Welcome guest, Sign in

Home

X
加载中

β-galactosidase and β-glucuronidase enzymes are commonly used as reporters for gene expression from gene promoter-lacZ or uidA fusions (respectively). The protocol described here is a high-throughput alternative to the commonly used Miller assay (Miller, 1972) that utilises a fluorogenic substrate (Fiksdal et al., 1994) and 96-well plate format. The fluorogenic substrates 4-Methylumbelliferyl β-D-galactoside (for β-galactosidase assays) (Ramsay et al., 2013) or 4-Methylumbelliferyl β-D-glucuronide (for β-glucuronidase assays) (Ramsay et al., 2011) are cleaved to produce the fluorescent product 4-methylumbelliferone. Cells are permeabilized by freeze-thawing and lysozyme, and the production of 4-methylumbelliferone is monitored continuously by a fluorescence microplate reader as a kinetic assay. The rate of increase in fluorescence is then calculated, from which relative gene-expression levels are extrapolated. Due to the high sensitivity fluorescence-based detection of 4-methylumbelliferone and the high density of time points collected, this assay may offer increased accuracy in the quantification of low-level gene expression. The assay requires small sample volumes and minimal preparation time. The permeabilisation conditions outlined in this protocol have been optimised for Gram-negative bacteria (specifically Escherichia coli and Serratia), but is likely suitable for other organisms with minimal optimisation.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

High-throughput β-galactosidase and β-glucuronidase Assays Using Fluorogenic Substrates

Microbiology > Microbial cell biology > Cell-based analysis > Reporter assay
Author: Joshua P. Ramsay
Joshua P. RamsayAffiliation: School of Biomedical Sciences, Curtin University, Perth, Australia
For correspondence: josh.ramsay@curtin.edu.au
Bio-protocol author page: a450
Vol 3, Iss 14, 7/20/2013, 4202 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.827

[Abstract] β-galactosidase and β-glucuronidase enzymes are commonly used as reporters for gene expression from gene promoter-lacZ or uidA fusions (respectively). The protocol described here is a high-throughput alternative to the commonly used Miller assay (Miller, 1972) that utilises a fluorogenic substrate (Fiksdal et al., 1994) and 96-well plate format. The fluorogenic substrates 4-Methylumbelliferyl β-D-galactoside (for β-galactosidase assays) (Ramsay et al., 2013) or 4-Methylumbelliferyl β-D-glucuronide (for β-glucuronidase assays) (Ramsay et al., 2011) are cleaved to produce the fluorescent product 4-methylumbelliferone. Cells are permeabilized by freeze-thawing and lysozyme, and the production of 4-methylumbelliferone is monitored continuously by a fluorescence microplate reader as a kinetic assay. The rate of increase in fluorescence is then calculated, from which relative gene-expression levels are extrapolated. Due to the high sensitivity fluorescence-based detection of 4-methylumbelliferone and the high density of time points collected, this assay may offer increased accuracy in the quantification of low-level gene expression. The assay requires small sample volumes and minimal preparation time. The permeabilisation conditions outlined in this protocol have been optimised for Gram-negative bacteria (specifically Escherichia coli and Serratia), but is likely suitable for other organisms with minimal optimisation.

Keywords: LacZ, UidA, Galactosidase, Glucuronidase, Miller assay

Materials and Reagents

  1. Bacteria cell culture
  2. 4-Methylumbelliferyl β-D-galactoside (Life Technologies, catalog number: M-1489MP) (for β-galactosidase assays)
  3. 4-Methylumbelliferyl β-D-glucuronide (Life Technologies, catalog number: M-1490) (for β-glucuronidase assays)
  4. Phosphate-Buffered Saline Tablets (Life Technologies, InvitrogenTM, catalog number: 00-3002)
  5. Lysozyme from chicken egg white (Sigma-Aldrich, catalog number: L7651)
  6. 200x stock solution for 4-Methylumbelliferyl β-D-galactoside or 4-Methylumbelliferyl β-D-glucuronide (see Recipes)
  7. Final working reagent of 4-Methylumbelliferyl β-D-galactoside or 4-Methylumbelliferyl β-D-glucuronide (see Recipes)

Equipment

  1. Fluorescence microplate reader, for example: Gemini XPS Fluorescence Microplate Reader (Molecular Devices) or Infinite 200 PRO (Tecan Group Ltd.)
  2. 96-Well microplates (Thermo Fisher Scientific, catalog number: 269787)
  3. Flat-bottomed clear 96-well microplates (low autofluorescence and/or absorbance at 365 and 445 nm is preferable)
  4. Multi-channel pipette(s) (12 or 8 channel) capable of dispensing 10 μl and 100 μl
  5. Ultra-low temperature freezer (-70 °C)

Procedure

  1. Record the OD600 of the samples to be assayed. It is recommended that the OD600 of the samples to be analysed are in the 0.1-1 OD600 range. Dilute the samples in growth media to give an OD600 in the 0.1-1 range. Depending on the range of gene expression levels observed, the dilution factor may need to be optimised by analysing multiple dilutions.
  2. Collect 100 μl of the diluted samples (0.1-1 OD600 range) to be assayed and freeze at -70 °C in a 96-well microplate (the "master plate"). Once all samples are collected, return plate to the -70 °C and leave to freeze overnight.
    For example: For a time-course experiment in E. coli, 100 μl samples could be collected every hour for 12 h in a single microplate, returning the microplate to the -70 °C after collecting each sample. This microplate now acts as the "master plate", from which replicate assays can be carried out at a later date if desired. Negative media-only controls can also be added to the master plate in free wells.
  3. After all samples are collected (make sure all samples have been fully frozen at -70 °C), defrost the master plate in the 37 °C incubator with the lid off (to avoid cross-contamination via condensation).
  4. When the master plate has fully defrosted, aliquot 10 μl from each well into a new microplate (the "assay plate") using a multichannel pipette and place the assay plate in the -70 °C for at least 15 min.
  5. Pre-warm the microplate reader to 37 °C prior to carrying out the assay and initialise the microplate reader software so it is ready to go (see Notes about microplate reader settings for example settings).
  6. Prepare the final working reagent.
    Note: Prepare immediately prior to use and avoid prolonged exposure to light (i.e. Use the same day). Make enough for 100 μl per reaction.
  7. Defrost the assay plate in the 37 °C incubator with the lid off (to avoid cross-contamination via condensation) for at least 10 min and/or until any visible ice crystals have dissolved in all wells.
  8. Place yourself within close distance of the microplate reader. Dispense 100 μl of working reagent into each well of the 96-well microplate (or just wells containing samples) using a multichannel pipette. Try to minimize the time between dispensing reagent to each well. Immediately place the microplate in the plate reader with the lid off and start the program. Samples containing large amounts of β-galactosidase and β-glucuronidase can saturate the assay within the first 10 min, therefore it is important to capture reads quickly after the addition of substrate.
    Note: For highly expressing samples that saturate the detector early, data is still recoverable. However as there are likely to be fewer timepoints with a linear increase in fluorescence the rate estimation may be less accurate. Sample dilution will improve accurate measurement of these samples.
  9. Extract the rate of increase in fluorescence from the reads using the microplate software or export to excel (see Figure 1):
    1. Plot the relative fluorescence intensity (the machine carries out internal fluorescence normalisation, hence it is "relative" fluorescence intensity) over time (per second is usually convenient). Choose a linear portion of the graph with the steepest slope (Vmax) from which to extrapolate the rate (in excel, the equation = SLOPE() can be used). This will provide relative fluorescence units per second (RFU/sec). Some platereader software will calculate Vmax automatically and in real time during the assay. If the graph is curved or very noisy, you may have problems with cell permeabilization or the fluorescence detector gain settings, respectively (see Troubleshooting below).
    2. Optional: Subtract the average RFU/sec of negative control wells (media-only with final working reagent) from all samples (in practice this value is usually zero or very small and so this step is usually not necessary).
    3. Normalise to optical cell density (OD600) recorded in step 1. This will give you units of RFU/sec/OD600.
    4. Values can be normalized to account for sample volume used (i.e. Divided by 0.01 ml to give values as RFU/sec/ml/OD600), however this normalization is somewhat arbitrary if the same volumes (10 μl) are always used. Alternatively, standard curves with known concentrations of purified β-galactosidase or β-glucuronidase can be used to estimate actual units of enzyme concentration.


      Figure 1. Plot of raw relative fluorescence units over time. Biological triplicate samples were analysed by the β-galactosidase assay over a 30-min period, as described above. Mean slope and standard deviation for the three replicates are indicated next to each group. The highly-expressed samples saturate the detector after 600 sec. Statistically significant low-level expression is also detected from the lowly-expressed sample compared to the negative media-only (with final reagent mix) control. Final values for publication/presentation are normalised to OD600.

Notes about microplate reader settings

  1. Universal settings:
    Temperature
    37 °C
    Excitation wavelength
    360-365 nm
    Emmission wavelength
    445-460 nm
    Total assay time    
    10-30 min depending on expression level
    Additional settings may be optional on some machines, however shaking between each read is recommended if avaliable.
  2. Specific machine settings:
    Gemini XPS Fluorescence Microplate Reader
    Temperature: 
    37 °C
    Autoshake: 
    on
    Read type:
    kinetic read of 30 min, read intervals 1 min
    Excitation:
    360 nm
    Emission: 
    450 nm
    cut-off:
    435 nm
    Reads:  
    8 reads/well
    PMT:
    High
    Calibrate: 
    On


    Infinite 200 PRO

    Select full plate read
    Nuclon flat-bottom transparent
    lid mode
    off
    Temperature
    37 °C
    Number of cycles
    30
    Kinetic time interval
    1 min
    Shaking duration
    5 sec
    Shaking amplitude
    1 mm
    Shaking mode
    Linear
    Excitation
    360 nm
    Emission
    460 nm
    Plate read mode
    Top
    Gain
    Manual Gain mode
    Gain
    85
    Lagtime
    0
    Integration time
    20
    Multiple reads per well
    No

Troubleshooting

  1. Excitation/emission
    4-methylumbelliferone has an excitation peak at 365 nm and emission peak at 448 nm. However the peak absorption and emission spectra observed on your particular machine and the settings available to you may vary slightly. Additionally, autofluorescence and/or absorbance from the media and/or microplate may require you to select wavelengths outside the peaks. It is best to optimize for your setup by carrying out an emission/excitation wavelength scan with a positive control, such as a sample with known β-galactosidase or β-glucuronidase activity. In our experience, LB, TY and minimal medium and Nunc MicroWell Flat-bottomed 96-Well Microplates do not generate any problems with autofluorescence. It is possible that a particular organism may generate products that fluoresce in this assay. Excitation/emission wavelength scans of samples containing appropriate negative control strains (lacking lacZ or uidA) may reveal if this is the case.
  2. Cell permeabilization
    In our hands this assay is very sensitive to the degree of cell permeabilization. In this protocol, optimized for Gram-negative bacteria, efficient cell permeabilization is achieved by freeze-thawing the samples twice between -70 and 37 °C and through the addition of high concentrations of lysozyme in the assay buffer. If cells are not completely permeabilized or become increasingly permeabilized throughout the assay, the RFU/sec plot will appear curved rather than linear. You may need to optimise the protocol to allow efficient permeabilization of your samples.
  3. Microplate reader gain
    Some microplate readers automatically adjust the detector gain over the entire plate or for each individual well. This can increase the dynamic range of the reader, as ideally both very lowly and very highly fluorescent samples can be analysed in the same plate. However for some microplate readers, the gain may need to be set manually. If fluorescence is not detected at all then the gain is likely too low. If the gain is too high, samples will saturate the detector almost immediately after the assay begins. To determine the optimal gain for your assay and machine, take samples from your most highly expressed samples and your most lowly expressed samples and carry out trial assays, adjusting the gain with each iteration. Choose a level at which lowly expressed samples have a detectible increase and the highest expressed sample doesn't saturate the detector before 10 min.

Recipes

  1. 200x stock solution for 4-Methylumbelliferyl β-D-galactoside or
    4-Methylumbelliferyl β-D-glucuronide Dissolve 4-Methylumbelliferyl β-D-galactoside or 4-Methylumbelliferyl β-D-glucuronide in DMSO at 50 mg ml-1.
    Store in small aliquots away from light at -70 °C. Avoid repeated freeze-thawing.
  2. Final working reagent of 4-Methylumbelliferyl β-D-galactoside or 4-Methylumbelliferyl β-D-glucuronide
    Dilute 200x stock to 1x in PBS buffer containing 2 mg ml-1 lysozyme

References

  1. Fiksdal, L., Pommepuy, M., Caprais, M.-P. and Midttun, I. (1994). Monitoring of fecal pollution in coastal waters by use of rapid enzymatic techniques. Appl Environ Microbiol 60(5): 1581-1584.
  2. Miller, J. H. (1972). Experiments in molecular genetics, Cold Spring Harbor Laboratory Cold Spring Harbor, New York. p. 352-355.
  3. Ramsay, J. P., Major, A. S., Komarovsky, V. M., Sullivan, J. T., Dy, R. L., Hynes, M. F., Salmond, G. P. and Ronson, C. W. (2013). A widely conserved molecular switch controls quorum sensing and symbiosis island transfer in Mesorhizobium loti through expression of a novel antiactivator. Mol Microbiol 87(1): 1-13.


How to cite this protocol: Ramsay, J. P. (2013). High-throughput β-galactosidase and β-glucuronidase Assays Using Fluorogenic Substrates. Bio-protocol 3(14): e827. DOI: 10.21769/BioProtoc.827; Full Text



Share Your Feedback:

  • Add Photo
  • Add Video

Bio-protocol's major goal is to make reproducing an experiment an easier task. If you have used this protocol, it would be great if you could share your experience by leaving some comments, uploading images or even sharing some videos. Please login to post your feedback.

Q&A and Troubleshooting:

  • Add Photo
  • Add Video

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.


Login | Register
Share
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook