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High-throughput β-galactosidase and β-glucuronidase Assays Using Fluorogenic Substrates
利用荧光底物高通量分析β半乳糖苷酶和β葡萄糖醛酸酶   

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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(LacZ), UidA(葡糖苷酸酶), Galactosidase(半乳糖苷酶), Glucuronidase(葡萄糖醛酸酶), Miller assay(Miller法)

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.

简介

β-半乳糖苷酶和β-葡糖醛酸糖苷酶通常用作从基因启动子 - lacZ/em或uidA/em融合(分别)的基因表达的报道分子。本文所述的方案是利用荧光底物(Fiksdal等人,1994)和96孔板形式的通常使用的Miller测定法(Miller,1972)的高通量替代物。荧光底物4-甲基伞形基β-D-半乳糖苷(用于β-半乳糖苷酶测定)(Ramsay等人,2013)或4-甲基伞形基β-D-葡萄糖醛酸苷(用于β-葡糖醛酸糖苷酶测定)( Ramsay等人,2011)切割以产生荧光产物4-甲基伞形酮。通过冷冻 - 融化和溶菌酶使细胞透化,并且通过荧光微板读数器作为动力学测定连续监测4-甲基伞形酮的产生。然后计算荧光增加的速率,从中推断相对基因表达水平。由于高灵敏度基于荧光的4-甲基伞形酮的检测和所收集的高密度时间点,该测定可以在低水平基因表达的定量中提供增加的准确度。该测定需要小的样品体积和最小的制备时间。本方案中概述的透化条件已经针对革兰氏阴性细菌(特别是大肠杆菌和沙雷氏菌)进行了优化,但是可能适合于具有最小优化的其他生物体。

关键字:LacZ, 葡糖苷酸酶, 半乳糖苷酶, 葡萄糖醛酸酶, Miller法

材料和试剂

  1. 细菌培养
  2. 4-甲基伞形基β-D-半乳糖苷(Life Technologies,目录号:M-1489MP)(用于β-半乳糖苷酶测定)
  3. 4-甲基伞形基β-D-葡糖苷酸(Life Technologies,目录号:M-1490)(用于β-葡糖醛酸糖苷酶测定)
  4. 磷酸盐缓冲盐水片(Life Technologies,Invitrogen TM ,目录号:00-3002)
  5. 来自鸡蛋白的溶菌酶(Sigma-Aldrich,目录号:L7651)
  6. 200x 4-甲基伞形基β-D-半乳糖苷或4-甲基伞形基β-D-葡萄糖醛酸的储备溶液(参见配方)
  7. 4-甲基伞形基β-D-半乳糖苷或4-甲基伞形基β-D-葡糖苷酸的最终工作试剂(参见配方)

设备

  1. 荧光微板读数器,例如:Gemini XPS荧光微板读数器(Molecular Devices)或Infinite 200 PRO(Tecan Group Ltd.)
  2. 96孔微孔板(Thermo Fisher Scientific,目录号:269787)
  3. 平底透明96孔微孔板(低自发荧光和/或在365和445 nm的吸光度是优选的)
  4. 能够分配10μl和100μl的多通道移液器(12或8通道)
  5. 超低温冰箱(-70℃)

程序

  1. 记录待测样品的OD 600。 推荐待分析的样品的OD 600位于0.1-1OD 600范围内。 在生长培养基中稀释样品,得到0.1-1范围内的OD 600。 根据观察到的基因表达水平的范围,稀释因子可能需要通过分析多重稀释来优化
  2. 收集100μl稀释的样品(0.1-1OD 600范围),并在-70℃下在96孔微量培养板("主板")中冷冻。 收集所有样品后,将板放回-70°C,并过夜冷冻。
    例如:对于大肠杆菌中的时间过程实验,在单个微孔板中每小时收集100μl样品12小时,在收集每个样品后将微孔板返回至-70℃。此微孔板现在充当"主板",如果需要,可以在稍后的日期从中进行重复测定。只有负介质的对照也可以在免费孔中添加到主平板。
  3. 在收集所有样品(确保所有样品已经在-70℃下完全冷冻)之后,在37℃孵育器中将盖板除霜(以避免通过冷凝交叉污染)除霜母板。
  4. 当母板已完全解冻时,使用多通道移液器将每孔中的10μl等分试样加入新的微量培养板("测定板")中,并将测定板置于-70℃至少15分钟。
  5. 在进行测定前将微孔板读数器预温至37℃,并初始化微孔板读数器软件,以便准备去(参见关于微孔板读数器设置的说明,例如设置)。
  6. 准备最终工作试剂。
    注意:在使用前立即准备,避免长时间暴露于光(即使用当天)。使每个反应100μl足够。
  7. 在37°C培养箱中关闭测定板(避免通过冷凝交叉污染)至少10分钟,和/或直到任何可见的冰晶体溶解在所有孔中。
  8. 将自己置于微孔板读数器的近距离内。使用多通道移液器分配100微升工作试剂到96孔微板(或只是含有样品的孔)的每个孔。尽量缩短向每个孔分配试剂之间的时间。立即将微量培养板放在读板器中,盖子关闭,然后启动程序。含有大量β-半乳糖苷酶和β-葡糖醛酸糖苷酶的样品可在最初10分钟内使测定法饱和,因此在加入底物后快速捕获读取是重要的。
    注意:对于高度表达早期饱和检测器的样品,数据仍然可恢复。然而,由于可能具有较少的具有荧光线性增加的时间点,速率估计可能不太准确。样品稀释可提高这些样品的准确测量。
  9. 使用微孔板软件提取读数的荧光增加速率或导出到excel(参见图1):
    1. 绘制相对荧光强度(机器进行内部荧光标准化,因此它是"相对"荧光强度)随时间(每秒通常是方便的)。选择具有最陡的斜率(V max)的图的线性部分,从中推断速率(在excel中,可以使用等式= SLOPE())。这将提供每秒的相对荧光单位(RFU /秒)。一些平板仪软件将在测定期间自动地和实时地计算V max max。如果曲线图是曲线或非常嘈杂,您可能分别有细胞透化或荧光检测器增益设置的问题(请参见下面的疑难解答)。
    2. 可选:从所有样品中减去阴性对照孔(仅含最终工作试剂的培养基)的平均RFU /秒(实际上,该值通常为零或非常小,因此通常不需要此步骤)。
    3. 归一化为步骤1中记录的光学细胞密度(OD <600>)。这将给出RFU/sec/OD <600>的单位。
    4. 可以对值进行归一化以考虑所使用的样品体积(除以0.01ml,得到值为RFU/sec/ml/OD 600),然而,该归一化有些任意如果总是使用相同的体积(10μl)。或者,已知浓度的纯化β-半乳糖苷酶或β-葡糖醛酸糖苷酶的标准曲线可用于估计酶浓度的实际单位。


      图1.原始相对荧光单位随时间的图。 如上所述,通过β-半乳糖苷酶测定在30分钟时间内分析生物三份样品。在每组旁边指示三次重复的平均斜率和标准偏差。高度表达的样品在600秒后使检测器饱和。与仅负的培养基(具有最终试剂混合物)对照相比,也从低表达的样品中检测到统计学显着的低水平表达。发布/呈现的最终值被归一化为OD <600>。

有关酶标仪设置的注意事项

  1. 通用设置:
    温度
    37°C
    激发波长
    360-365 nm
    发射波长
    445-460 nm
    总测定时间    
    10-30分钟,取决于表达水平
    在某些机器上,其他设置可能是可选的,但如果可用,建议每次阅读之间发生抖动。
  2. 具体机器设置:
    Gemini XPS荧光微孔板读数器
    温度:
    37°C
    自动拍摄:

    阅读类型:
    动力学读数为30分钟,读取间隔为1分钟
    激励:
    360 nm
    排放:
    450 nm
    截止:
    435 nm
    阅读:  
    8次/井
    PMT:

    校准:



    无限 200 PRO

    选择全版读取
    Nuclon平底透明
    盖模式
    关闭
    温度
    37°C
    周期数
    30
    动力学时间间隔
    1分钟
    振动持续时间
    5秒
    振幅
    1毫米
    振动模式
    线性
    激励
    360 nm
    发光
    460 nm
    读板模式
    顶部
    增益
    手动增益模式
    增益
    85
    滞后时间
    0
    积分时间
    20
    每个孔多次读取
    没有

故障排除

  1. 激发/发射
    4-甲基伞形酮具有在365nm的激发峰和在448nm的发射峰。然而,在您的特定机器上观察到的峰值吸收和发射光谱以及可用的设置可能略有不同。此外,来自培养基和/或微板的自发荧光和/或吸光度可能需要选择峰外的波长。最好通过用阳性对照(例如具有已知的β-半乳糖苷酶或β-葡糖醛酸糖苷酶活性的样品)进行发射/激发波长扫描来优化您的设置。根据我们的经验,LB,TY和基本培养基和Nunc MicroWell平底96孔微孔板不产生任何自身荧光的问题。特定生物体可能产生在该测定中发荧光的产物。含有适当的阴性对照菌株(缺少lacZ 或 uidA )的样品的激发/发射波长扫描可以揭示是否是这种情况。
  2. 细胞透化
    在我们手中,该测定对细胞透化程度非常敏感。在该方案中,针对革兰氏阴性细菌优化,通过在-70和37℃之间将样品冻融两次并通过在测定缓冲液中添加高浓度的溶菌酶来实现有效的细胞透化。如果细胞在整个测定中未完全透化或变得越来越透化,则RFU/sec曲线将呈现弯曲而不是线性。您可能需要优化方案以允许样品的有效透化
  3. 酶标仪读数增益
    一些微孔板读数器在整个板或每个单独的孔上自动调节检测器增益。这可以增加读取器的动态范围,因为理想地,非常低和非常高荧光的样品可以 在同一板中分析。然而,对于一些酶标仪,增益可能需要手动设置。如果根本没有检测到荧光,则增益可能太低。如果增益太高,样品将在测定开始后立即饱和检测器。为了确定您的测定和机器的最佳增益,从您最高表达的样品和您最低表达的样品中取样,并进行试验测定,调整每次迭代的增益。选择低表达的样品具有可检测的增加的水平,并且最高表达的样品在10分钟之前不使检测器饱和。

食谱

  1. 200x 4-甲基伞形酮β-D-半乳糖贮存液或
    4-甲基伞形基β-D-葡萄糖醛酸苷以50mg/ml溶于DMSO中的4-甲基伞形基β-D-半乳糖苷或4-甲基伞形基β-D-葡萄糖醛酸苷。
    以小等分试样从-70°C的光下存储。避免反复冻融。
  2. 4-甲基伞形基β-D-半乳糖苷或4-甲基伞形基β-D-葡糖醛酸的最终工作试剂
    在含有2mg ml溶菌酶的PBS缓冲液中稀释200x储液至1×

参考文献

  1. Fiksdal,L.,Pommepuy,M.,Caprais,M.-P.和Midttun,I。(1994)。 使用快速酶技术监测沿海水域的粪便污染。 Appl Environ Microbiol 60(5):1581-1584。
  2. Miller,J.H。(1972)。 Experiments in molecular geneticetics,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)。 广泛保守的分子开关通过表达新型抗活化剂控制在中生根瘤菌中的群体感应和共生岛转移 。 Mol Microbiol 87(1):1-13。
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引用: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.
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