Method Article

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

DOI:

10.3791/56600

⸱

December 21st, 2017

 ,  , 
1Department of Physics of Complex Systems, Weizmann Institute of Science

In This Article

Summary

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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.

Abstract

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A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Introduction

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Stochasticity is a fundamental and unavoidable aspect of gene expression and gives rise to cell-cell heterogeneity1, both at the level of transcripts and proteins2,3. Quantifying the variability between cells under well-defined conditions offers a unique window into the basic processes that underlie gene expression and its regulation. One important source of cell-cell heterogeneity in bacteria takes place at the transcriptional level. Transcript numbers vary not only due to the stochasticity of transcription, but also to post-transcriptional processes such as regulation by small RNAs an....

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Protocol

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1. Probe Design

NOTE: This protocol uses commercially available oligonucleotide probes already tagged with fluorophores. The probes consist of a set of specific sequences complementary to a target mRNA, each probe being conjugated to a single fluorescent molecule. Alternatively, it is possible to attach fluorescent markers to probes, as described elsewhere5,16.

  1. Design smFISH probes using the Probe Designer16 algorithm developed by Arjun Raj (van Oudenaarden Lab, Massachusetts Institute of Technology); sequences of smFISH probes used in this m....

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Results

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We carried out smFISH measurements of galK and sodB transcripts in E. coli cells. The transcripts were hybridized with a set of specific sequences complementary to the target sequence, each probe being conjugated to a single fluorescent molecule (see table of materials). Fluorescence and phase contrast images of MG1655 wild-type E. coli strain (WT) or a JW0740 (Keio collection)19 galK-deleted strain (ΔgalK) were exposed to 2 mM D-fucose a.......

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Discussion

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We have measured in our laboratory the transcript number of different genes in E. coli cells using the smFISH method2. In brief, this procedure consists of the following steps: cell fixation, permeabilization of membranes to allow for probe penetration, probe hybridization, and sample imaging using a standard fluorescence microscope. This procedure is based on previously published ones with some modifications6,7,

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This work was supported by an Israel Science Foundation grant 514415 (to J.S.) and a BSF-NSF (MCB) grant 2016707 (to J.S.). Support from a Siegfried and Irma Ullman Professorial Chair (to J.S.) is also acknowledged.

....

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Dextran sulfate sodium saltSigma-AldrichD8906
Pure Ethanol, 99.5%, ACS reagent, absoluteMallinckrodt Baker - Avantor8025.25
Diethylpyrocarbonate (DEPC)Sigma-AldrichD5758
RNase-free 20X SSCLife Technologies/AmbionAM9763
RNase-free 10X PBSLife Technologies/AmbionAM9625
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) BioUltra, for molecular biologySigma-Aldrich93283
nuclease-free waterThermo Fisher Scientific10977035
Formaldehyde solution for molecular biology, 36.5-38% in waterSigma-AldrichF8775
Deionized formamide, nuclease freeThermo Fisher Scientific/AmbionAM9342
E. coli tRNA (ribonucleic acid, transfer type xx from escherichia)Sigma-AldrichR1753-500UN
UltraPure BSA (50mg/ml)Thermo Fisher Scientific/AmbionAM2616
Vanadyl-ribonucleoside complex,VRC, 200 mMNew England BiolabS1402S
Poly-L-LysineSigmaP4707
Cysteamine and oxygenSigma-Aldrich30070
Glucose Oxidase from Aspergillus niger, Type VII, 50KUSigma-AldrichG2133
CatalaseSigma-AldrichC40
D-glucoseSigma-AldrichG8270
D-fucoseSigma-AldrichF8150
Vybrant DiO Cell-Labeling SolutionLife TechnologiesV2286
Agarose,low melting reagentSigma-AldrichA9414
Adhesive silicone isolator 24-2mm Dia. X 1.8 mm depth JTR24R-A2-2.0Grace Bio-LabsJTR24R-A2-2.0 666208
poly-D-lysine-coated glass bottom Glass Bottom Culture DishesMatTek CorporationP35GC-1.5-14-C
Super life nitrile powder free examination glovesSupermaxTC-N-9889
Brand sterilization incubator tapeSigma-AldrichBR61750
Microcentrifuge tubes (1.8 ml)Axygen - Corning Life SciencesMCT-175C
Falcon round-bottom polypropylene tubes (14 ml)BD Biosciences352059
Conical-bottom centrifuge polypropylene tubes (50 ml)Corning430828
Serological pipettes (Corning 5 ml)Corning Life Sciences4051
Serological pipettes (Corning 10 ml)Corning Life Sciences4488
Serological pipettes (Corning 25 ml)Corning Life Sciences4251
Spectrophotometer cuvettesSarstedt67.742
RNase-free pipette tips 0.2 - 20 μlFroggaBioFT20
RNase-free pipette tips 10 - 200 μlAxigene/corningTF-200
RNase-free pipette tips 100 - 1000 μlFroggaBioFT1000
RNase-free pipette tips 100 - 1000 μlSorenson14200
Syringe disposile 10 mL needle G-21Becton Dickinson, BiosciencesBD-309643
Minisart 0.2 um Syringe FilterSartorius16534 K
Nikon instruments microscope type A immersion oil A, 8ccNikonMXA20233
Microscope slides 76 x26, 3"x1"x1mmThermo Fisher Scientific421-004ET
#0 coverslip slide 24x60Thermo Fisher Scientific/MenzelBNBB024060A0
Orbital shakerM.R.CTOU-50
Hot blockM.R.C
VortexFried Electric CompanyG-560-E
MicrocentrifugeEppendorf5427R
CentrifugeEppendorf5810R
Portable Pipet-Aid XP2, Pipette ControllerDrummond Scientific Company4-000-501-I
OD600 Spectrophotometer for Bacterial Growth Rates DiluPhotometerMidsciOD600-10
iXon X3 EMCCD cameraAndorDU-897E-CS0-#BV
Eclipse Ti microscopeNikonMEA53100
CFI plan apochromat DM 100X oil objective lambda PH-3 N.A 1.45 WD 0.13NikonMRD31905
Filter set (TRITC/CY3): EX - ET545/30X; EM - ET620/60M; BS - T570LPNikon49005
Filter set (CY5): EX - ET640/30X; EM - ET690/50M; BS - T660PNikon49009
Nis-elements AR auto reaserch softwareNikonMQS31000
STORM microscopeVutaraSR-200
NA 1.2 water immersion objective Olympus
SRX image acquisition and analysis softwareVutara
Evolve 512 EMCCD cameraPhotometrics
Stellaris® FISH Probes, Custom Assay with CAL Fluor® Red 590 DyeBiosearch Technologies IncSMF-1083-5
Probe Sequence for galK mRNA:
gtgttttttctttcagactc
tagccaaatgcgttggcaaa
ctgaatggtgtgagtggcag
caccaatcaaattcacgcgg
acgaaaccgtcgttgtagtc
tgataatcaatcgcgcaggg
gtggtgcacaactgatcacg
acgcgaactttacggtcatc
gctgattttcataatcggct
gcatcgagggaaaactcgtc
gttttcatgtgcgacaatgg
cacgccacgaacgtagttag
ttacgcagttgcagatgttt
tccagtgaagcggaagaact
tgctgcaatacggttccgac
gtccagcggcagatgataaa
tgaccgttaagcgcgatttg
agcctacaaactggttttct
gcggaaattagctgatccat
aaggcatgatctttcttgcc
cagtgagcggcaatcgatca
tgggcatggaaactgctttg
gatgatgacgacagccacac
gggtacgtttgaagttactg
gtgttgtattcgctgccaac
ggtttcgcactgttcacgac
tggctgctggaagaaacgcg
ttcaatggtgacatcacgca
catgcgcaacagcgttgaac
ggcgttttcagtcagtatat
atacgtttcaggtcgccttg
tgagactccgccatcaactc
gaaatcatcgcgcatagagg
caatttgcggcacggtgatt
ttgacgatttctaccagagt
acctttgtcgccaatcacag
ggatcagcgcgacgatacag
atattgttcagcgacagctt
gtctctttaatacctgtttt
ctccttgtgatggtttacaa
Stellaris® FISH Probes, Custom Assay with Quaser Fluor® Red 670 DyeBiosearch Technologies IncSMF-1083-5
Probe Sequence for sodB mRNA:
gtgttttttctttcagactc
tagccaaatgcgttggcaaa
ctgaatggtgtgagtggcag
caccaatcaaattcacgcgg
acgaaaccgtcgttgtagtc
tgataatcaatcgcgcaggg
gtggtgcacaactgatcacg
acgcgaactttacggtcatc
gctgattttcataatcggct
gcatcgagggaaaactcgtc
gttttcatgtgcgacaatgg
cacgccacgaacgtagttag
ttacgcagttgcagatgttt
tccagtgaagcggaagaact
tgctgcaatacggttccgac
gtccagcggcagatgataaa
tgaccgttaagcgcgatttg
agcctacaaactggttttct
gcggaaattagctgatccat
aaggcatgatctttcttgcc
cagtgagcggcaatcgatca
tgggcatggaaactgctttg
gatgatgacgacagccacac
gggtacgtttgaagttactg
gtgttgtattcgctgccaac
ggtttcgcactgttcacgac
tggctgctggaagaaacgcg
ttcaatggtgacatcacgca
catgcgcaacagcgttgaac
ggcgttttcagtcagtatat
atacgtttcaggtcgccttg
tgagactccgccatcaactc
gaaatcatcgcgcatagagg
caatttgcggcacggtgatt
ttgacgatttctaccagagt
acctttgtcgccaatcacag
ggatcagcgcgacgatacag
atattgttcagcgacagctt
gtctctttaatacctgtttt
ctccttgtgatggtttacaa

References

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  1. Tsimring, L. S. Noise in biology. Reports Prog. Phys. 77 (2), 26601(2014).
  2. Arbel-Goren, R., et al. Transcript degradation and noise of small RNA-controlled genes in a switch activated network in Escherichia coli. Nucleic Acids Res. 44 (14)....

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Tags

Single molecule FISHE coli mRNA labelingvisualizationFluorescent oligonucleotide probesBacterial cell fixationPermeabilization protocolHybridization bufferAgarose gel mountingFluorescence microscopySTORM imaging

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