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Determination of Polyhydroxybutyrate (PHB) Content in Ralstonia eutropha Using Gas Chromatography and Nile Red Staining
采用气相色谱法和尼罗红染色法测定富养罗尔斯通菌中聚羟基丁酸酯(PHB)的含量   

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FEMS Microbiology Letters
Dec 2016

Abstract

Ralstonia eutropha H16 produces and mobilizes (re-utilizes) intracellular polyhydroxybutyrate (PHB) granules during growth. This protocol describes the visualization of intracellular Nile red stained PHB granules and the quantification of PHB by gas chromatography. Our first method describes how to analyze PHB granules by fluorescence microscopy qualitatively. Our second approach enables the conversion of PHB to volatile hydroxycarboxylic acid methyl esters by acidic methanolysis and their quantification by gas chromatography. Through this method, it is possible to obtain an absolute quantification of PHB, e.g., per cell dry weight.

Keywords: Polyhydroxybutyrate (PHB) (聚羟基丁酸酯(PHB)), Gas chromatography (气相色谱法), Nile red (尼罗红), Acidic methanolysis (酸性甲醇分解), Ralstonia eutropha (富养罗尔斯通菌)

Background

Polyhydroxyalkanoates (PHA), especially polyhydroxybutyrate (PHB), are energy and carbon storage compounds in many prokaryotic species, ensuring bacterial survival under stress conditions (Anderson and Dawes, 1990; Pötter and Steinbüchel, 2006; Jendrossek and Pfeiffer, 2014; Bresan et al., 2016). An industrial application of these biopolymers is the production of biodegradable plastic (Chen, 2009; Riedel et al., 2015) and the research on potential medicinal components (Wu, 2009; Zonari et al., 2015; Pacheco et al., 2015; Giretova et al., 2016). Ralstonia eutropha H16, a Gram-negative facultative chemolithoautotrophic β-proteobacterium, is a model organism for PHB accumulation as it can accumulate up to 80% of its cell dry weight of PHB. Within the cells, PHB forms granules or so-called carbonosomes covered with different surface proteins (Jendrossek and Pfeiffer, 2014; Bresan et al., 2016). PHB is synthesized from its parent substance acetyl-CoA in a 3-step reaction. The first step is a condensation reaction of two acetyl-CoA molecules by the acetyl-CoA-acetyltransferase PhaA. Acetoacetyl-CoA is then reduced to (R)-3-hydroxybutyryl-CoA by the acetoacetyl-CoA-reductase PhaB. The last step includes an essential non-redundant reaction: the polymerization of (R)-3-hydroxybutyryl-CoA to PHB by the PHB synthase called PhaC (Figure 1).


Figure 1. Biosynthesis of PHB

A fast and easy way to detect intracellular PHB is a microscopy approach using Nile red staining. Nile red (also known as Nile blue oxazone) is a lipophilic fluorescent dye used to visualize hydrophobic cell structures such as membranes or lipid-like inclusions (PHB, triacyl-glycerides) (Spiekermann et al., 1999). Nile red binds to PHB granules and can easily be detected by fluorescence microscopy. Its colors (i.e., fluorescent emission wave lengths) vary from dark red (for binding to polar membrane lipids) to an intense yellow-gold emission (for binding to neutral lipids in intracellular storages). The emission (> 590 nm) and excitation (560 nm) wavelengths characteristic of the Nile red hydrophobic compound adducts also depend on solvent polarity (Spiekermann et al., 1999); in most polar solvents Nile red shows no or only little fluorescence.

Gas chromatography (GC) can be used to quantify PHB and to determine its monomeric composition. PHB decomposes at temperatures below its boiling point. Therefore, PHB must be converted into products that are stable and volatile at the temperature of the GC-column. This is achieved by conversion of PHB into volatile hydroxycarboxylic acid methyl esters, hereafter, methyl esters (Figure 2) (Brandl et al., 1988). The methyl esters interact specifically with the solid phase thereby allowing a separation of different hydroxyalkanoate methyl esters in case co-polyesters of different hydroxyalkanoates have to be analyzed. Measuring the time point of appearance and the area under the resulting compound peak of the detector signals in the chromatogram enable its quantitative and qualitative determination.


Figure 2. Acidic methanolysis of PHA

Materials and Reagents

  1. Microscope slides (e.g., Carl Roth, catalog number: H868.1 )
  2. Cover slips (e.g., Carl Roth, catalog number: H873.2 )
  3. 2 ml reaction tubes (e.g., SARSTEDT, catalog number: 72.695.500 )
  4. 50 ml Falcon tubes (e.g., SARSTEDT, catalog number: 62.559.001 )
  5. 6 ml culture tubes with screw-cap with chloroform resistant PTFE seal (e.g., DWK Life Sciences, DURAN, catalog number: 26 135 11 5 )
  6. GC glass vial (e.g., Brown, catalog number: 155710 )
  7. 50 ml Omnifix® Syringes (e.g., B.Braun Medical, catalog number: 4591281 )
  8. Sterile filter Filtropur S 0.2 (e.g., SARSTEDT, catalog number: 83.1826.001 )
  9. Scalpel blade (e.g., Gebrüder Martin, KLS Martin, catalog number: 10-155-24-04 )
  10. Pipette tips 1,000 μl (e.g., SARSTEDT, catalog number: 70.762.010 )
  11. Pipette tips 200 μl (e.g., SARSTEDT, catalog number: 70.760.002 )
  12. Pipette tips 10 μl (e.g., VWR, catalog number: 53509-070 )
  13. 1.5 ml tubes (e.g., SARSTEDT, catalog number: 72.690.001 )
  14. 0.3 ml limited volume inserts (e.g., Brown, catalog number: 155650 )
  15. Septa (e.g., Brown, catalog number: 155615 )
  16. Organisms
    1. Ralstonia eutropha H16 (alternative strain designations: Hydrogenomonas eutropha H16, Alcaligenes eutrophus H16, Wautersia eutropha H16, Cupriavidus necator H16). DSM 428 (Deutsche Sammlung für Mikroorganismen and Zellkulturen GmbH, https://www.dsmz.de/). Wild type strain produces PHB and related short-chain-length PHA
    2. Ralstonia eutropha H16-PHB-4 (DSM 541), PHB negative mutant of strain H16 because of mutation G320A in the PHB synthase (phaC) gene (Raberg et al., 2014)
  17. PHB (e.g., Sigma-Aldrich, catalog number: 363502 )
  18. Agarose standard (e.g., Carl Roth, catalog number: 3810.4 )
  19. Nitrogen gas (e.g., Air Liquide, ALPHAGAZTM 1 Stickstoff, catalog number: P0271L50R2A001 )
  20. Helium gas (e.g., Air Liquide, ALPHAGAZTM 1 Helium, AIR LIQUIDE Deutschland, catalog number: P0251L50R2A001 )
  21. Synthetic air (e.g., Air Liquide, ALPHAGAZTM 1 Luft, AIR LIQUIDE Deutschland, catalog number: P0291L50R2A001 )
  22. Octane (e.g., Sigma-Aldrich, catalog number: 74821 )
  23. Nile red (e.g., Sigma-Aldrich, catalog number: N3013 )
  24. DMSO (e.g., Carl-Roth, catalog number: 7029.2 )
  25. Trichloromethane/Chloroform (e.g., Carl Roth, catalog number: 6340.2 )
  26. Methanol for GC (e.g., VWR, catalog number: 20864.320 )
  27. Methyl benzoate (e.g., Sigma-Aldrich, catalog number: M29908 )
  28. Sulphuric acid 96% (e.g., Carl Roth, catalog number: 4623.1 )
  29. Fructose
  30. Nutrient broth (e.g., BD, DifcoTM, catalog number: 231000 )
  31. Na2HPO4·12H2O
  32. KH2PO4
  33. NH4Cl
  34. MgSO4·7H2O
  35. CaCl2·7H2O
  36. Ferric ammonium citrate
  37. ZnSO4
  38. MnCl2·4H2O
  39. H3BO3
  40. CoCl2·6H2O
  41. CuCl2·2H2O
  42. NiCl2·6H2O
  43. NaMoO4·2H2O
  44. D-Gluconic acid sodium salt (e.g., Sigma-Aldrich, catalog number: G9005 )
  45. NB medium (see Recipes)
  46. Mineral salts medium (see Recipes)
  47. D-Gluconic acid sodium salt solution (20% stock solution) (see Recipes)
  48. Nile red solution (see Recipes)

Equipment

  1. 100 ml Erlenmeyer flasks (e.g., DWK Life Sciences, DURAN, catalog number: 21 216 24 )
  2. 500 ml Erlenmeyer flasks (e.g., DWK Life Sciences, DURAN, catalog number: 21 216 44 )
  3. 3 L Erlenmeyer flasks (e.g., DWK Life Sciences, DURAN, catalog number: 21 216 68 )
  4. Incubation shaker (e.g., INFORS HT)
  5. Pipettes (e.g., Thermo Scientific)
  6. Spatula
  7. Centrifuge (e.g., Eppendorf, model: 5417 C )
  8. Freeze-dryer (e.g., Christ, model: Alpha 1-2 LDplus )
  9. Rotary vane pumps (e.g., Pfeiffer Vacuum, model: DUO 5 M )
  10. Analytical balance (e.g., Sartorius, model: A 200 S )
  11. Fume hood
  12. Oil bath (e.g., Memmert)
  13. Gas chromatograph (e.g., Agilent Technologies, model: Agilent 7890A ; flame ionization detector (FID))
  14. Gastight syringe for GC (e.g., VWR, catalog number: 5490572)
    Manufacturer: Hamilton, model: 1701 SN CTC .
  15. CTC automated sample injector (e.g., Agilent Technologies, catalog number: G6501-CTC )
  16. GC column DB-WAX (e.g., Agilent Technologies, catalog number: 122-7032 )
  17. Fluorescence microscope with a Plan Apo objective (100x/1.4 oil) (e.g., Nikon Instruments, model: Eclipse Ti-E )
  18. Nile red-Filter (Excitation: 562/40 nm/Emission: 594 (long pass), e.g., AHF Analysentechnik AG, Tübingen, Germany, www.ahf.de/)
  19. Liner 4 mm ID LPD (e.g., Agilent Technologies, catalog number: 5183-4647 )
  20. Freezer
  21. Sterile bench (e.g., HERA safe)
  22. Refrigerated Falcon centrifuge (e.g., Sigma Zentrifugen, model: 4K15 )
  23. Vortex
  24. Laboratory glass bottles (e.g., DWK Life Sciences, DURAN, catalog number: 21 801 54 5 )
  25. Autoclave

Software

  1. GC ChemStation Rev. B.04.01 SP1, Agilent
  2. Excel, Microsoft, Redmont, USA
  3. Nikon imaging software
  4. ImageJ Fiji vl.50c

Procedure

Note: Ensure that all safety instructions for the handling of hazardous compounds and for waste management are properly considered; since these may vary in different countries the following protocol does not provide any instruction on these issues.

Part I. Nile red staining

See Figure 3 for the outline of Nile red staining procedure.


Figure 3. Flow chart the Nile red staining

Note: Nile red staining can be performed with cells taken either from cultures for gas chromatography analysis or from independent cultures prepared for microscopy experiments.

  1. Preparations of cells
    1. Inoculate a first seed culture of 10 ml NB medium (or of a culture medium that allows good growth of the species to investigate) with a single colony of R. eutropha H16 cells in a 100 ml flask and incubate the flask for 24 h on a shaker at 150 rpm and 30 °C.
    2. Inoculate a second seed culture of 9 ml NB medium with 1 ml of the first seed culture (1:10 dilution) in a 100 ml flask. Incubate the cells for 24-30 h on a shaker at 150 rpm and 30 °C. The procedure of two subsequent seed cultures on NB medium provides R. eutropha cells that are in the stationary growth phase as revealed by the presence of mainly short-rod-shaped or almost coccoid cells. Most of the cells (> 95%) have mobilized any previously accumulated PHB and the cells appear to be ‘empty’ after Nile red staining.
    3. Inoculate the main culture by transferring 1 ml of the second pre-culture to 9 ml of fresh NB medium (in a 100 ml flask) supplemented with 0.2% D-gluconic acid sodium salt (100 μl of 20% stock solution) and incubate the cells on a shaker at 150 rpm and 30 °C. Gluconate increases the C to N ratio of the medium and promotes accumulation of PHB. If different species are investigated other C-sources that a metabolized via acetyl-coenzyme A (precursor of PHB) may be added to promote the formation of PHB, e.g., acetate or glucose.

  2. Preparation of microscope slides
    1. Label the microscope slides.
    2. Prepare agarose pads by pipetting 100 µl of hot (~60 °C) agarose solution (1% [w/v] in H2O) on the slide and immediately place the cover slip on the agarose (Figure 4).


      Figure 4. Preparation of an agarose pad for microscopy

    3. Let it solidify (≥ 2 min) and carefully remove the cover slip using a blade. Having removed the cover slip the sample should be applied within the next two minutes. Otherwise, the agar surface will become dry.

  3. Nile red staining of cells
    1. Take samples every 2 h after inoculation of the main culture during the accumulation and mobilization phase of PHB. Remaining cells of the second pre-culture can be used as a negative control and represent the time point ‘0’ (T0). These cells (> 95%) should have no accumulated PHB. Alternatively, cells of R. eutropha PHB-4 can be used as a negative control for PHB granule accumulation. Due to a mutation in the PHB synthase gene cells of R. eutropha PHB-4 are unable to synthesize storage PHB (Raberg et al., 2014).
    2. Harvest 1 ml of culture by centrifugation (60 sec, 13,000 x g), discard the supernatant and resuspend the pellet in the remaining ~30-50 µl medium that congregates from the tube walls at the bottom within ~1 min.
    3. Add 4 µl of cells to 1 µl of Nile red (working solution: 10 µg/ml in DMSO, light sensitive, stable for several months at 4 °C) in a reaction tube.
    4. Drop 1 or 2 µl of the stained cell suspension on the agarose pad, let it dry for a few seconds and carefully place the cover slip on the agarose.

  4. Microscopy analysis
    1. Image the samples using an appropriate fluorescence filter for Nile red (excitation: 562/40 nm, emission 594 (long pass), AHF Analysentechnik AG, Tübingen, Germany, www.ahf.de). Image the cells also under bright field.
    2. Analyze the pictures with the Nikon imaging software or ImageJ Fiji vl.50c.

Part II. Determination of PHB content using gas chromatography

See Figure 5 for the outline of the procedure to determine the PHB content using gas chromatography.


Figure 5. Flow chart of the determination of PHB content using gas chromatography

  1. Cultivation and harvesting of cells
    1. Three independent colonies and cultures should be used for biological triplicates.
    2. Calculate the appropriate amount of culture volume depending on how many samples (data points) will be taken. 100 ml of culture is necessary for a data point when the culture has an OD600 ≤ 0.6. 50 ml of culture is necessary for a data point when the culture has an OD600 ≥ 0.6. We usually take a sample every 4 h over a period of 48 h.
    3. Inoculate the first seed cultures with 10 ml NB medium, the appropriate antibiotics if necessary and a colony of R. eutropha H16 cells and incubate it overnight in a shaker at 30 °C up to 24 h.
    4. Inoculate the second seed cultures with a dilution of 1:10 in NB medium and the appropriate antibiotics if necessary. Incubate it for at least 24-30 h in a shaker at 30 °C to get rid of all previously accumulated PHB. The culture should have a volume of at least 50 ml to have enough cells to harvest the T0 time point from the seed culture and to inoculate the main culture.
    5. Inoculate the main cultures with a dilution of 1:20 in NB medium, 0.2% sodium gluconate and the appropriate antibiotics if necessary and incubate it in a shaker at 30 °C.
    6. Take 50-100 ml samples every 4 h during the accumulation and mobilization phase of PHB. T0 is represented by the second pre culture. The volume of the main culture should be 50 ml higher than the sum of the volumes harvested for all data points.
    7. Harvest 50-100 ml of culture in 50 ml Falcon tubes by centrifugation for 20 min at 5,000 x g and 4 °C.
    8. Discard the supernatant and freeze the pellet for at least 2 h at -20 °C.

  2. Lyophilization and weighing of samples
    1. In order to freeze-dry the cell pellets, slightly open the lids of the Falcon tubes or use lids with a prepared hole.
    2. Place the Falcon tubes in the freeze-dryer and start the drying process by switching on the (rotary) vacuum pump. Freeze-dry the samples for at least 24 h. Lyophilization of the samples is finished when the cell pellets are dry. The absence of any residual water/moisture is important.
    3. Disrupt all cell pellets into small clumps using a spatula.
    4. Weigh approximately 10 mg of the freeze-dried cell pellets on an analytical balance into screw-capped culture tubes with chloroform resistant PTFE seal. If the size of the pellet is limited, it is possible to use fewer amounts of freeze-dried cells but the (mass) weight should always be between 5-10 mg.
      Note: It is important to determine the exact cell mass (weight) for later calculations.
    5. In order to generate a PHB calibration graph, weigh 2 mg, 4 mg, 6 mg and 8 mg of pure PHB into culture tubes with screw-caps, respectively.

  3. Acidic methanolysis and preparation of GC samples (Figure 6)
    Note: All steps should be performed in a fume hood.
    1. Add 1 ml of chloroform to the culture tubes.
    2. Add 1 ml of methanol supplemented with 15% (v/v) H2SO4 (150 μl H2SO4 + 850 μl methanol per sample) to the special culture tubes containing the weighted dried cells. Close the tubes tightly and vortex for three seconds. All pellet clumps should be in the solvent mixture.
    3. Incubate the tubes for 2 h 30 min at 100 °C in a thermostat-equipped oil bath. Follow the local safety instructions as the applied temperature is above the boiling point of methanol and chloroform. Intact sealing and the absence of any fissures in the glass tubes are essential.
    4. Cool down the samples on ice for 5 min.
    5. Add 1 ml of deionized water and 1 ml of chloroform containing 0.2% (v/v) methyl benzoate (2 μl methyl benzoate + 998 μl chloroform per sample) as an internal standard.
      Note: Use the same chloroform-methyl benzoate mixture for all samples to avoid dilution errors.
    6. Close the tubes tightly and vortex vigorously for 30 sec.
    7. Let the tubes stand for a minute to allow phase separation.
    8. Pipette 150 µl of the organic (bottom) phase into 0.3 ml limited volume inserts in GC glass vials.
    9. Close the screw cap of the GC vials.
    10. Prepare a GC vial with 150 µl pure chloroform as equilibration solvent for the column.
    11. Pipette 1 ml of pure chloroform and 1 ml of the chloroform methyl benzoate mixture into a culture tube to prepare an external standard. Transfer 150 µl of the mixture into a GC vial.


      Figure 6. Images of the acidic methanolysis steps of PHB. Weighted cells (A), complete methanolysis sample before oil bath (B), sample after oil bath (C) and phase-separated sample with the bottom organic phase (white arrow, D).

  4. GC measurement
    1. Place all GC vials in a GC tray.
    2. Refill all glass vials which are used to rinse the syringe three times between different samples with octane.
    3. Empty all waste vials.
    4. Set up the GC apparatus and the CTC automated sample injector according to the instructions supplied by the manufacturer and install a gastight syringe as well as the appropriate liner.
    5. Use a DB-WAX column as stationary phase and the inert gas helium as mobile phase for the separation of the methyl esters.
    6. Program an injection volume of 1 µl, a split mode with a split ratio of 1:8 and an injection temperature of 250 °C.
    7. Program a flow rate of 0.7 ml/min.
    8. Program the following temperature gradient for the separation of the methyl esters:


    9. Detection is performed with a flame ionization detector (FID) at 275 °C.

Data analysis

Part I. Nile red staining–Image analysis

Analyze the taken pictures using the Nikon imaging software or ImageJ Fiji vl.5 as shown in Figure 7.


Figure 7. Fluorescence micrographs of cells with and without PHB. From left to right: bright field, Nile red, merged channels of bright field and Nile red. Upper panel: cells with PHB granules, Lower panel: cells without PHB granules.
Note: That cells can have ‘dark’ inclusions (e.g., most upper cell in lower panel) that are not stained with Nile red. Such inclusions might represent other inclusion bodies/structures (e.g., polyphosphate granules). 

Part II. Determination of PHB content using gas chromatography

  1. Double click on the sample name of interest to open the chromatogram.
  2. The 3-hydroxybutyryl methyl ester (3HBME) should elute at 7.0-7.1 min. The internal standard (methyl benzoate) should elute at 8.7-8.8 min. The exact retention times are apparatus- and condition-specific and might vary. If a copolymer of 3HB and 3-hydroxyvalerate (3HV) is accumulated by the cells the retention times of 3HV methylester (3HVME) is around 7.9-8.0 min. The same protocol can also be applied for the analysis and detection of medium-chain-length PHAs. The methyl esters of the 3-hydroxyalkanoic acids with 6, 8, 10 and 12 C-atoms elute at ~9.0 min (C6), ~11.4 min (C8), ~13.5 min (C10) and ~15.5 min (C12), respectively (not shown).
  3. Click on the integration button (Red square in Figure 8).
  4. In the event that the peak is too small to be automatically integrated, it can be manually integrated by pressing the manual integration button (Green box in Figure 8) and drawing the pink line under the peak with the mouse cursor.
  5. The values of the area under the curve will be listed in a table under the chromatogram (Orange box in Figure 8).


    Figure 8. Screenshot of a chromatogram and results tables (A) and representative chromatograms for 3HVME and 3HBME (B)

  6. Copy the values of the area under the 3-hydroxybutyryl methyl ester (3-HBME) peak and the methyl benzoate peak to an excel table for all samples of the PHB standard curve and for all samples to be analyzed.
  7. Calculate the normalized area of the 3-HBME peaks using the formula:

  8. Create a standard curve by plotting the normalized areas of the PHB standard samples on the y-axis against the weighted amounts of PHB on the x-axis (Figure 9) and determine the equation of the function (y = a*x + b).


    Figure 9. Standard curve for the determination of PHB weight. One line connects the data points, the second line is the trend line used to generate the equation of the line.

  9. Calculate the weight of PHB in your sample using the formula:

  10. Calculate the percentage of PHB per cell dry weight using the formula:

  11. Represent the PHB content in the graph with the program of your choice (Figure 10).


    Figure 10. Example of the growth (A) and PHB production (B) of R. eutropha H16 on NB-medium with 0.2% Na-gluconate (complex medium) or on mineral salts medium with 2% fructose

Recipes

  1. NB medium
    1. Weigh 8 g of NB powder, fill it into a 1,000 ml glass bottle and add 1,000 ml of deionized water. Mix it and autoclave it at 121 °C for 20 min
    2. Store the NB medium at room temperature
  2. Mineral salts medium
    Any mineral salt medium that is suited for growth of the strain of interest can be used. For Ralstonia eutropha H16, we use the following medium. For preparation of 1,000 ml medium:
    1. Dissolve 9 g Na2HPO4·12H2O, 1.5 g KH2PO4 and 1 g NH4Cl in ≈ 950 ml deionized water using a magnetic stirrer
    2. Add 1 ml each of the following 1,000x stock solutions:
      Mg-solution (200 g/L MgSO4·7H2O)
      Ca-solution (20 g/L CaCl2·7H2O)
      Fe-solution (1.2 g/L ferric ammonium citrate)
      Note: It is important that a magnetic stirrer is operating during the addition of Mg-, Ca-, and Fe- solutions. Otherwise white precipitates might form.
    3. Finally, add 100 µl of a trace element solution
      Values per liter trace element stock solution: 1 g ZnSO4, 0.3 g MnCl2·4H2O, 3 g H3BO3, 2 g CoCl2·6H2O, 0.1 g CuCl2·2H2O, 0.2 g NiCl2·6H2O, 0.3 g NaMoO4·2H2O and fill up to the end volume of 1,000 ml with deionized water
    4. The pH of the mineral salts medium should be at pH 7.0 ± 0.1.
    5. Sterilize the medium at 121 °C for 20 min
    Notes:
    1. Minor amounts of precipitates will form upon sterilization that will partially re-dissolve upon cooling and stirring. Remaining small amounts of precipitates can be tolerated.
    2. Add the desired amount of carbon source before inoculation with the strain of interest (e.g., with sodium-gluconate stock solution up to 2% in case that high amounts of PHB should accumulate).
    3. Optionally, the amount of nitrogen source (NH4Cl) can be reduced to 0.5 g/L. This will lead to a higher C to N ratio and cause a reduction of the yield in cell biomass but will increase the % accumulated PHB per g cellular dry weight to ≈ 80%.
  3. D-Gluconic acid sodium salt solution (20% stock solution)
    1. Prepare a 20% wt/vol of D-gluconic acid sodium salt stock solution by dissolving 20 g sodium gluconate in 100 ml of deionized water
    2. Sterilize the solution by filtration through a sterile filter (0.2 µm) or by heat-sterilization (20 min 121 °C)
    3. The stock solution is stable at room temperature
  4. Nile red solution
    1. Prepare a Nile red stock solution of 1 mg/ml and dissolve it in DMSO
    2. Dilute the stock solution with DMSO to obtain a working solution of 10 µg/ml
    3. The Nile red solutions can be stored at 4 °C for several months
    4. Protect the solutions from light

Acknowledgments

We thank Deutsche Forschungsgemeinschaft (DFG Je152/17-1) for funding. The authors declare no conflicts of interests.

References

  1. Anderson, A. J. and Dawes, E. A. (1990). Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54(4): 450-472.
  2. Brandl, H., Gross, R. A. and Lenz, R. W. (1988). Pseudomonas oleovorans, as a source of poly(beta-hydroxyalkanoates) for potential applications. Appl Env Microbiol 54:1977-1982.
  3. Bresan, S., Sznajder, A., Hauf, W., Forchhammer, K., Pfeiffer, D. and Jendrossek, D. (2016). Polyhydroxyalkanoate (PHA) granules have no phospholipids. Sci Rep 6: 26612.
  4. Chen, G. Q. (2009). A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry. Chem Soc Rev 38:2434-2446.
  5. Giretova, M., Medvecky, L., Stulajterova, R., Sopcak, T., Briancin, J. and Tatarkova, M. (2016). Effect of enzymatic degradation of chitosan in polyhydroxybutyrate/chitosan/calcium phosphate composites on in vitro osteoblast response. J Mater Sci Mater Med 27:181.
  6. Jendrossek, D. and Pfeiffer, D. (2014). New insights in the formation of polyhydroxyalkanoate granules (carbonosomes) and novel functions of poly(3-hydroxybutyrate). Environ Microbiol 16(8): 2357-2373.
  7. Pacheco, D. P., Amaral, M. H., Reis, R. L., Marques, A. P. and Correlo, V. M. (2015). Development of an injectable PHBV microparticles-GG hydrogel hybrid system for regenerative medicine. Int J Pharm 478:398-408.
  8. Raberg, M., Voigt, B., Hecker, M. and Steinbüchel, A. (2014). A closer look on the polyhydroxybutyrate- (PHB-) negative phenotype of Ralstonia eutropha PHB-4. PLoS One 9:e95907.
  9. Riedel, S. L, Jahns, S., Koenig, S., Bock, M. C. E., Brigham, C. J., Bader, J. and Stahl, U. (2015). Polyhydroxyalkanoates production with Ralstonia eutropha from low quality waste animal fats. J Biotechnol 214:119-127.
  10. Spiekermann, P., Rehm, B. H., Kalscheuer, R., Baumeister, D. and Steinbüchel, A. (1999). A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 171:73-80.
  11. Wu, Q., Wang, Y. and Chen, G. Q. (2009). Medical application of microbial biopolyesters polyhydroxyalkanoates. Artif Cells Blood Substitutes Immobil Biotechnol 37:1-12.
  12. Zonari, A., Martins, T. M. M., Paula, A. C. C., Boeloni, J. N., Novikoff, S., Marques, A. P., Correlo, V. M., Reis, R. L. and Goes, A. M. (2015). Polyhydroxybutyrate-co-hydroxyvalerate structures loaded with adipose stem cells promote skin healing with reduced scarring. Acta Biomater 17:170-181.

简介

在生长过程中,富养罗尔斯通氏菌H16产生和动员(重新利用)细胞内聚羟基丁酸酯(PHB)颗粒。 该协议描述了细胞内尼罗红染色的PHB颗粒的可视化和通过气相色谱定量PHB。 我们的第一种方法描述了如何通过荧光显微镜定性分析PHB颗粒。 我们的第二种方法可以通过酸性甲醇分解和气相色谱定量法将PHB转化为挥发性羟基羧酸甲酯。 通过该方法,可以获得PHB的绝对定量,例如,每细胞干重。

【背景】聚羟基脂肪酸酯(PHA),尤其是聚羟基丁酸酯(PHB),是许多原核生物物种中的能量和碳储存化合物,确保细菌在压力条件下存活(Anderson和Dawes,1990;Pötter和Steinbüchel,2006; Jendrossek和Pfeiffer,2014; Bresan, >等。,2016)。这些生物聚合物的工业应用是生物降解塑料的生产(Chen,2009; Riedel等人,2015)和潜在药物成分的研究(Wu,2009; Zonari等人, ,2015; Pacheco et al。,2015; Giretova et。,2016)。富养罗尔斯通氏菌H16是一种革兰氏阴性兼性化学自养营养型β-变形杆菌,是一种PHB积累的模式生物,因为它可以累积其PHB细胞干重的80%。在细胞内,PHB形成被不同表面蛋白覆盖的颗粒或所谓的碳纳米颗粒(Jendrossek和Pfeiffer,2014; Bresan等人,2016)。 PHB是通过3步反应由其母体乙酰辅酶A合成的。第一步是乙酰-CoA-乙酰转移酶PhaA对两个乙酰-CoA分子的缩合反应。乙酰乙酰-CoA然后通过乙酰乙酰-CoA-还原酶PhaB还原为(R-R)-3-羟丁酰-CoA。最后一步包括一个基本的非冗余反应:PHB合成酶称为PhaC(图1)将(R) - 3-羟基丁酰-CoA聚合成PHB。

“”src
图1. PHB的生物合成

检测细胞内PHB的快速而简单的方法是使用尼罗红染色的显微镜方法。尼罗红(也称为尼罗蓝oxazone)是一种亲脂性荧光染料,用于使疏水性细胞结构(如膜或脂质样包涵体(PHB,三酰甘油酯))可视化(Spiekermann等人。,1999)。尼罗红与PHB颗粒结合,可通过荧光显微镜轻松检测。它的颜色(即,荧光发射波长)从深红色(用于结合极性膜脂质)变为强烈的黄色金色发射(用于结合细胞内储存中的中性脂质)。尼罗红疏水化合物加合物的发射波长(> 590nm)和激发波长(560nm)特征也取决于溶剂极性(Spiekermann等人,1999);在大多数极性溶剂中,尼罗红显示没有或只有很少的荧光。

气相色谱(GC)可用于定量PHB并确定其单体组成。 PHB在低于其沸点的温度下分解。因此,必须将PHB转换成在GC色谱柱温度下稳定且易挥发的产品。这通过将PHB转化为挥发性羟基羧酸甲酯(以下称为甲酯)来实现(图2)(Brandl等人,1988)。甲基酯与固相特异性相互作用,从而允许在不同的羟基链烷酸酯的共聚酯必须分析的情况下分离不同的羟基链烷酸酯甲酯。测量色谱图中出现的时间点和检测器信号产生的复合峰下面积可以进行定量和定性测定。

“”src
图2. PHA的酸性甲醇分解

关键字:聚羟基丁酸酯(PHB), 气相色谱法, 尼罗红, 酸性甲醇分解, 富养罗尔斯通菌

材料和试剂

  1. 显微镜幻灯片(,例如,Carl Roth,目录号:H868.1)
  2. 封页(例如,Carl Roth,产品目录号:H873.2)
  3. 2毫升反应管(例如,,SARSTEDT,目录号:72.695.500)
  4. 50毫升猎鹰管(,例如,SARSTEDT,目录号:62.559.001)
  5. 6毫升带螺纹盖的培养管,带耐氯仿的聚四氟乙烯密封(例如,DWK生命科学公司,DURAN,目录号:26135115)
  6. GC玻璃小瓶(如,Brown,目录号:155710)
  7. 50毫升Omnifix注射器(例如,B.Braun Medical,目录号:4591281)
  8. 无菌过滤器Filtropur S 0.2(例如,SARSTEDT,目录号:83.1826.001)
  9. 手术刀刀片( ,GebrüderMartin,KLS Martin,目录号:10-155-24-04)
  10. 移液器吸头1,000μl(,例如,SARSTEDT,目录号:70.762.010)
  11. 移液器吸头200μl(,例如,SARSTEDT,目录号:70.760.002)
  12. 移液器吸头10μl(,例如,VWR,目录号:53509-070)
  13. 1.5毫升试管(,例如,SARSTEDT,目录号:72.690.001)

  14. 0.3毫升有限容积的插入物(,例如,布朗,目录号:155650)
  15. (如,Brown,产品目录号:155615)
  16. 生物
    1. 富养罗尔斯通氏菌H16(替代品系命名:富养氢氧化氢真菌H16,真养产碱杆菌H16,Wautersia eutropha H16, > Cupriavidus necator H16)。 DSM 428(Deutsche SammlungfürMikroorganismen和Zellkulturen GmbH, https://www.dsmz.de/ )。野生型菌株产生PHB和相关的短链PHA
    2. 由于PHB合酶(phaC )基因中的突变G320A(Raberg等人,H16-PHB-4(DSM 541),菌株H16的PHB阴性突变体,2014)
  17. PHB(例如,Sigma-Aldrich,目录号:363502)
  18. 琼脂糖标准品(如,Carl Roth,目录号:3810.4)
  19. 氮气(例如,液化空气,ALPHAGAZ TM 1 Stickstoff,目录号:P0271L50R2A001)
  20. 氦气(例如,液化空气,ALPHAGAZ TM 1 Helium,AIR LIQUIDE Deutschland,目录号:P0251L50R2A001)
  21. 合成空气(例如,液化空气,ALPHAGAZ TM 1 Luft,AIR LIQUIDE Deutschland,目录号:P0291L50R2A001)
  22. 辛烷(例如,Sigma-Aldrich,目录号:74821)
  23. 尼罗红(例如,Sigma-Aldrich,目录号:N3013)
  24. DMSO(例如,Carl-Roth,目录号:7029.2)
  25. 三氯甲烷/氯仿(如,Carl Roth,目录号:6340.2)
  26. 用于GC的甲醇(如,VWR,目录号:20864.320)
  27. 苯甲酸甲酯(例如,Sigma-Aldrich,目录号:M29908)
  28. 硫酸96%(例如,Carl Roth,目录号:4623.1)
  29. 果糖
  30. 营养肉汤(例如,BD,Difco TM,产品目录号:231000)
  31. Na 2 HPO 4•12H 2 O
  32. KH <2> PO <4>
  33. NH 4 Cl
  34. MgSO 4•7H 2 O。
  35. CaCl 2•7H 2 O O 0
  36. 柠檬酸铁铵
  37. ZnSO 4
  38. MnCl 2•4H 2 O•O
  39. H 3 BO 3
  40. CoCl 2•6H 2 O•O 0
  41. CuCl 2 2•2H 2 O
  42. NiCl 2•6H 2 O•O
  43. NaMoO•4•2H 2 O•O
  44. D-葡糖酸钠盐(例如,Sigma-Aldrich,目录号:G9005)
  45. NB媒介(见食谱)
  46. 无机盐培养基(见食谱)
  47. D-葡糖酸钠盐溶液(20%原液)(见食谱)
  48. 尼罗红溶液(见食谱)

设备

  1. 100ml锥形瓶(例如,DWK Life Sciences,DURAN,目录号:21 216 24)
  2. 500毫升锥形瓶(例如,DWK生命科学公司,DURAN,目录号:21 216 44)
  3. 3升锥形烧瓶(如,DWK Life Sciences,DURAN,目录号:21 216 68)
  4. 孵化振动筛(,例如,INFORS HT)
  5. 移液器(如,Thermo Scientific)
  6. 刮刀
  7. 离心机(例如,Eppendorf,型号:5417C)
  8. 冷冻干燥机(,例如,Christ,型号:Alpha 1-2 LDplus)
  9. 旋转叶片泵(例如,Pfeiffer Vacuum,型号:DUO 5 M)
  10. 分析天平(例如,,Sartorius,型号:A 200 S)
  11. 通风橱
  12. 油浴(如,Memmert)
  13. 气相色谱仪(例如,Agilent Technologies,型号:Agilent 7890A;火焰离子化检测器(FID))
  14. 用于GC的气密注射器(如,VWR,目录号:5490572)
    制造商:Hamilton,型号:1701 SN CTC。
  15. CTC自动进样器(例如,安捷伦科技,产品目录号:G6501-CTC)
  16. GC色谱柱DB-WAX( ,Agilent Technologies,产品目录号:122-7032)
  17. 具有Plan Apo物镜(100x / 1.4油)的荧光显微镜(例如,Nikon Instruments,型号:Eclipse Ti-E)
  18. 尼罗红滤色片(激发:562/40纳米/发射:594(长传), eg ,AHF Analysentechnik AG,德国图宾根, www.ahf.de/
  19. 内衬4mm ID LPD(例如,,Agilent Technologies,目录号:5183-4647)
  20. 冷冻机
  21. 无菌工作台(,例如,HERA安全)
  22. 冷藏猎鹰离心机(例如,,Sigma Zentrifugen,型号:4K15)
  23. 涡流
  24. 实验室玻璃瓶(例如,DWK Life Sciences,DURAN,目录号:21 801 54 5)
  25. 高压灭菌器

软件

  1. GC化学工作站B.04.01版SP1,安捷伦
  2. Excel,Microsoft,Redmont,美国
  3. 尼康成像软件
  4. ImageJ Fiji vl.50c

程序

注意:确保妥善考虑处理危险化合物和废物管理的所有安全说明;因为这些可能在不同的国家有所不同,下面的协议不提供任何有关这些问题的指导。

尼罗红染色


见尼罗红染色程序概述图3

图3.尼罗河红染色流程图

注意:尼罗红染色可以使用培养的细胞进行气相色谱分析,也可以从为显微镜实验准备的独立培养物中进行细胞染色。

  1. 细胞的制备
    1. 在100ml烧瓶中接种10ml NB培养基(或允许物种良好生长以进行研究的培养基)的第一次种子培养物与单一菌落的富养罗尔斯通氏菌H16细胞并孵育在150rpm和30℃的摇床上振荡24小时。
    2. 用1ml第一次种子培养物(1:10稀释)在100ml烧瓶中接种9ml NB培养基的第二次种子培养物。在150rpm和30℃的摇床上孵育细胞24-30小时。在NB培养基上的两个随后的种子培养的过程提供了R。由于存在主要为短杆状或几乎球状的细胞,所以处于静止生长期的富营养细胞。大部分细胞(> 95%)已经动员了任何先前积聚的PHB,并且细胞在尼罗红染色后看起来是“空的”。
    3. 通过将1ml第二预培养物转移到补充有0.2%D-葡萄糖酸钠盐(100μl20%储备溶液)的9ml新鲜NB培养基(在100ml烧瓶中)接种主培养物并孵育细胞在150rpm和30℃的摇床上培养。葡萄糖酸盐增加培养基的C与N比并促进PHB的积累。如果研究不同的物种,可以加入其他通过乙酰辅酶A(PHB前体)代谢的C源以促进PHB,例如乙酸或葡萄糖的形成。

  2. 显微镜载玻片的制备
    1. 标签显微镜幻灯片。
    2. 在载玻片上吸取100μl热(〜60℃)琼脂糖溶液(H 2 O中1%[w / v]),准备琼脂糖垫,并立即将盖玻片置于琼脂糖上图4)。


      图4.用于显微镜的琼脂糖垫的制备

    3. 让它凝固(≥2分钟),并用刀片小心地取下盖玻片。取下盖玻片后,应在接下来的两分钟内使用样品。否则,琼脂表面会变干。

  3. 尼罗红染色的细胞
    1. 在PHB的积累和动员阶段期间,在主培养物接种后每2小时采样一次。第二次预培养的剩余细胞可以用作阴性对照,并表示时间点'0'(T0)。这些细胞(> 95%)应该没有累积的PHB。或者, R的单元格。富养菌体PHB-4可用作PHB颗粒积累的阴性对照。由于在R的PHB合酶基因细胞中发生突变,富营养化石PHB-4无法合成储存PHB(Raberg等人,2014年)。
    2. 通过离心(60秒,13,000×gg)收获1ml培养物,丢弃上清液,并将沉淀重悬于剩余的约30-50μl培养基中,该培养基从底部的管壁聚集在〜1分钟。

    3. 在反应管中加入4μl细胞到1μl尼罗红(工作溶液:DMSO中10μg/ ml,光敏,在4℃下稳定几个月)。
    4. 将1μl或2μl染色的细胞悬液放在琼脂糖垫上,让其干燥几秒钟,小心地将盖玻片放在琼脂糖上。

  4. 显微镜分析
    1. 使用合适的荧光过滤器对尼罗红(激发:562 / 40nm,发射594(长通道),AHF Analysentechnik AG,Tübingen,Germany, www.ahf.de )。
      在明亮的场地下显示细胞。
    2. 使用尼康成像软件或ImageJ Fiji v1.50c分析照片。

第二部分。使用气相色谱法测定PHB含量


请参见图5以了解使用气相色谱法确定PHB含量的程序大纲

图5.使用气相色谱法测定PHB含量的流程图

  1. 培养和收获细胞

    1. 三个独立的殖民地和文化应该用于生物三重复
    2. 根据将采取多少个样品(数据点)计算适当的培养体积量。当培养物的OD 600≤0.6时,对于数据点需要100ml的培养物。当培养物的OD600≥0.6时,对于数据点需要50ml的培养物。我们通常在48小时内每4小时采样一次。
    3. 用10ml NB培养基接种第一次种子培养物,必要时使用适当的抗生素和em菌落。 eutropha H16细胞,并在30°C摇床中培养过夜至24小时。
    4. 在NB培养基中稀释1:10接种第二次种子培养物,必要时接受适当的抗生素。在30°C摇床中孵育至少24-30小时,以除去所有先前积聚的PHB。培养物的体积应至少为50毫升,以便有足够的细胞从种子培养物中收获T0时间点并接种主要培养物。
    5. 用NB培养基,0.2%葡萄糖酸钠和适当的抗生素按1:20的稀释度接种主要培养物,必要时将其在30℃的摇床中培养。
    6. 在PHB的积聚和动员阶段期间,每4小时取50-100毫升样品。 T0代表第二次预培养。主要培养物的体积应比所有数据点收获的总量高50毫升。
    7. 通过在5,000×g和4℃下离心20分钟,在50ml Falcon管中收获50-100ml培养物。
    8. 弃去上清液并在-20°C冻结沉淀至少2小时。

  2. 冻干和称量样品
    1. 为了冻干细胞颗粒,稍微打开Falcon管的盖子或使用带有预留孔的盖子。
    2. 将Falcon管置于冷冻干燥器中,通过打开(旋转)真空泵启动干燥过程。将样品冷冻干燥至少24小时。当细胞团块干燥时,冻干样品完成。没有任何残留水分/湿气是重要的。

    3. 使用刮刀将所有细胞团粒破碎成小团块。
    4. 在分析天平上称取约10mg冷冻干燥的细胞团块放入带耐氯仿PTFE密封的带螺旋盖的培养管中。如果颗粒大小有限,可以使用更少量的冻干细胞,但(质量)重量应始终在5-10毫克之间。
      注意:确定准确的细胞质量(重量)用于以后的计算很重要。
    5. 为了生成PHB校准曲线图,分别将2 mg,4 mg,6 mg和8 mg纯PHB分别称重到带有螺帽的培养管中。

  3. 酸性甲醇分解和GC样品制备(图6)
    注意:所有步骤应在通风橱中进行。

    1. 加入1毫升氯仿到培养管中
    2. 加入1ml补充有15%(v / v)H 2 SO 4(150μlH 2 SO 4) /每个样品+850μl甲醇)到含有加权干细胞的特殊培养管中。紧紧闭合管并涡旋三秒钟。
      所有颗粒团应该在溶剂混合物中。
    3. 在配有恒温器的油浴中,在100°C孵育管2小时30分钟。应用温度高于甲醇和氯仿的沸点时,请按照当地安全说明进行操作。
      完整的密封和玻璃管中没有任何裂缝是必不可少的。
    4. 在冰上冷却样品5分钟。
    5. 加入1ml去离子水和1ml含0.2%(v / v)苯甲酸甲酯(2μl苯甲酸甲酯+每份样品998μl氯仿)的氯仿作为内标。
      注意:所有样品都使用相同的氯仿 - 甲基苯甲酸酯混合物,以避免稀释错误。
    6. 紧紧闭合管并剧烈漩涡30秒。
    7. 让管子静置一分钟以使相分离。
    8. 移取150微升有机(底部)相到0.3毫升有限容量的GC玻璃小瓶中。
    9. 关闭GC瓶的螺帽。
    10. 用150μl纯氯仿作为色谱柱的平衡溶剂准备一个GC小瓶。
    11. 将1ml纯氯仿和1ml氯仿甲苯甲酸酯混合物移入培养管中以制备外标。将150μl混合物转移到GC小瓶中。


      图6. PHB酸性甲醇分解步骤的图像。 (A),油浴(B)之前的完全甲醇分解样品,油浴(C)之后的样品和具有底部有机相的相分离样品(白色箭头D)。

  4. GC测量
    1. 将所有GC样品瓶放入GC托盘。
    2. 用不同的样品用辛烷重新装满用于冲洗注射器三次的所有玻璃瓶。
    3. 清空所有废液瓶。
    4. 根据制造商提供的说明安装气相色谱仪和CTC自动进样器,并安装气密注射器和适当的衬管。
    5. 使用DB-WAX柱作为固定相,惰性气体氦气作为流动相分离甲酯。
    6. 编程1μl的注射体积,分流比为1:8,注射温度为250℃的分流模式。
    7. 编程流量为0.7毫升/分钟。
    8. 为以下温度梯度编程分离甲酯:



    9. 使用火焰离子化检测器(FID)在275°C进行检测

数据分析

第一部分尼罗红染色 - 图像分析

使用Nikon成像软件或ImageJ Fiji v1.5分析拍摄的照片,如图7所示。


图7.具有和不具有PHB的细胞的荧光显微照片。从左到右:明亮的场地,尼罗河红色,明亮的场地和尼罗河红色的合并渠道。上图:具有PHB颗粒的细胞,下图:没有PHB颗粒的细胞。
注意:细胞可能含有“黑色”内含物(例如,下图中的大多数上层细胞),不会被尼罗红染色。这些内含物可能代表其他包涵体/结构(如聚磷酸盐颗粒)。

第二部分。用气相色谱法测定PHB含量

  1. 双击感兴趣的样品名称打开色谱图。
  2. 3-羟基丁酰甲基酯(3HBME)应在7.0-7.1分钟洗脱。内标(苯甲酸甲酯)应在8.7-8.8分钟洗脱。确切的保留时间是设备和条件特定的,可能会有所不同。如果细胞累积3HB和3-羟基戊酸(3HV)的共聚物,3HV甲酯(3HVME)的保留时间约为7.9-8.0分钟。同样的方案也可以应用于中链长度PHA的分析和检测。具有6,8,10和12个C原子的3-羟基链烷酸的甲酯在约9.0分钟(C6),约11.4分钟(C8),约13.5分钟(C10)和约15.5分钟(C12)洗脱, (未显示)。
  3. 点击集成按钮(图8中的红色方块)。
  4. 如果峰值太小而无法自动集成,可以通过按下手动集成按钮(图8中的绿色框)并使用鼠标光标在峰值下绘制粉红线来手动集成它。
  5. 曲线下面积的值将列在色谱图下方的表格中(图8中的橙色框)。


    图8. 3HVME和3HBME的色谱图和结果表(A)和代表性色谱图的截图(B)

  6. 将PHB标准曲线的所有样品和待分析的所有样品的3-羟基丁酰甲基酯(3-HBME)峰和苯甲酸甲酯峰下的面积值复制到excel表中。
  7. 使用公式计算3-HBME峰的归一化面积:

  8. 通过绘制PHB标准样品的标准化区域来创建标准曲线 在y轴上对抗x轴上PHB的加权量(图9),并确定函数的方程(y = a * x + b)。


    图9.测定PHB重量的标准曲线。 一条线连接数据点,第二条线是用于生成线的方程的趋势线。

  9. 使用公式计算样本中PHB的重量:

  10. 使用公式计算PHB每细胞干重的百分比:

  11. 用您选择的程序表示图表中的PHB内容(图10)。


    图10. R的增长(A)和PHB生产(B)的例子。在富含0.2%葡糖酸钠(复合培养基)的NB培养基上或富含2%果糖的矿物盐培养基上的富营养化石H16

食谱

  1. NB媒介
    1. 称取8克NB粉末,装入1000毫升玻璃瓶中,加入1000毫升去离子水。将其混合并在121°C高压灭菌20分钟
    2. 在室温下储存NB培养基
  2. 矿物盐介质
    可以使用适合感兴趣菌株生长的任何无机盐培养基。对于Ralstonia eutropha H16,我们使用以下培养基。为了制备1,000ml培养基:
    1. 将9克Na 2 HPO 4•12H 2 O,1.5克KH 2 PO 4,使用磁力搅拌器在≈950ml去离子水中的1g NH 4 Cl和1g NH 4 Cl。

    2. 添加1毫升以下的1,000x储备溶液 镁溶液(200g / L MgSO 4•7H 2 O)
      Ca溶液(20g / L CaCl 2•7H 2 O)
      铁溶液(1.2克/升柠檬酸铁铵)
      注意:在加入Mg,Ca和Fe溶液过程中,磁力搅拌器的运行非常重要。否则可能会形成白色沉淀。
    3. 最后,加入100μl微量元素溶液
      每升微量元素原液的数值:1g ZnSO 4,0.3g MnCl 2•4H 2 O,3g H 3 BO 3,2克CoCl 2•6H 2 O,0.1克CuCl 2•2H 2 O 2,0.2g NiCl 2•6H 2 O,0.3g NaMoO 4•2H 2 O用去离子水补足1000毫升的最终体积
    4. 矿物盐介质的pH值应为pH 7.0±0.1。
    5. 将培养基在121°C灭菌20分钟
    备注:
    1. 一旦灭菌就会形成少量的沉淀,在冷却和搅拌时会部分再溶解。剩余的少量沉淀物是可以容忍的。
    2. 在感兴趣的菌株接种之前,添加所需量的碳源(例如,在高PHB应该积累的情况下,葡萄糖酸钠储备液高达2%)。
    3. 可选地,氮源(NH 4 Cl:em)的量可以减少到0.5g / L。这将导致更高的碳氮比,并导致细胞生物量产量的降低,但会使每克细胞干重的PHB积累百分比增加到80%。
  3. D-葡糖酸钠盐溶液(20%原液)
    1. 通过将20g葡萄糖酸钠溶解在100ml去离子水中制备20%wt / vol的D-葡萄糖酸钠盐储备液
    2. 通过无菌过滤器(0.2μm)过滤或加热灭菌(20分钟121°C)消毒溶液。
    3. 储备溶液在室温下稳定
  4. 尼罗河红色解决方案
    1. 准备1毫克/毫升的尼罗红储备溶液并将其溶解在DMSO中。
    2. 用DMSO稀释原液,得到10μg/ ml的工作溶液
    3. 尼罗河红色溶液可以在4°C下储存几个月
    4. 保护光线下的解决方案

致谢

我们感谢Deutsche Forschungsgemeinschaft(DFG Je152 / 17-1)的资助。作者声明不存在利益冲突。

参考

  1. Anderson,A.J。和Dawes,E.A。(1990)。 细菌聚羟基链烷酸酯的发生,代谢,代谢作用和工业用途。 Microbiol Rev 54(4):450-472。
  2. Brandl,H.,Gross,R.A。和Lenz,R.W。(1988)。 假单胞菌 oleovorans,作为聚(β-羟基链烷酸酯)的来源潜在的应用。 Appl Env Microbiol 54:1977-1982。
  3. Bresan,S.,Sznajder,A.,Hauf,W.,Forchhammer,K.,Pfeiffer,D.和Jendrossek,D。(2016)。 聚羟基链烷酸酯(PHA)颗粒不含磷脂。 Sci Rep 6:26612.
  4. Chen,G.Q.(2009)。 基于微生物聚羟基链烷酸酯(PHA)的生物和材料工业。 Chem Soc Rev 38:2434-2446。
  5. Giretova,M.,Medvecky,L.,Stulajterova,R.,Sopcak,T.,Briancin,J。和Tatarkova,M.(2016)。 聚羟基丁酸酯/壳聚糖/磷酸钙复合物中壳聚糖的酶促降解对体外成骨细胞反应。 J Mater Mater Mater Med 27:181。
  6. Jendrossek,D。和Pfeiffer,D。(2014)。 聚羟基链烷酸酯颗粒(碳纳米颗粒)形成的新见解和聚(3-羟基丁酸酯) 。
  7. Pacheco,D. P.,Amaral,M. H.,Reis,R. L.,Marques,A. P.和Correlo,V. M.(2015)。 开发用于再生医学的可注射PHBV微粒-GG水凝胶杂交系统 Int J Pharm 478:398-408。
  8. Raberg,M.,Voigt,B.,Hecker,M.和Steinbüchel,A。(2014)。 详细了解真养罗尔斯通氏菌的聚羟基丁酸酯(PHB-)阴性表型, PHB-4。 PLoS One 9:e95907。
  9. Riedel,S.L,Jahns,S.,Koenig,S.,Bock,M.C.E.,Brigham,C.J。,Bader,J。和Stahl,U。(2015)。 采用低质量废弃动物脂肪制备富含Ralstonia eutropha的聚羟基脂肪酸酯。 a> J Biotechnol 214:119-127。
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  11. Wu,Q.,Wang,Y.和Chen,G.Q.(2009)。 微生物生物聚酯聚羟基链烷酸酯的医学应用 Artif Cells血液替代品Immobil Biotechnol 37:1-12。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Juengert, J. R., Bresan, S. and Jendrossek, D. (2018). Determination of Polyhydroxybutyrate (PHB) Content in Ralstonia eutropha Using Gas Chromatography and Nile Red Staining. Bio-protocol 8(5): e2748. DOI: 10.21769/BioProtoc.2748.
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