搜索

Isolation of Outer Membrane Vesicles from Phytopathogenic Xanthomonas campestris pv. campestris
从野油菜黄单胞菌野油菜致病变种分离外膜囊泡   

下载 PDF 引用 收藏 1 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Gram-negative bacteria naturally release outer membrane vesicles (OMVs) to the surrounding environment. OMVs contribute to multiple processes, such as cell-cell communication, delivery of enzymes and toxins, resistance to environmental stresses and pathogenesis. Little is known about OMVs produced by plant-pathogenic bacteria, and their interactions with host plants. The protocol described below discusses the isolation process of OMVs from Xanthomonas campestris pv. campestris strain 33913, a bacterial pathogen of Crucifiers. Nevertheless, this protocol can be used and/or adapted for isolation of OMVs from other phytopathogenic bacteria to promote the study of OMVs in the context of plant-microbe interactions.

Keywords: OMVs(OMVs), Outer membrane vesicles(外膜囊泡), Extracellular vesicles(胞外囊泡), Isolation(分离), Purification(纯化), Xanthomonas campestris pv. campestris(野油菜黄单胞菌), Plant-microbe interactions(植物-微生物相互作用)

Background

Extracellular vesicle (EV) release is a process shared by many organisms from all domains of life. In Gram-negative bacteria, most EVs are a result of the outer membrane blebbing and eventually pinching off the bacterial cell wall, and are hence referred to as outer membrane vesicles (OMVs). The study of OMVs focuses on OMV biogenesis, cargo, functions and interactions with host organisms. To date, most of the study on OMVs focused on bacterial pathogens of humans and environmental bacteria, however very little research has been done on OMVs from phytopathogenic bacteria. The protocol described here was adapted from the protocol described by Chutkan et al. (2013) with slight modification, and presented here with the phytopathogen X. campestris pv. campestris. To our understanding this is the first, fully detailed, protocol for isolation of OMVs from phytopathogenic bacteria and we hope it could serve as a guiding protocol for other research groups interested in this topic.

Materials and Reagents

  1. Sterile single-use inoculating loop
  2. 200 μl pipette tips (such as: Corning, Axygen®, catalog number: # T-200-C )
  3. Parafilm (Bemis, catalog number: PM996 )
  4. Syringe-mounted 0.45-µM filter (EMD Millipore, catalog number: SLHV033RS )
  5. Polyethersulfone (PES) filter, 0.45-µM, Ø75-mM membrane diameter, with an attached 500-ml bottle. Operated by a vacuum pump (e.g., Guangzhou Jet Bio-Filtration, catalog number: FPE404500 )
  6. 1 ml and 10 ml sterile syringes (without needle)
    1 ml syringes (such as: KDL Medical Product, catalog number [as written on the package]: 7290010993604)
    10 ml syringes (such as: Medisposables, catalog number [as written on the package]: 9304-10/16933506093041 )
  7. Sterile 15-ml Falcon tube (such as: SPL Life Sciences, catalog number: 50015 )
  8. 12 ml polypropylene/polyallomer ultracentrifuge tubes (such as: Beckman Coulter, catalog number: 331372 ) – make sure that they are fit for the ultracentrifuge’s speed
  9. Syringe-mounted 0.22-µM filter (Axiva Sichem Biotech, catalog number: SFPV13 R )
  10. Sterile 1.5-ml tube (1.7-ml clear tube) (Corning, Axygen®, catalog number: # MCT-175-C )
  11. 1 ml pipette tips (such as: Corning, Axygen®, catalog number: # T-1000-C )
  12. Polystyrene (PS) aseptic Petri dishes, 90 x 15-mm (Miniplast Ein-Shemer, catalog number: 820-090-01-017 )
  13. Xanthomonas strain in glycerol stock stored at -80 °C – for the preparation of this protocol we used X. campestris pv. campestris (Xcc) 33913, which was obtained from American Type Culture Collection (ATCC)
  14. Difco nutrient agar (BD, catalog number: 21300 )
    Note: It contains 3 g/L of beef extract, 5 g/L of peptone, and 15 g/L of agar, with a final pH of 6.8 ± 0.2 (as written on the powder’s container).
  15. Cephalexin hydrate (Sigma-Aldrich, catalog number: C4895 ) – stock solution at concentration of 10 mg/ml
  16. Sterile dH2O
  17. 10x phosphate buffered saline (PBS) buffer (optional) (such as: Sigma-Aldrich, catalog number: P5493 )
  18. OptiPrep density gradient medium (Sigma-Aldrich, catalog number: D1556 )
  19. ‘Coomassie Plus – The Better Bradford Assay Kit’ (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23236 ) (optional)
  20. Lipid dye FM4-64FX (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: F34653 ) (optional)
  21. Materials and reagents required for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as indicated in the following protocol (optional): http://www.bio-protocol.org/e80 (‘[Bio101] Laemmli-SDS-PAGE’)
  22. Yeast extract powder (EMD Millipore, catalog number: 61931105001730 )
  23. Bacto tryptone (BD, Bacto, catalog number: 211705 )
  24. Sodium chloride (NaCl) (EMD Millipore, catalog number: 7647-14-5 )
  25. Sucrose crystallized (Duchefa Biochemie, catalog number: S0809 )
  26. Magnesium sulfate heptahydrate (MgSO4·7H2O) (EMD Millipore, catalog number: 105886 )
  27. NaOH or HCl (in order to adjust pH)
  28. Peptone (EMD Millipore, catalog number: 61930705001730 )
  29. L(+)-glutamic acid (Acros Organics, catalog number: 6106-04-3 )
  30. HEPES (Sigma-Aldrich, catalog number: H3375 )
  31. YEB medium (see Recipes)
  32. PSB medium (see Recipes)
  33. OptiPrep diluent buffer (see Recipes)

Note: (optional) – not necessary for the vesicles’ isolation process.

Equipment

  1. Biological hood
  2. Temperature controlled incubator shaker (28 °C), with a capability to reach 200 RPM (such as: Thermo Fisher Scientific, Thermo ScientificTM, model: MaxQTM 6000 )
  3. Two 2-L Erlenmeyer flasks
  4. Cuvette  
  5. Polypropylene Copolymer (PPCO) 250-ml centrifuge bottles (such as: Thermo Fisher Scientific, Thermo ScientificTM, model: NalgeneTM PPCO Centrifuge Bottle ) – make sure that they are fit for the centrifuge’s speed
  6. High-speed centrifuge (e.g., Beckman Coulter, model: Avanti J-E , catalog number: 369003; or Thermo Fisher Scientific, model: SorvallTM RC-6 , catalog number: 74804*) and 6 x 250-ml rotor (such as: Beckman Coulter, model: JLA-16.250 , catalog number: 363930, dual-locking lid; or Thermo Fisher Scientific, SorvallTM, model: SLA-1500 , catalog number: 46615* [old model]) at a speed of approx. 32,000 x g
  7. Ultracentrifuge (e.g., Thermo Fisher Scientific, SorvallTM, model: Discovery 90SE , catalog number: 40130867*, Kendro Laboratory Products) and a 6 x 12-ml swinging bucket ultracentrifuge rotor such as: Thermo Fisher Scientific, SorvallTM, model: TH-641 , catalog number: 54295* (old model, Kendro Laboratory Products), or Thermo Fisher Scientific, Thermo ScientificTM, model: TH-641 , catalog number: 54295 (new model,) at a speed of approx. 175,000 x g
  8. Autoclave
  9. NanoSight device (Malvern) (optional)
  10. Transmission electron microscopy (optional)
  11. Equipment required for SDS-PAGE as indicated in the following protocol (optional): http://www.bio-protocol.org/e80 (‘[Bio101] Laemmli-SDS-PAGE’)

Notes

  1. *Catalog number of an old model.
  2. (optional) – not necessary for the vesicles’ isolation process.
  3. Important note: the centrifuge speeds mentioned above, were calculated with Science Gateway's centrifuge rotor speed calculator (http://www.sciencegateway.org/tools/rotor.htm) – if the centrifuge/ultracentrifuge did not give such option.

Procedure

  1. Preparations prior to OMV isolation
    1. Preparation of 1 L of YEB (for starters, see Recipes).
    2. Preparation of 1 L of PSB (the main medium, see Recipes).
    3. Plating the bacteria
      Plate Xcc 33913 (from glycerol stock) on Difco nutrient agar plates, by using a sterile single-use inoculating loop.
      Note: It is recommended to plate the bacteria 2-5 days before making the starters with Xcc.
    4. Making Xcc starters (2 sterile tubes with 3 ml of sterile YEB in each)
      1. To every tube, add 3 µl of cephalexin hydrate 10 mg/ml (final concentration will be 10 µg/ml of medium).
      2. Using a sterile 200-µl pipette tip, scrub 3-5 Xcc colonies from the plate, and add them to the medium in the tube. Must be done in a biological hood.
      3. Put the tubes in 28 °C shaker for overnight (the speed is 180-200 rpm).
    5. Inoculation of the main medium (PSB)
      1. To every 500 ml of PSB medium in each 2-L Erlenmeyer flask, add 500 µl of cephalexin hydrate 10 mg/ml (final concentration will be 10 µg/ml of medium).
      2. Then, from one of the Erlenmeyer flasks, transfer 1 ml of medium (+ cephalexin) into a clean, new cuvette (will serve as a blank for the spectrophotometer). Cover the cuvette with 2 layers of Parafilm, and keep the cuvette at 4 °C.
      3. Then, to each Erlenmeyer flask, add 300 µl from the same starter (has to be very cloudy).
      4. Put the Erlenmeyer flasks into 28 °C shaker, at the speed of 180-200 rpm, for overnight (Video 1).
      Notes:
      1. Must be done in a biological hood.
      2. The ‘cloudiness’ of the starter might affect the time needed for the inoculated PSB media, to reach the desired OD600 nm.
      3. Temperature above 30 °C might inhibit bacterial growth in the Erlenmeyer flasks.

        Video 1. Culture incubation

    6. On the next day – object density measurement
      1. The cuvette stored at 4 °C (with the PSB medium and cephalexin hydrate) serves as a blank. This cuvette should be kept at 4 °C between measurements.
      2. At the ‘normal range’ described in Table 1, the concentration of Xcc 33913 is approximately 108 cfu/ml, and the cells are at around the mid log phase of the growth curve. Just before cell harvest, a small sample of the culture is taken, serially diluted and plated to determine cell number and to assess the amount of purified vesicles per cell.

        Table 1. The object density ranges


      Notes:
      1. Must be done in a biological hood. Since different spectrophotometers may give different reads for the same sample, it is recommended that first a proper bacterial growth curve experiment will be conducted using OD measurements and cell counts on plates, to accurately define the different growth phases and their respective ODs with the instrument used.
      2. About the required OD600 nm: Ideally, cells should be harvested at mid-log phase. Harvesting at a later time point might result in cell death and cell debris, and/or release of other substances that could contaminate the preparation and/or clog the filtering membrane. The typical incubation time, that is required in order to get to the desired OD600 nm, is 17-21 h. 

  2. The isolation process
    1. Removal of bacteria
      1. Pour the cultures into 250-ml bottles, and centrifuge them at approx. 13,200 x g for 22 min, at the temperature of 4 °C to pellet bacterial cells. Rotors that can be used: GSA (Sorvall), JLA-16.250 (Beckman-Coulter) (Video 2).

        Video 2. Removal of bacteria

      2. Then, filter the supernatant through a 0.45-µM PES filter with Ø75-mM membrane diameter (operated by a vacuum pump).
        Optional: It is possible to filter the entire 1 L of the culture through such filter; thus, you can connect it to a 1-L bottle (if the filter fits). The filtered supernatant can be stored at 4 °C up to 1 day.
    2. Pelleting the vesicles
      1. Pour the filtered supernatant into 250-ml bottles, and centrifuge them at approx. 32,000 x g for 2 h, at 4 °C. The vesicles will be pelleted (Figure 1) in the outward direction from the center of the rotor. Rotors that can be used: SLA-1500 (Sorvall), JLA-16.250 (Beckman-Coulter) (Video 3).
      2. Discard the supernatant and resuspend the pelleted vesicles, using 5-8 ml of sterile dH2O, by pipetting.
        Optional: Filter the resuspended vesicles through 0.45-µM (operated by a 10-ml syringe) into a sterile 15-ml Falcon tube, so you can keep them at 4 °C for a few days. Must be done in a biological hood.


        Figure 1. The pelleted OMVs with a translucent yellow color can be seen in the bottom of the tubes (within the marked area of each tube), after centrifugation at approx. 32,000 x g for 2 h
         
        Video 3. Pelleting the vesicles

    3. Ultracentrifugation
      1. Pour the resuspended vesicles, from the previous step, into two 12-ml ultracentrifuge tubes, and use sterile dH2O to fill the remaining volume of the tubes.
      2. Ultracentrifuge the resuspended vesicles, at approx. 175,000 x g, for 1 h and 35 min, at 4 °C. Rotor that can be used: Sorvall TH-641. Ultracentrifuge that can be used: Sorvall Discovery 90SE. The vesicles will be pelleted in the bottom of the tubes (Figure 2, Video 4).


        Figure 2. The pelleted OMVs seen in the bottom of the 12-ml tubes, after ultracentrifugation at approx. 175,000 x g for 1 h and 35 min

        Video 4. Ultracentrifugation

    4. Final resuspension and storage
      1. Discard the supernatant and resuspend the pelleted vesicles with sterile dH2O or with PBS buffer by pipetting, using a volume that is 1/1,000 of original medium volume. For example: 1 ml of sterile dH2O for 1 L of PSB.
      2. Filter the resuspended vesicles through a 0.22-µM filter (operated by a syringe), into a sterile 1.5-ml tube. Must be done in a biological hood. The filtered OMV preparation can be stored at 4 °C as is or further purified by density gradient centrifugation.
    5. Density gradient centrifugation: In order to purify the OMV further, for analyses that require highly-purified samples (e.g., protein analysis in LC-MS/MS), a density gradient centrifugation step is required (Chutkan et al., 2013).
      1. For density gradient centrifugation, use OptiPrep density gradient medium 60% stock solution (Sigma-Aldrich), and make 45%, 40%, 35%, 30%, 25%, and 20% OptiPrep solutions (using OptiPrep diluent buffer, see Recipes).
      2. Carefully layer 1 ml of each diluted OptiPrep solution on the top of the other, starting from 45% OptiPrep solution at the bottom. On the top of the 20% OptiPrep solution, carefully layer the OMV prep and sterile dH2O.
        Notes:
        1. If you have the entire OMV sample layered in one tube, you must have another, balancing tube, with the same diluted OptiPrep layers, but a sterile dH2O layered on them (instead of OMV).
        2. It’s recommended to use equally-weighted tubes, even when empty, to avoid imbalance, and to keep equal volumes of the top layer (OMV or dH2O) between the tubes.
      3. Spin the samples at approx. 246,000 x g for 6 h, at 4 °C. After the ultracentrifugation, a layer/s of the vesicles will be visible in the tube, due to Xanthomonas yellow pigment Xanthomonadin (Figure 3; Video 5).


        Figure 3. OMVs (the yellow line within the tube) are suspended within one of the fractions of the density gradient, after ultracentrifugation at approx. 246,000 x g for 6 h

        Video 5. Purification by a density gradient

      4. With a 1-ml pipette tip laid against the side of tube, slowly and carefully collect each 1 ml fraction from top to bottom. Each 1 ml collected should be transferred into a separate 12-ml ultracentrifuge tube. Change the pipette tip between the fractions.
      5. To each 12-ml ultracentrifuge tube containing a 1 ml fraction from the previous step (from the OptiPrep layers), add 11 ml of sterile dH2O to dilute the OptiPrep, and spin at approx. 170,000 x g for 2 h, at 4 °C (rotor that can be used: Sorvall TH-641; Ultracentrifuge: Sorvall Discovery 90SE).The vesicles will be pelleted
      6. Then, the pellet can be resuspended in 0.2-0.5 ml of sterile dH2O or PBS buffer and filtered (by a syringe-mounted 0.22-µM filter), or the pelleted OMV can be scrubbed from the bottom of the tubes by using two clean thin spatulas. This can be achieved by collecting (scrubbing) the pellet using one of the spatulas.
      7. Then, use the other spatula to remove the pellet from the first spatula and stick it, or smear it, on the sterile 1.5-ml tube’s wall. The 1.5-ml tube containing the pelleted OMVs can be then stored at -80 °C.
        Note: Must be done in a biological hood.

        Table 2. Isolation and purification process summary


  3. OMV preparation quality and quantity checks
    1. Checking if the preparation is sterile
      1. Pipette 10 µl of OMV preparation on a plate with Difco nutrient agar, and leave open until the 10 µl drop dries out. Must be done in a biological hood.
      2. Then, place the plate in a 28 °C incubator for 2-5 days, and check if colonies are formed where the drop was placed.
    2. Quantification of the OMVs
      1. ‘Coomassie Plus – The Better Bradford Assay Kit’, can be used to quantify the protein cargo of the vesicles.
      2. A NanoSight device, can be used to test the size distribution of the vesicles and their quantity.
      3. Another way to estimate the vesicles’ concentration, provided that the OMVs are from Xanthomonas, is by measuring the object density of Xanthomonadin. Xanthomonadin maximum absorbance is at 445 nm. When measuring OD445 nm, use sterile dH2O as blank (Munhoz et al., 2011; Goel et al., 2001).
        Note: The OD445 nm of the OMV preparation differs between a filtered and a non-filtered preparation.
      4. The lipid dye FM4-64FX can be used to estimate the concentration of the vesicles, based on lipid amount.
    3. Transmission electron microscopy (TEM)
      TEM can be used to view OMV (Figure 4) and estimate size range and concentration as well as examine the purity of the preparation.


      Figure 4. TEM image of an OMV preparation, diluted in sterile dH2O by a ratio of 1:20. OMV diameter may range from 20-250. A scale bar is at the bottom left.

    4. SDS-PAGE
      In order to get the profile of the proteinaceous cargo of OMV, run an OMV sample, which was denatured in a sample buffer containing sodium dodecyl sulfate (SDS) and dithiothreitol (DTT)/β-mercaptoethanol, on a protein gel. If you are searching for a specific protein associated with OMV, you can also run a Western blot or a dot blot assays. SDS-PAGE is also a way to compare the proteinaceous profiles of OMV isolated from different bacteria. 

Data analysis

Perhaps one of the main challenges in purifying OMVs from bacterial culture is to prove its purity. Since OMVs are purified from culture supernatants the risk of contamination by surface, extracellular bacterial appendages, such as flagella, pili, fimbriae, must be taken into consideration. Carefully examining multiple TEM grid samples and determining whether such appendages had co-purified with the OMVs or not is highly recommended to assure purity. Another possible contaminant of OMV preparations are broken cell debris and large protein complexes. To minimize the chances of broken cell contamination cultures should be harvested during the logarithmic growth phase when cell death and breakage is less likely to occur. Additionally, we highly recommend that the density gradient centrifugation step will be carried out for any down-stream studies, especially for analytical studies such as proteomics, to avoid co-purification of large protein or protein complexes from the supernatant.

Recipes

  1. YEB (1 L, for starters)
    1. Add:
      5 g of yeast extract powder
      10 g of Bacto tryptone
      5 g of NaCl
      5 g of sucrose crystallized
      5 g of MgSO4·7H2O
      to 900 ml of dH2O
    2. Adjust the pH to 7.3 using NaOH or HCl
      Note: Do not heat while measuring pH. 
    3. Bring to 1 L by adding more dH2O, and autoclave at 121 °C for 20 min
  2. PSB (1 L, the main medium)
    1. Add:
      10 g of peptone
      10 g sucrose crystallized
      1 g of L(+)-glutamic acid
      to 900 ml of dH2O
      A slight heating while stirring might assist in dissolving the components, yet it’s not compulsory. Turn off the heat, and transfer the beaker to another, colder, stirrer
    2. Adjust the pH to 7.3 using NaOH or HCl
      Note: Do not heat while checking pH.
    3. Add more dH2O in order to reach 1 L
    4. Mix by passing the medium between the beaker and a measuring cylinder
    5. Divide the medium between two 2-L Erlenmeyer flasks (500 ml in each), and autoclave at 121 °C for 20 min
  3. OptiPrep diluent buffer (1 L)
    1. Add:
      8.5 g of NaCl
      2.38 g of HEPES
      to approx. 750 ml of dH2O in a beaker
    2. Adjust the pH to 7.4 with NaOH
      Note: Do not heat while checking pH.
    3. Bring to 1 L with dH2O and filter-sterilize with 0.45-μM filter (operated by a vacuum pump)
    4. Suggestion: keep the buffer at 4 °C

Acknowledgments

Work at O. Bahar lab was supported by the German-Israeli Foundation for Scientific Research and Development (GIF), grant No. I-2392-203.13/2015 and by the Israel Science Foundation, grant No. 2025/16. We would like also to thank our lab members for every help given, S. Burdman’s lab, M. Levy’s lab, and N. Sela, for collaboration in experiments, and also M. Mawassi’s lab, V. Gaba’s lab, A. Dombrovsky’s lab, and S. Manulis-Sasson’s lab, for some of the equipment needed for the experiments.

References

  1. Chutkan, H., Macdonald, I., Manning, A. and Kuehn, M. J. (2013). Quantitative and qualitative preparations of bacterial outer membrane vesicles. Methods Mol Biol 966: 259-272.
  2. Goel, A. K., Rajagopal, L. and Sonti, R. V. (2001). Pigment and virulence deficiencies associated with mutations in the aroE gene of Xanthomonas oryzae pv. oryzae. Appl Environ Microbiol 67(1): 245-250.
  3. Munhoz, C. F., Weiss, B., Hanai, L. R., Zucchi, M. I., Fungaro, M. H., Oliveira, A. L., Monteiro-Vitorello, C. B. and Vieira, M. L. (2011). Genetic diversity and a PCR-based method for Xanthomonas axonopodis detection in passion fruit. Phytopathology 101(4): 416-424.

简介

革兰氏阴性细菌自然释放外膜囊泡(OMVs)到周围环境。 OMV有助于多个过程,如细胞通讯,酶和毒素的传递,对环境的压力和发病机制的抵抗。对植物病原菌产生的OMV及其与宿主植物的相互作用知之甚少。下面描述的协议讨论了野生黄单胞菌(Manthomonas campestris)的OMV的隔离过程。菌种33913(一种Crucizer的细菌病原体)。然而,该方案可以用于和/或适于将OMV与其他植物病原细菌分离,以促进在植物 - 微生物相互作用的背景下对OMV的研究。

背景 细胞外泡(EV)释放是许多生物从生命各个领域共享的过程。在革兰氏阴性细菌中,大多数EV是外膜起泡的结果,最终夹住细菌细胞壁,因此被称为外膜囊泡(OMV)。 OMV的研究重点是OMV生物发生,货物,功能和与宿主生物的相互作用。迄今为止,大多数关于OMV的研究集中在人类和环境细菌的细菌病原体上,然而对植物病原菌的OMV研究很少。这里描述的协议是由Chutkan等人描述的方案改编的。 (2013)稍作修改,并在此介绍植物病原体X。 campestris pv。 campestris 。为了我们的理解,这是第一个完全详细的OMV与植物生长细菌分离的方案,我们希望它可以作为对这一主题感兴趣的其他研究组的指​​导性协议。

关键字:OMVs, 外膜囊泡, 胞外囊泡, 分离, 纯化, 野油菜黄单胞菌, 植物-微生物相互作用

材料和试剂

  1. 无菌一次性接种环
  2. 200μl移液器吸头(如:Corning,Axygen ®,目录号:#T-200-C)
  3. 石蜡膜(Bemis,目录号:PM996)
  4. 注射器安装的0.45μM过滤器(EMD Millipore,目录号:SLHV033RS)
  5. 聚醚砜(PES)过滤器,0.45μM,Ø75-mM膜直径,附带500毫升瓶。使用真空泵(例如,广州喷气生物过滤,目录号:FPE404500)
  6. 1 ml和10ml无菌注射器(无针)
    1 ml注射器(如:KDL医疗产品,目录号[包装上的]:7290010993604)
    10 ml注射器(如:Medisposables,目录号[以书面形式出现]:9304-10/16933506093041)
  7. 无菌15-ml Falcon管(如:SPL Life Sciences,目录号:50015)
  8. 12毫升聚丙烯/聚乙二醇超速离心管(如:Beckman Coulter,目录号:331372) - 确保它们适合超速离心机的速度
  9. 注射器安装的0.22μM过滤器(Axiva Sichem Biotech,目录号:SFPV13 R)
  10. 无菌1.5ml管(1.7ml透明管)(Corning,Axygen ,目录号:#MCT-175-C)
  11. 1 ml移液器吸头(如:Corning,Axygen ®,目录号:#T-1000-C)
  12. 聚苯乙烯(PS)无菌培养皿,90 x 15 mm(Miniplast Ein-Shemer,目录号:820-090-01-017)
  13. 甘油存在于-80℃的黄单胞菌属菌株 - 为了制备我们使用的这个方案。 campestris pv。从美国典型培养物保藏中心(ATCC)获得的野蛮士( Xcc )33913
  14. Difco营养琼脂(BD,目录号:21300)
    注意:它含有3 g/L牛肉提取物,5 g/L蛋白胨和15 g/L琼脂,最终pH为6.8±0.2(如粉末容器上所写)。 >
  15. 头孢氨苄水合物(Sigma-Aldrich,目录号:C4895) - 浓度为10mg/ml的储备溶液
  16. 无菌dH 2 O
  17. 10x磷酸缓冲盐水(PBS)缓冲液(可选)(如:Sigma-Aldrich,目录号:P5493)
  18. OptiPrep密度梯度培养基(Sigma-Aldrich,目录号:D1556)
  19. "考马斯加 - 更好的Bradford测定试剂盒"(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:23236)(可选)
  20. 脂质染料FM4-64FX(Thermo Fisher Scientific,Molecular Probes TM,目录号:F34653)(可选)
  21. 如以下协议(可选)所示,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)所需的材料和试剂: http://www.bio-protocol.org/e80 ('[Bio101] Laemmli-SDS-PAGE')
  22. 酵母提取物粉末(EMD Millipore,目录号:61931105001730)
  23. Bacto胰蛋白胨(BD,Bacto,目录号:211705)
  24. 氯化钠(NaCl)(EMD Millipore,目录号:7647-14-5)
  25. 蔗糖结晶(Duchefa Biochemie,目录号:S0809)
  26. 七水硫酸镁(MgSO 4·7H 2 O)(EMD Millipore,目录号:105886)
  27. NaOH或HCl(为了调节pH)
  28. 蛋白胨(EMD Millipore,目录号:61930705001730)
  29. L(+) - 谷氨酸(Acros Organics,目录号:6106-04-3)
  30. HEPES(Sigma-Aldrich,目录号:H3375)
  31. YEB培养基(见食谱)
  32. PSB培养基(见食谱)
  33. OptiPrep稀释缓冲液(见配方)

注意:(可选) - 小泡隔离过程不需要。

设备

  1. 生物罩
  2. 温度控制的孵化器振荡器(28℃),能够达到200RPM(例如:Thermo Fisher Scientific,Thermo Scientific TM,型号:MaxQ TM 6000) br />
  3. 两个2升Erlenmeyer烧瓶
  4. 比维埃特
  5. 聚丙烯共聚物(PPCO)250毫升离心机瓶(如:Thermo Fisher Scientific,Thermo Scientific TM,型号:Nalgene TM PPCO离心瓶) - 确保它们是适合离心机的速度
  6. 高速离心机(例如,Beckman Coulter,型号:Avanti JE,目录号:369003;或Thermo Fisher Scientific,型号:Sorvall TM, RC-6,目录号:74804 * )和6×250ml转子(例如:Beckman Coulter,型号:JLA-16.250,目录号:363930,双锁盖;或Thermo Fisher Scientific,Sorvall TM 型号:SLA-1500,目录号:46615 * [old model]) 32,000 x g
  7. 超速离心机(例如,Thermo Fisher Scientific,Sorvall TM,型号:Discovery 90SE,目录号:40130867 * ,Kendro Laboratory Products)和a 6 x 12-ml摆动式超速离心机转子,如:Thermo Fisher Scientific,Sorvall TM,型号:TH-641,目录号:54295 * (旧型号,Kendro Laboratory产品)或Thermo Fisher Scientific,Thermo Scientific TM,型号:TH-641,目录号:54295(新型号),速度约为175,000 x g
  8. 高压灭菌器
  9. NanoSight设备(Malvern)(可选)
  10. 透射电子显微镜(可选)
  11. SDS-PAGE所需的设备,如以下协议(可选)所示: http://www.bio-protocol.org/e80 ('[Bio101] Laemmli-SDS-PAGE')

  1. *旧型号的目录号。
  2. (可选) - 泡囊隔离过程不需要。
  3. 重要提示:使用Science Gateway的离心转子速度计算器计算上述离心机速度( http://www.sciencegateway.org/tools/rotor.htm 选项。

程序

  1. OMV隔离前的准备
    1. 准备1升YEB(初学者参见食谱)
    2. 制备1升PSB(主要介质见食谱)。
    3. 电镀细菌
      通过使用无菌一次性接种回路,在Difco营养琼脂平板上从板(XIII)33913(来自甘油储备液)。
      注意:建议在使用Xcc启动器前2-5天对细菌进行平铺。
    4. 制作Xcc 起始器(2个无菌管,每个3ml无菌YEB)
      1. 向每个管中加入3μl头孢氨苄水合物10mg/ml(终浓度为10μg/ml培养基)。
      2. 使用无菌的200μl移液器吸头,从板上擦洗3-5个xcc菌落,并将其加入管中的培养基。必须在生物罩上完成。
      3. 将试管置于28℃振荡器中过夜(速度为180-200rpm)。
    5. 主要介质(PSB)的接种
      1. 在每个2升三角烧瓶中每500ml的PSB培养基中加入500μl头孢氨苄水合物10mg/ml(最终浓度为10μg/ml培养基)。
      2. 然后,从其中一个锥形瓶中,将1ml培养基(+头孢氨苄)转移到干净的新的比色杯中(作为分光光度计的空白)。用2层Parafilm覆盖比色皿,并将比色皿保持在4°C。
      3. 然后,向每个锥形瓶中加入300μl来自同一台起动器(必须非常多云)。
      4. 将锥形瓶放入28℃振荡器中,速度为180-200rpm,过夜(视频1)。
      注意:
      1. 必须在生物罩中进行。
      2. 启动器的"混浊"可能会影响接种的PSB介质所需的时间,达到所需的OD 600 nm 。
      3. 30℃以上的温度可能会抑制锥形瓶中的细菌生长。

        <! - flashid2160v181开始 - >
        视频1.文化孵化
        <! - [if!IE]> - > <! - <![endif] - >

        要播放视频,您需要安装较新版本的Adobe Flash Player。

        Get Adobe Flash Player

        - ! - [if!IE]> - >
        <! - <![endif] - >
        <! - flashid2160v181结束 - >
    6. 第二天 - 物体密度测量
      1. 然后,从其中一个锥形瓶中,将1ml培养基(+头孢氨苄)转移到干净的新的比色杯中(作为分光光度计的空白)。用2层Parafilm覆盖比色皿,并将比色皿保持在4°C。
      2. 然后,向每个锥形瓶中加入300μl来自同一台起动器(必须非常多云)。
      3. 将锥形瓶放入28℃振荡器中,速度为180-200rpm,过夜(视频1)。
      注意:
      1. 必须在生物罩中进行。
      2. 启动器的"混浊"可能会影响接种的PSB介质所需的时间,达到所需的OD 600 nm 。
      3. 30℃以上的温度可能会抑制锥形瓶中的细菌生长。

        <! - flashid2160v181开始 - >
        视频1.文化孵化
        <! - [if!IE]> - > <! - <![endif] - >

        要播放视频,您需要安装较新版本的Adobe Flash Player。

        Get Adobe Flash Player

        - ! - [if!IE]> - >
        <! - <![endif] - >
        <! - flashid2160v181结束 - >
    7. 第二天 - 物体密度测量
      1. ...


        - ! - [if!IE]> - ><! - <![endif] - > <! - flashid2160v183结束 - >
      2. 然后,通过0.45μm膜直径(由真空泵操作)的0.45微米PES过滤器过滤上清液。
        可选:可以通过这种过滤器过滤整个1升培养物;因此,您可以将其连接到1升瓶(如果过滤器适合)。过滤的上清液可以在4℃下储存1天。
    8. 造粒囊泡
      1. 将过滤的上清液倒入250ml瓶中,并将其离心。 32,000 x g 2 h,4°C。小泡将从转子中心向外的方向沉淀(图1)。可以使用的转子:SLA-1500(Sorvall),JLA-16.250(Beckman-Coulter)(视频3)。
      2. 弃去上清液,重新悬浮沉淀的囊泡,使用5-8毫升无菌dH 2 O,通过压片法。
        可选:通过0.45-μM(由10ml注射器操作)将重悬的囊泡过滤到无菌的15-ml Falcon管中,以便将其保持在4℃下几天。必须在生物罩上完成。


        图1.具有半透明黄色的颗粒状OMV可以在管的底部(每个管的标记区域内)看到,大约在离心后。 32,000 x g 2小时
         
        <! - flashid2160v184开始 - >
        视频3.起泡囊泡
        <! - [if!IE]> - > <! - <![endif] - >

        要播放视频,您需要安装较新版本的Adobe Flash Player。

        Get Adobe Flash Player

        - ! - [if!IE]> - >
        <! - <![endif] - >
        <! - flashid2160v184结束 - >
    9. 超速离心
      1. 将从前一步骤中重悬的囊泡倒入两个12-ml超离心管中,并使用无菌dH 2 O以填充管的剩余体积。
      2. 超重离心重悬的囊泡, 175,000 x g,在4℃下1小时35分钟。可以使用的转子:Sorvall TH-641。可以使用的超速离心机:Sorvall Discovery 90SE。小泡将在管的底部沉淀(图2,视频4)。


        图2.在12 ml管底部看到的颗粒状OMV,超声离心后, 175,000 x g 1小时35分钟

        <! - flashid2160v185开始 - >
        视频4.超速离心
        <! - [if!IE]> - > <! - <![endif] - >

        要播放视频,您需要安装较新版本的Adobe Flash Player。

        Get Adobe Flash Player

        - ! - [if!IE]> - >
        <! - <![endif] - >
        <! - flashid2160v185结束 - >
    10. 最终重新悬浮和储存
      1. 弃去上清液,用无水dH 2 O或PBS缓冲液通过移液,使用体积为原始培养基体积的1/1000的重悬浮颗粒状囊泡。例如:对于1L PSB,1ml无菌dH 2 O。
      2. 通过0.22μM过滤器(由注射器操作)将重悬的囊泡过滤到无菌的1.5-ml管中。必须在生物罩上完成。过滤的OMV制剂可以按照4℃储存或通过密度梯度离心进一步纯化。
    11. 密度梯度离心:为了进一步纯化OMV,对于需要高纯化样品(例如LC-MS/MS中的蛋白质分析)的分析,需要密度梯度离心步骤(Chutkan ,2013)。
      1. 对于密度梯度离心,使用OptiPrep密度梯度培养基60%储备溶液(Sigma-Aldrich),并制备45%,40%,35%,30%,25%和20%OptiPrep溶液(使用OptiPrep稀释缓冲液,参见食谱)。
      2. 从底部的45%OptiPrep溶液开始,将1ml每种稀释的OptiPrep溶液顶部放在另一个顶部。在20%OptiPrep溶液的顶部,仔细地层层OMV制备和无菌dH 2 O。
        注意:
        1. 如果您将整个OMV样品分层在一个管中,您必须使用另一个平衡管,使用相同的稀释OptiPrep层,但在其上层叠无菌的dH 2 O 2(而不是OMV )。
        2. 建议使用同等重量的管,即使是空的,以避免不平衡,并在管之间保持等体积的顶层(OMV或dH 2 O 2)。
      3. 旋转样品约246,000 x g 6 h,4°C。在超速离心后,由于黄单胞菌属黄色素xanthomonadin (图3;视频5),在管中将有一层囊泡可见。


        图3. OMV(管中的黄线)悬浮在密度梯度的一个分数之内,超声离心后约246,000 x g 6小时

        <! - flashid2160v186开始 - >
        视频5.通过密度梯度净化
        <! - [if!IE]> - > <! - <![endif] - >

        要播放视频,您需要安装较新版本的Adobe Flash Player。

        Get Adobe Flash Player

        - ! - [if!IE]> - >
        <! - <![endif] - >
        <! - flashid2160v186结束 - >
      4. 用1毫升移液管尖端放置在管的侧面,慢慢地,仔细收集从上到下的每个1毫升级分。收集的每1 ml应转移到单独的12 ml超速离心管中。更改分数之间的移液器尖端。
      5. 向每个含有前一步1ml级分的12ml超速离心管(来自OptiPrep层)中加入11ml无菌dH 2 O以稀释OptiPrep,并在约170,000 x g 2 h,4°C(可使用的转子:Sorvall TH-641;超速离心机:Sorvall Discovery 90SE)。囊泡将被颗粒化。
      6. 然后,将沉淀物重新悬浮于0.2-0.5ml无菌dH 2 O或PBS缓冲液中,并过滤(通过注射器安装的0.22-μM过滤器),或者可以将沉淀的OMV从通过使用两个干净的薄抹布管的底部。这可以通过使用其中一个刮刀收集(擦洗)沉淀物来实现。
      7. 然后,使用另一个刮刀从第一把铲上取下颗粒,将其粘贴,或将其抹去无菌的1.5毫升管壁上。然后将含有沉淀的OMV的1.5ml管储存在-80℃。
        注意:必须在生物罩上完成。

        表2.分离和纯化过程摘要


  2. OMV准备质量和数量检查
    1. 检查制剂是否无菌
      1. 用Difco营养琼脂吸取10μlOMV制剂,放开直至10μl滴入。必须在生物罩上完成。
      2. 然后,将板放在28℃的培养箱中2-5天,并检查是否形成了放置滴液的菌落。
    2. OMV的量化
      1. "考马斯加 - 更好的布拉德福德测定试剂盒"可用于量化囊泡的蛋白质货物。
      2. NanoSight装置可用于测试囊泡的尺寸分布及其数量。
      3. 通过测量Xanthomonadin的物体密度,另外一种估计囊泡浓度的方法,只要OMV来自是通过测量。 Xanthomonadin 最大吸光度为445nm。当测量OD 445nm时,使用无菌dH 2 O作为空白(Munhoz等人,2011; Goel等人, em>。,2001)。
        注意:OMV制剂的OD 445nm在过滤和未过滤的制剂之间不同。
      4. 脂质染料FM4-64FX可用于根据脂质量估算囊泡的浓度。
    3. 透射电子显微镜(TEM)
      TEM可用于观察OMV(图4)并估算大小范围和浓度以及检查制剂的纯度。


      图4.在无菌dH 2 O中以1:20的比例稀释的OMV制剂的TEM图像。 OMV直径可能在20-250之间。比例尺位于左下角。

    4. SDS-PAGE
      为了获得OMV的蛋白质货物的轮廓,运行OMV样品,其在含有十二烷基硫酸钠(SDS)和二硫苏糖醇(DTT)/β-巯基乙醇的样品缓冲液中在蛋白质凝胶上变性。如果您正在寻找与OMV相关的特定蛋白质,您还可以运行Western印迹或斑点印迹测定。 SDS-PAGE也是比较从不同细菌分离的OMV的蛋白质谱的方法

数据分析

也许从细菌培养中纯化OMV的主要挑战之一就是证明其纯度。由于OMVs是从培养上清液中纯化的,所以必须考虑表面污染的风险,细菌细菌附属物如鞭毛,皮毛,菌毛等。仔细检查多个TEM网格样品,并确定是否将这些附属物与OMV共同纯化,以确保纯度。 OMV制剂的另一种可能的污染物是破碎的细胞碎片和大的蛋白质复合物。为了尽可能减少破碎细胞污染的机会,在细胞死亡和断裂不太可能发生的对数生长阶段应该收获培养物。此外,我们强烈建议对任何下游研究进行密度梯度离心步骤,特别是对蛋白质组学等分析研究,以避免从上清液中共同纯化大蛋白质或蛋白质复合物。

食谱

  1. YEB(1 L,初学者)
    1. 添加:
      5克酵母提取物粉末 10克Bacto胰蛋白胨
      5克NaCl
      5克蔗糖结晶
      5g MgSO 4·7H 2 O→// 至900ml的dH O O
    2. 使用NaOH或HCl调节pH至7.3 注意:不要在测量pH时加热。 
    3. 通过加入更多的dH 2 O使1升,并在121℃下高压灭菌20分钟
  2. PSB(1 L,主要介质)
    1. 添加:
      10g蛋白胨
      10g蔗糖结晶
      1克L(+) - 谷氨酸 至900ml的dH O O
      搅拌时稍微加热可能有助于溶解组分,但不是强制性的。关闭热量,并将烧杯转移到另一个较冷的搅拌器上
    2. 使用NaOH或HCl调节pH至7.3 注意:请勿在检查pH值时加热。
    3. 添加更多dH 2 O以达到1L
    4. 将介质通过烧杯和量筒之间的介质混合
    5. 将培养基分成两个2-L锥形瓶(每个500ml),121℃高压灭菌20分钟。
  3. OptiPrep稀释缓冲液(1升)
    1. 添加:
      8.5克NaCl
      2.38克HEPES
      到约750毫升dH 2 O在烧杯中
    2. 用NaOH调节pH至7.4 注意:请勿在检查pH值时加热。
    3. 带有dH 2 O的1 L,用0.45μM过滤器(由真空泵操作)进行过滤灭菌
    4. 建议:将缓冲液保持在4°C

致谢

在巴哈尔实验室的工作得到了德黑两国科学研究与发展基金会(GIF),第I-2392-203.13/2015号授权和以色列科学基金会授予的第2025/16号资助。我们还要感谢我们的实验室成员,S. Burdman的实验室,M. Levy的实验室和N. Sela,在实验中的协作,以及M. Mawassi的实验室V.Gaba的实验室,A. Dombrovsky的实验室和S. Manulis-Sasson的实验室,用于实验所需的一些设备。

参考文献

  1. Chutkan,H.,Macdonald,I.,Manning,A.and Kuehn,MJ(2013)。  细菌外膜囊泡的定性和定性制剂。方法Mol Biol 966:259-272。
  2. Goel,AK,Rajagopal,L。和Sonti,RV(2001)。与xanthomonas oryzae pv的 aroE 基因突变相关的颜料和毒力缺陷。 oryzae Appl Environ Microbiol 67(1):245-250。
  3. Munhoz,CF,Weiss,B.,Hanai,LR,Zucchi,MI,Fungaro,MH,Oliveira,AL,Monteiro-Vitorello,CB和Vieira,ML(2011)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/21077774"target ="_ blank">在百香果中检测黄单胞菌轴突的基因多样性和基于PCR的方法 植物病理学 101(4):416-424。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mordukhovich, G. and Bahar, O. (2017). Isolation of Outer Membrane Vesicles from Phytopathogenic Xanthomonas campestris pv. campestris. Bio-protocol 7(5): e2160. DOI: 10.21769/BioProtoc.2160.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。

Tiffany Lowe-Power
UW-Madison
Why did you choose to use 0.22 um filters? OMVs can range in size from 30-300 nm, so these filters might exclude the larger vesicles.
6/23/2017 7:53:04 AM Reply
Ofir Bahar
Agricultural Research Organization, Volcani Center

Your point is absolutely true.We had to decide whether we want to be more stringent with the filtering by using the 0.22uM, on the cost of loosing some large OMV, or use 0.45uM and then risk that some contaminants will go through the filter. Since we had some contamination issues in the past while using the 0.45uM we decided be on the safe side with the filtering.

6/25/2017 12:14:45 AM