots are seen in the absence of extra-cellular D-fucose. Another example for the application of this protocol is furnished by the sodB gene, with fluorescence and phase contrast images of smFiSH in a wild type or a JW1648 sodB deleted strain. Samples prepared with this protocol may also be imaged using STORM which allows accurate precisioning of transcripts in the cell.
After its development, this technique paved the way for researchers in the field of systems biology to explore the statistics of transcription events in individual cells.
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Overview
This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. This technique allows for the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.
Key Study Components
Area of Science
- Neuroscience
- Systems Biology
- Cell Biology
Background
- Single-molecule fluorescence in situ hybridization (smFISH) is a powerful visualization method.
- This method enables the study of transcriptional processes at the single-cell level.
- Understanding mRNA localization and quantification is crucial for systems biology.
- E. coli serves as a model organism for these experiments.
Purpose of Study
- To label individual mRNA transcripts in E. coli.
- To visualize mRNA localization using fluorescently labeled DNA probes.
- To enhance understanding of transcriptional variability among cells.
Methods Used
- Grow E. coli MG1655 in LB medium overnight.
- Dilute the overnight culture in fresh medium.
- Measure optical density to ensure proper growth conditions.
- Apply fluorescently labeled DNA probes for smFISH analysis.
Main Results
- Successful labeling of individual mRNA transcripts.
- Visualization of mRNA localization within fixed cells.
- Quantification of transcriptional processes at the single-molecule level.
- Insights into cell-to-cell variability in transcript expression.
Conclusions
- The method provides a robust approach for studying mRNA dynamics.
- It contributes valuable information to the field of systems biology.
- This technique can be applied to various research questions regarding gene expression.
Frequently Asked Questions
Executive Industry Relevance
Single-molecule fluorescence in situ hybridization (smFISH) in E. coli enables direct quantification and spatial mapping of mRNA transcripts at single-cell resolution, addressing key challenges in understanding transcriptional heterogeneity. This capability supports predictive confidence in early discovery by revealing cell-to-cell variability and subcellular transcript localization. The method is strategically positioned for target validation and mechanistic de-risking in microbial systems biology and synthetic biology pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables quantitative interrogation of gene expression variability across individual bacterial cells.
- Supports mechanistic de-risking by mapping transcript localization and abundance at single-molecule resolution.
- Facilitates functional target validation by distinguishing true biological effects from population-averaged artifacts.
- Provides high-content data for hypothesis-driven pathway analysis in microbial models.
Screening & Assay Development
- Prepares validated bacterial systems for downstream screening of gene regulation or synthetic constructs.
- Delivers reproducible, quantitative mRNA copy number outputs for assay standardization.
- Enables multiplexed transcript detection within single cells, limited by spectral overlap of fluorophores.
- Supports robust evaluation of gene induction or repression in response to experimental perturbations.
Translational & Preclinical Research
- Aligns with translational biomarker strategies by enabling precise quantification of gene expression changes in microbial models.
- Provides continuity from discovery to preclinical validation for engineered strains or pathway interventions.
- Reduces risk in advancing microbial targets by supplying single-cell resolution data on transcript dynamics.
Pipeline & Workflow Integration
This smFISH labeling method integrates into the discovery-to-preclinical continuum for microbial systems, supporting both hypothesis testing and quantitative analytics.
- Discovery Biology: Delivers direct measurement of transcriptional events and spatial transcript distribution for pathway clarification.
- Screening: Supplies standardized, quantitative readouts for evaluating gene expression modulation in engineered or wild-type strains.
- Analytics: Enables statistical analysis of mRNA copy number distributions and cell-to-cell variability using open-source or custom software.
- Translational Research: Bridges discovery findings to preclinical models by validating gene expression changes at single-cell resolution.
- Enterprise Reuse: Offers a reusable workflow for transcript quantification in diverse bacterial systems and experimental contexts.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in microbial gene expression studies.
- Operational Value: Standardizes transcript labeling and imaging for reproducible, scalable data generation.
- Strategic Value: Improves go/no-go decisions for microbial targets and synthetic constructs by providing high-resolution expression data.
- Portfolio Impact: Enables risk-adjusted prioritization of microbial targets and engineered pathways based on quantitative single-cell analytics.
Implementation Considerations
- Requires expertise in bacterial cell handling, fixation, and fluorescence microscopy.
- Needs access to fluorescence imaging platforms with low-intensity detection and appropriate filter sets.
- Demands cross-team standardization of probe design and imaging protocols for reproducibility.
- Adaptation to other bacterial strains or gene targets may require probe redesign and spectral optimization.
- Multiplexing is limited by spectral overlap of available fluorophores, constraining simultaneous gene detection.
