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Glycogen and Extracellular Glucose Estimation from Cyanobacteria Synechocystis sp. PCC 6803
蓝藻集胞藻PCC 6803中糖原和胞外葡萄糖估测   

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本实验方案简略版
Scientific Reports
Sep 2016

Abstract

Cyanobacteria, which have the extraordinary ability to grow using sunlight and carbon dioxide, are emerging as a green host to produce value-added products. Exploitation of this highly promising host to make products may depend on the ability to modulate the glucose metabolic pathway; it is the key metabolic pathway that generates intermediates that feed many industrially important pathways. Thus, before cyanobacteria can be considered as a leading source to produce value-added products, we must understand the interaction between glucose metabolism and other important cellular activities such as photosynthesis and chlorophyll metabolism. Here we describe reproducible and reliable methods for measuring extracellular glucose and glycogen levels from cyanobacteria.

Keywords: Extracellular glucose (胞外葡萄糖), Glycogen (糖原), Cyanobacteria (蓝藻), Synechocystis sp. PCC 6803 (集胞藻PCC 6803)

Background

Cyanobacteria have a light-dark cycle in their natural habitat. In the light, their metabolism is centered on photosynthesis, the Calvin cycle, glycolysis and the TCA cycle with N-assimilation; carbon is stored as glycogen. In the dark, glycogen is metabolized through glycolysis and the oxidative pentose phosphate (OPP) pathway, the oxidative and reductive branches of the TCA cycle, and the C4 cycle (Nagarajan et al., 2014). Thus, the shift from dark to light or light to dark drives metabolic reprogramming.

In the laboratory, the addition of glucose to the culture media also impacts cyanobacteria metabolic programs. For example, nutritional and environmental conditions influence how the cyanobacterium Synechocystis metabolizes glucose; Synechocystis metabolizes glucose differently in photoautotrophic, heterotrophic and mixotrophic conditions. Previous studies reported that some strains of Synechocystis are light-dependent and glucose tolerant (Anderson and McIntosh, 1991). Light-activated heterotrophic growth (LAHG) conditions are characterized by the presence of glucose and growth in the dark with a pulse of white or blue light for at least 5-15 min per day. However, some strains of Synechocystis are glucose intolerant, meaning that they cannot grow in the presence of glucose in the dark. In summary, the addition of glucose to the culture media of Synechocystis has been reported to bring physiological and metabolic changes such as pigmentation (Ryu et al., 2004), carbon metabolism (Lee et al., 2007; Takahashi et al., 2008), phosphorylation patterns (Bloye et al., 1992), carbon dioxide uptake (Kaplan and Reinhold, 1999), and oxidative stress generation (Narainsamy et al., 2013).

To identify the utility of cyanobacteria to produce natural product, growing cyanobacteria in large-scale is a prerequisite. For growing cyanobacteria efficiently, it’s important to characterize the direct impact of common environmental factors such as light and temperature on glucose metabolism. Here, we present an accurate, reproducible, and reliable method to quantify extracellular glucose and glycogen levels of cyanobacteria, we belelive that this method will help determine the utility of cyanobacteria as a source for engineering natural products.

Materials and Reagents

  1. Pipette tips (20 µl-1 ml, autoclaved)
  2. Aluminum foil
  3. 1.5 and 2 ml Eppendorf tubes (autoclaved)
  4. 0.45 µm filter
  5. Cyanobacteria Synechocystis sp. PCC 6803 (WT, mutant D95 & C95)
    Note: For more information about these strains, please see Data analysis A4.
  6. Sulfuric acid (ACS reagent, Sigma-Aldrich, catalog number: S1526 )
  7. Nitrogen
  8. Ethanol (Sigma-Aldrich, catalog number: 362808-1L )
  9. Glycogen (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0561 )
  10. Amyloglucosidase (Sigma-Aldrich, catalog number: 10115-1G-F )
  11. Sodium carbonate (Na2CO3) (Sangon Biotech, catalog number: ST0840 )
  12. Sodium nitrate (NaNO3) (Sinopharm Chemical Reagent, catalog number: 10019918 )
  13. Hydrochloric acid (HCl) (Sinopharm Chemical Reagent, catalog number: 10011018 )
  14. Sodium hydroxide (NaOH) (Sangon Biotech, catalog number: SB0617 )
  15. Potassium phosphate dibasic (K2HPO4) (Sangon Biotech, catalog number: PB0447 )
  16. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sangon Biotech, catalog number: MT0864 )
  17. Ferric ammonium sulphate (Sangon Biotech, catalog number: A502657 )
  18. Citric acid (Sangon Biotech, catalog number: C0529 )
  19. Calcium chloride dihydrate (CaCl2·2H2O) (Sangon Biotech, catalog number: CT1331 )
  20. EDTA-Na2 (Sangon Biotech, catalog number: E0105 )
  21. Boric acid (H3BO3) (Sangon Biotech, catalog number: BB0044 )
  22. Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sangon Biotech, catalog number: A500331 )
  23. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sangon Biotech, catalog number: A602906 )
  24. Sodium molybdate dehydrate (Na2MoO4·2H2O) (Sangon Biotech, catalog number: SB0865 )
  25. Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Sangon Biotech, catalog number: A501425 )
  26. Cobalt(II) nitrate hexahydrate (Co(NO3)2·6H2O) (Sangon Biotech, catalog number: CB7774 )
  27. Sodium thiosulfate anhydrous (Na2S2O3) (Sangon Biotech, catalog number: S1712 )
  28. D-glucose (Sangon Biotech, catalog number: 501991 )
  29. Glucose standard solution (Sigma-Aldrich, catalog number: G3285 )
  30. Benzoic acid (Sinopharm Chemical Reagent, catalog number: 30018615 )
  31. Glucose oxidase/peroxidase (Sigma-Aldrich, catalog number: G3660 )
  32. o-Dianisidine reagent (Sigma-Aldrich, catalog number: D2679 )
  33. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P6310 )
  34. Sodium acetate (Sigma-Aldrich, catalog number: S2889 )
  35. TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (Sangon Biotech, catalog number: TB0927 )
  36. BG-11 media (see Recipes)
  37. D-Glucose (see Recipes)
  38. Assay reagent (see Recipes)
  39. 30% (w/v) KOH (see Recipes)
  40. 100 mM sodium acetate (pH 4.5) (see Recipes)

Equipment

  1. Conical flasks (100, 200, 500 ml) (SHUNIU, Chengdu, China)
  2. Vortex (FINE PCR, model: Finevortex )
  3. Centrifuge machine, unrefrigerated, maximum speed 17,000 x g, with rotor for microtubes (Thermo Fisher Scientific, model: HeraeuesTM PicoTM 17 )
  4. Glass test tubes 18 x 150 mm
  5. Spectrophotometer (METASH, model: V-5600 )
  6. 1 ml glass cuvettes (METASH)
  7. Pipettes (20 µl, 200 µl, 1 ml, 5 ml) (Gilson, France)
  8. Water baths (temperature set at 37 ± 1 °C; 60 °C; 92 °C) (Meier, model: XMTD-204 )
  9. Shaking light-incubator (light:up to 150 µmoles/m2/sec; temperature: 20-50 °C) (Shanghai Zhichu, model: ZQWY-200G )
  10. Freeze dryer (Labconco, model: FreeZone Plus 6 )
  11. Oven, 60 °C (Boxun, model: GZX-9140MBE )
  12. Autoclave (Boxun, model: YXQ-LS-100SII )
  13. pH meter (Mettler Toledo, model: FE 20 )
  14. Swimming holders

Software

  1. GraphPad PRISM (Version 5.01)

Procedure

  1. Cultivation of cyanobacteria
    1. Cyanobacterium (Synechocystis) cell stocks are kept at -80 °C in 20% glycerol. Add ~100 µl of the frozen stock to 50 ml BG-11 media (see Recipe 1) in a 100 ml conical flask. Grow a starter culture for 3-4 days (until OD730 is around 0.5) under white light (25 μmol m-2 sec-1) in a light-incubator at 30 °C with shaking at 150 rmp. To conduct glucose consumption and glycogen estimation experiments, make a fresh culture in 100 ml BG-11 media in a 200 ml conical flask with an initial OD730 of 0.1 and incubate in a photo-incubator under the same conditions mentioned above.
    2. When the OD730 is around 0.4 (it usually takes ~36 h), 1 ml (0.5 M) sterile D-glucose solution (see Recipe 2) is added to the 100 ml culture media to get a final glucose concentration of 5 mM. Grow cultures in two conditions: (1) Dark-glucose condition, in which the conical flask is wrapped with aluminum foil and (2) Light-glucose condition in which the conical flask is not wrapped in foil. Collect a sample for glucose and glycogen measurement every 24 h from both conditions.
    3. For each 2 ml collection, transfer it to a 2 ml Eppendorf tube, measure the OD730 using a spectrophotometer and record it. Centrifuge the 2 ml samples at 17,000 x g for 2 min at room temperature and transfer the supernatant to a separate 2 ml Eppendorf tube. The supernatant is used for assaying glucose and the rest is used for assaying glycogen (more details in glycogen measurement section).

  2. Extracellular glucose measurement
    1. The sample from Step A3 is used to assay for glucose (Table 1)

      Table 1. Experimental design for extracellular glucose test

      Note: We take 5 biological & 3 technical replicates for each experiment.

    2. Open the spectrophotometer. Add 2 ml assay reagent (see Recipe 3) to each tube. Mix thoroughly by vortexing. Add 500 µl of samples and standard to cuvettes and measure the absorbance at 540 nm against the blank. Record as ‘initial reading’.
    3. Place the tubes in a water bath at 37 °C for exactly 30 min. Stop the reaction by adding 2 ml of 12 N H2SO4 (add the acid in the chemical hood for safety) and mix thoroughly with extra care by vortexing. To get the exact incubation duration, keep a 30-60 sec interval of pipetting of blank, standard and test samples. The presence of glucose results in the development of pink color (Figures 1A and 1B). The more glucose in the sample, the stronger the pink color looks.
    4. The OD at 540 is measured after 30 min against the blank and recorded as ‘final reading’.
      Note: The Step B4 should be performed immediately after Step B3. The time interval should be maintained strictly equal.


    Figure 1. Quantitative assay of extracellular glucose from growth medium. Three replicates are shown for WT (panel A) and a mutant cyanobacterium (panel B). In principle, glucose is oxidized to gluconic acid and hydrogen peroxide by glucose oxidase. Hydrogen peroxide then reacts with reduced o-Dianisidine in the presence of peroxidase to form a brown colored oxidized o-Dianisidine. Oxidized o-Dianisidine reacts with sulfuric acid to form a more stable pink-colored product. The intensity of the pink color measured at 540 nm is proportional to the original glucose concentration. Thus, the pink color indicates the presence of glucose, whereas the white/clear color indicates very little glucose. The results are also available in Khan et al., 2016.

  3. Glycogen measurement
    1. Use the sample collected in Step A3. Calculate the approximate cell number from the OD730 value as follows; OD730 value of 1.0 can be taken as 1.03 x 108 cells per ml. Centrifuge approximately 3 x 109 cells from wild type and mutant Synechocystis cultures each at 1,700 x g for 2 min. Remove the supernatant. Wash the pellet each with 1 ml sterile ddH2O two times. Immediately, move the cell pellet to -80 °C for 5 min to stop the metabolism.
    2. Next, place the cell pellet in a freeze dryer at -70 °C overnight under a continuous flow of nitrogen. On the next day, after drying ~14 h in the freeze dryer, weigh the dried cell pellet and record the weight.
    3. Resuspend the dried cell pellet in 1 ml of 30% (w/v) KOH (see Recipe 4). Mix completely by pipetting (3 to 5 min).
    4. Place the mixture in a 97 °C water bath for 2 h with swimming holder (set the water bath in advance to make sure the temperature is correct).
    5. Divide the 1 ml of 30% KOH-suspended sample into two separate Eppendorf tubes, each with 500 µl. Add ~1.3 ml ice cold ethanol to a final concentration of 70-75%. Incubate on ice for 2 h.
    6. Centrifuge the mixture at 17,000 x g for 2 min at room temperature. Remove the supernatant while being careful not to disturb the pellet at the bottom of the tube. Wash the pellet three times with 98% ethanol. The pellet seen at the bottom of the Eppendorf tube is glycogen.
    7. Open the lid of the Eppendorf tube and place in a 60 °C oven for 10-20 min (depends on how long it takes to dry completely).
    8. Resuspend the dried pellet in 0.5 ml 100 mM sodium acetate (pH 4.5) (see Recipe 5). If two tubes contain the same sample (divided in Step C5), they can be added together to get a volume of 1 ml. Dissolve a fixed amount of glycogen (0.005 mg) in 1 ml 100 mM sodium acetate (pH 4.5). This is the STDglyc.
    9. Add 2 mg amyloglucosidase to the 1 ml suspension obtained in the Step C8 (final concentration 2 mg/ml). Incubate in a 60 °C water bath for 2 h. Amyloglucosidase hydrolyzes glycogen into glucose.
    10. Measure the glucose following Steps B4 to B7 described in ‘Procedure B’.

Data analysis

  1. Extracellular glucose measurement
    1. The difference between the ‘final reading’ and ‘initial reading’ is calculated for the standard (ΔA540STD) and test samples (ΔA540Sample). The amount of glucose can be calculated using the equation below:
      Mg glucose = (ΔA540Sample) x (amount of glucose in 0.05 ml standard solution/ΔA540STD).
    2. The amount of glucose obtained is converted into mMol and normalized by dividing the OD730 recorded in Step A3. For each sample, we use five biological and three technical replicates.
    3. Open the GraphPad PRISM (Version 5.01) software (the interface of the software is shown in Figure 2). Select ‘grouped’ from the ‘New Graph & Table’ list. Select ‘start with an empty data table’ from the ‘Sample data’ option. Select ‘Interleaved Bar, Vertical’ from the “Choose a Graph’ option. From the ‘Y subcolumns for replicates or error bars’ option, select ‘Enter’ with 5 replicates. Select ‘Mean with SEM’ from the ‘Plot’ option and then click on ‘create’.


      Figure 2. The interface of the GraphPad PRISM software

    4. A page will open with 5 empty boxes on the top under the single title A (A:Y1, A:Y2, A:Y3, A:Y4, A:Y5). A series of titles will appear on the left marked with 1, 2, 3, 4, etc. Replace the label of title “A” with “WT”, “B” with “D95” and “C” with “C95”. The left title series are labeled with the corresponding time points: 0 h, 24 h, etc. The mean value of the three technical replicates is put in the box and there are five boxes for five biological replicates. Likewise, data for D95 and C95 are also placed in the boxes marked with title D95 and C95. D95 is a mutant in which the slr089 gene has been deleted, which results in this mutant having different responses to glucose metabolism compared to wild type. C95 is a strain where the endogenous slr0895 gene is deleted, but it is complemented with a wild type version of the slr089 gene linked to a different antibiotic resistance marker that was used to delete the slr089 gene in strain D95. C95 has wild type glucose metabolic activity. Analyses are performed by clicking the ‘Analysis’ option on the top of the page. The statistics can be viewed by clicking the ‘Result’ option on the left. The graph can be viewed by clicking on the ‘Graph’ option on the left of the page. Figure 3A can be copied by right clicking and selecting copy.


      Figure 3. Data analysis and presentation of extracellular glucose (A), and glycogen (B) estimation from wild type and different mutant strains of cyanobacteria, Synechocystis sp. PCC 6803 (see details in Data analysis A4). The results have been published in Scientific Report and more information on the mutants can be found in Khan et al. (2016).

    Schematic presentation of the reaction:


  2. Glycogen measurement from cyanobacteria
    1. The amount of glycogen is calculated using the formula below:
      Amount of glycogen (mg) in test sample = (amount of glucose of test sample) x (0.005/amount of glucose obtained from STDglyc).
      The amount of glycogen calculated is multiplied by 1,000 to convert the unit from mg to µg.
    2. The glycogen calculated in µg is normalized by dividing by the dry weight measured in Step C2 (Procedure section). To get reliable data, we use five biological and three technical replicates for each sample including the glycogen standard (STDglyc). We make an average of glucose obtained from STDglyc replicates before using in Step C1.
    3. Statistical analyses are performed following Steps A3 to A5 in the Data analysis section and sample results are shown in Figure 3B.

Recipes

  1. BG-11 media preparation
    1. Step 1: The stock solutions are prepared according to the composition given in Table 2 which were based on the solutions reported by Stainer et al. (1971). Autoclave the stock solutions. Wrap stock 3 with aluminum foil to protect from light. Store the stock solutions at room temperature

      Table 2. BG-11 media composition


    2. Step 2: To prepare 1 L of media, add 1 ml from each stock. Next, add 0.02 g/L of Na2CO3 and 1.5 g/L of NaNO3. The pH was adjusted to ~7.5 using hydrochloric acid (HCl) and sodium hydroxide (NaOH)
    3. Step 3: Pour the media into a conical flask and autoclave using the liquid media cycle
  2. D-Glucose
    Dissolve 1.350 g D-glucose in 15 ml of dH2O to make 0.5 M stock
    Sterilize using a 0.45 µm filter
  3. Assay reagent
    Add 0.8 ml of the o-Dianisidine reagent to an amber bottle containing 39.2 ml of glucose oxidase/peroxidase reagent
    Invert the bottle several times to mix
    Minimize exposure to light
    This solution is stable for up to 1 month at 2-8 °C
    Discard if turbidity develops or color forms
  4. 30% (w/v) KOH preparation
    Dissolve 15 g of KOH in 50 ml ddH2O to prepare a 30% (w/v) KOH solution
    Store at room temperature
  5. 100 mM sodium acetate preparation
    Dissolve 0.4102 g sodium acetate in 50 ml water to get a final concentration of 100 mM
    Adjust the pH to 4.5 using NaOH or HCl
    Store at room temperature

Acknowledgments

This protocol is an adapted version of the method described by Grundel et al. (2012). This work was supported by the 973 Program (2011CBA00803), the National Natural Science Foundation of China (31671504, 81421061), and the National Key Technology R&D Program (2012BAI01B09).
Authors declare no any conflicts of interest or competing interests.

References

  1. Anderson, S. L. and McIntosh, L. (1991). Light-activated heterotrophic growth of the cyanobacterium Synechocystis sp. strain PCC 6803: a blue-light-requiring process. J Bacteriol 173(9): 2761-2767.
  2. Bloye, S. A., Silman, N. J., Mann, N. H. and Carr, N. G. (1992). Bicarbonate concentration by Synechocystis PCC6803: Modulation of protein phosphorylation and inorganic carbon transport by glucose. Plant Physiol 99(2): 601-606.
  3. Grundel, M., Scheunemann, R., Lockau, W. and Zilliges, Y. (2012). Impaired glycogen synthesis causes metabolic overflow reactions and affects stress responses in the cyanobacterium Synechocystis sp. PCC 6803. Microbiology 158(Pt 12): 3032-3043.
  4. Kaplan, A. and Reinhold, L. (1999). CO2 concentrating mechanisms in photosynthetic microorganisms. Annu Rev Plant Physiol Plant Mol Biol 50: 539-570.
  5. Khan, R. I., Wang, Y. S., Afrin, S., Wang, B., Liu, Y., Zhang, X. Q., Chen, L., Zhang, W. W., He, L. and Ma, G. (2016). Transcriptional regulator PrqR plays a negative role in glucose metabolism and oxidative stress acclimation in Synechocystis sp. PCC 6803. Sci Rep 6: 32507.
  6. Lee, S., Ryu, J. Y., Kim, S. Y., Jeon, J. H., Song, J. Y., Cho, H. T., Choi, S. B., Choi, D., de Marsac, N. T. and Park, Y. I. (2007). Transcriptional regulation of the respiratory genes in the cyanobacterium Synechocystis sp. PCC 6803 during the early response to glucose feeding. Plant Physiol 145(3): 1018-1030.
  7. Nagarajan, S., Srivastava, S. and Sherman, L. A. (2014). Essential role of the plasmid hik31 operon in regulating central metabolism in the dark in Synechocystis sp. PCC 6803. Mol Microbiol 91(1): 79-97.
  8. Narainsamy, K., Cassier-Chauvat, C., Junot, C. and Chauvat, F. (2013). High performance analysis of the cyanobacterial metabolism via liquid chromatography coupled to a LTQ-Orbitrap mass spectrometer: evidence that glucose reprograms the whole carbon metabolism and triggers oxidative stress. Metabolomics 9(1): 21-32.
  9. Ryu, J. Y., Song, J. Y., Lee, J. M., Jeong, S. W., Chow, W. S., Choi, S. B., Pogson, B. J. and Park, Y. I. (2004). Glucose-induced expression of carotenoid biosynthesis genes in the dark is mediated by cytosolic pH in the cyanobacterium Synechocystis sp. PCC 6803. J Biol Chem 279(24): 25320-25325.
  10. Takahashi, H., Uchimiya, H. and Hihara, Y. (2008). Difference in metabolite levels between photoautotrophic and photomixotrophic cultures of Synechocystis sp. PCC 6803 examined by capillary electrophoresis electrospray ionization mass spectrometry. J Exp Bot 59(11): 3009-3018.
  11. Stanier, R. Y., Kunisawa, R., Mandel, M. and Cohen-Bazire, G. (1971). Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35(2): 171-205.

简介

具有使用阳光和二氧化碳生长的非凡能力的蓝细菌正在成为生产高附加值产品的绿色主机。 利用这种非常有希望的宿主来制造产品可能取决于调节葡萄糖代谢途径的能力; 它是产生中间产物的关键代谢途径,这些中间产物为许多工业上重要的途径提供了饲料。 因此,在蓝藻被认为是生产增值产品的主要来源之前,我们必须了解葡萄糖代谢与其他重要细胞活动如光合作用和叶绿素代谢之间的相互作用。 在这里我们描述了测量蓝细菌细胞外葡萄糖和糖原水平的可重复和可靠的方法。

【背景】蓝藻在自然栖息地有一个明暗周期。有鉴于此,他们的新陈代谢主要集中在光合作用,卡尔文循环,糖酵解和TCA循环中,同时进行N-同化;碳以糖原形式储存。在黑暗中,糖原通过糖酵解和氧化磷酸戊糖(OPP)途径,TCA循环的氧化和还原分支以及C4循环代谢(Nagarajan et al。,2014)。因此,从黑暗转变为光照或光照转变为黑暗推动了代谢重新编程。

在实验室中,向培养基中添加葡萄糖也会影响蓝藻的代谢程序。例如,营养和环境条件影响蓝藻集胞藻如何代谢葡萄糖;在光合自养,异养和混合营养条件下,集胞藻代谢葡萄糖的方式不同。先前的研究报道,一些菌株的集胞藻是轻度依赖的并且耐受葡萄糖(Anderson和McIntosh,1991)。光激活异养生长(LAHG)条件的特征在于存在葡萄糖并且在黑暗中用白光或蓝光脉冲生长至少5-15分钟/天。然而,一些集胞蓝细菌葡萄糖不耐受,这意味着它们在黑暗中不能生长在葡萄糖存在下。总之,已经报道在集胞藻的培养基中加入葡萄糖会带来生理和代谢变化,如色素沉着(Ryu等人,2004),碳代谢(Lee等人,2007; Takahashi等人,2008),磷酸化模式(Bloye等人,1992),二氧化碳摄取(Kaplan和Reinhold,1999)和氧化应激产生(Narainsamy等人,2013)。

为了确定蓝藻生产天然产品的效用,大规模生长蓝藻是一个先决条件。为了有效地生长蓝藻,重要的是要确定光照和温度等常见环境因素对葡萄糖代谢的直接影响。在这里,我们提供了一种准确,可重复和可靠的方法来量化蓝细菌的细胞外葡萄糖和糖原水平,我们相信这种方法将有助于确定蓝藻作为工程天然产物的来源的效用。

关键字:胞外葡萄糖, 糖原, 蓝藻, 集胞藻PCC 6803

材料和试剂

  1. 移液器吸头(20μl-1 ml,高压灭菌)
  2. 铝箔
  3. 1.5和2毫升Eppendorf管(高压灭菌)
  4. 0.45μm过滤器
  5. 蓝细菌<集胞藻> sp。 PCC 6803(WT,突变体D95和C95)
    注意:有关这些菌株的更多信息,请参阅数据分析A4。
  6. 硫酸(ACS试剂,Sigma-Aldrich,目录号:S1526)

  7. 乙醇(Sigma-Aldrich,目录号:362808-1L)
  8. 糖原(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0561)
  9. 淀粉葡糖苷酶(Sigma-Aldrich,目录号:10115-1G-F)
  10. 碳酸钠(Na 2 CO 3)(Sangon Biotech,目录号:ST0840)
  11. 硝酸钠(NaNO 3)(国药集团化学试剂,目录号:10019918)
  12. 盐酸(HCl)(国药集团化学试剂,目录号:10011018)
  13. 氢氧化钠(NaOH)(Sangon Biotech,目录号:SB0617)
  14. 磷酸氢二钾(K 2 HPO 4)(Sangon Biotech,目录号:PB0447)
  15. 硫酸镁七水合物(MgSO 4·7H 2 O)(Sangon Biotech,目录号:MT0864)
  16. 硫酸铁铵(Sangon Biotech,目录号:A502657)
  17. 柠檬酸(Sangon Biotech,目录号:C0529)
  18. 氯化钙二水合物(CaCl 2·2H 2 O)(Sangon Biotech,目录号:CT1331)
  19. EDTA-Na 2(Sangon Biotech,目录号:E0105)
  20. 硼酸(H 3 BO 3)(Sangon Biotech,目录号:BB0044)
  21. 氯化锰(II)四水合物(MnCl 2·4H 2 O)(Sangon Biotech,目录号:A500331)
  22. 硫酸锌七水合物(ZnSO 4·7H 2 O)(Sangon Biotech,目录号:A602906)
  23. 脱水钼酸钠(Na 2 MoO 4·2H 2 O)(Sangon Biotech,目录号:SB0865)
  24. 硫酸铜(II)五水合物(CuSO 4·5H 2 O)(Sangon Biotech,目录号:A501425)
  25. 硝酸钴(II)六水合物(Co(NO 3)2·6H 2 O)(Sangon Biotech,目录号:CB7774) />
  26. 无水硫代硫酸钠(Na 2 S 2 O 3)(Sangon Biotech,目录号:S1712)
  27. D-葡萄糖(Sangon Biotech,目录号:501991)
  28. 葡萄糖标准溶液(Sigma-Aldrich,目录号:G3285)
  29. 苯甲酸(国药集团化学试剂,目录号:30018615)
  30. 葡萄糖氧化酶/过氧化物酶(Sigma-Aldrich,目录号:G3660)
  31. - 茴香胺试剂(Sigma-Aldrich,目录号:D2679)
  32. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:P6310)
  33. 乙酸钠(Sigma-Aldrich,目录号:S2889)
  34. TES,N-三(羟甲基)甲基-2-氨基乙烷磺酸(Sangon Biotech,目录号:TB0927)
  35. BG-11媒体(见食谱)
  36. D-葡萄糖(见食谱)
  37. 分析试剂(见配方)
  38. 30%(w / v)KOH(见食谱)
  39. 100 mM乙酸钠(pH 4.5)(见食谱)

设备


  1. 锥形瓶(100,200,500毫升)(顺秋,成都,中国)
  2. 涡流(FINE PCR,型号:Finevortex)
  3. 离心机,未冷冻,最大速度17,000 emg,带微管转子(Thermo Fisher Scientific,型号:Heraeues TM Pico TM 17) />
  4. 玻璃试管18 x 150 mm
  5. 分光光度计(METASH,型号:V-5600)
  6. 1毫升玻璃比色皿(METASH)
  7. 移液器(20μl,200μl,1ml,5ml)(法国吉尔森)
  8. 水浴(温度设定在37±1℃; 60℃; 92℃)(Meier,型号:XMTD-204)
  9. 摇动光照培养箱(光照:150微摩尔/平方米/秒;温度:20-50℃)(上海知初,型号:ZQWY-200G)
  10. 冷冻干燥机(Labconco,型号:FreeZone Plus 6)
  11. 烤箱,60°C(Boxun,型号:GZX-9140MBE)
  12. 高压灭菌器(Boxun,型号:YXQ-LS-100SII)
  13. pH计(Mettler Toledo,型号:FE 20)
  14. 游泳池

软件

  1. GraphPad PRISM(版本5.01)

程序

  1. 蓝藻培养
    1. 蓝细菌(集胞蓝细菌)细胞原种保持在20%甘油中-80℃。在100ml锥形瓶中加入〜100μl冷冻原料至50ml BG-11培养基(见配方1)。在白光(25μmolm -2 s -1)下培养起始培养物3-4天(直到OD 730约为0.5) )在30℃的轻度培养箱中以150rpm振荡。为了进行葡萄糖消耗和糖原估计实验,在具有0.1的初始OD 730的200ml锥形瓶中在100ml BG-11培养基中进行新鲜培养,并在光孵化器中在相同温度下孵育上述条件。
    2. 当OD 730约为0.4(通常需要约36小时)时,向100ml培养基中加入1ml(0.5M)无菌D-葡萄糖溶液(参见配方2)以获得最终葡萄糖浓度为5mM。在两种条件下培养培养物:(1)暗葡萄糖条件,其中锥形瓶用铝箔包裹;以及(2)在光 - 葡萄糖条件下,锥形瓶不包裹在箔中。
      每24小时收集一次葡萄糖和糖原测量样本
    3. 对于每2ml收集物,将其转移到2ml Eppendorf管中,使用分光光度计测量OD 730,并记录它。在室温下将2ml样品在17,000gxg离心2分钟,并将上清液转移到单独的2ml Eppendorf管中。上清液用于测定葡萄糖,其余用于测定糖原(糖原测量部分更详细)。

  2. 胞外葡萄糖测量
    1. 来自步骤A3的样品用于测定葡萄糖(表1)

      表1.细胞外葡萄糖测试的实验设计

      注:我们采取5种生物&amp;每个实验3次技术重复。

    2. 打开分光光度计。向每个管中加入2 ml检测试剂(见配方3)。通过涡旋彻底混合。将500μl样品和标准品加入比色杯中,并测量540nm处的空白吸光度。记录为“初次阅读”。
    3. 将管放置在37℃的水浴中正好30分钟。加入2ml 12N H 2 SO 4 4以终止反应; (为了安全起见,在化学罩中添加酸),并通过涡流充分混合并充分混合。为了获得确切的孵育时间,保持空白,标准和测试样品的移液间隔为30-60秒。葡萄糖的存在导致粉红色的发展(图1A和1B)。样本中的葡萄糖越多,粉红色看起来就越强。
    4. 在对照空白30分钟后测量540处的OD,并记录为“最终读数”。
      注意:步骤B4应该在步骤B3之后立即执行。时间间隔应保持严格相等。


    图1.来自生长培养基的细胞外葡萄糖的定量测定对于WT(图A)和突变的蓝细菌(图B)显示了三个重复。原则上,葡萄糖被葡萄糖氧化酶氧化成葡萄糖酸和过氧化氢。过氧化氢然后在过氧化物酶的存在下与还原的茴香胺反应以形成棕色的氧化的茴香胺。氧化 - 茴香胺与硫酸反应形成更稳定的粉红色产品。在540nm测量的粉红色的强度与原始葡萄糖浓度成比例。因此,粉红色表示存在葡萄糖,而白色/透明颜色表示非常少的葡萄糖。结果也可在Khan et al。,2016中找到。

  3. 糖原测量
    1. 使用步骤A3中收集的样本。按照以下方式计算来自OD <730>值的近似细胞数目; 1.0的OD值730可以取为1.03×10 8个细胞/ ml。从野生型和突变体集胞蓝细菌培养物中分别以1700gxg离心约3×10 9个细胞2分钟。去除上清液。每次用1ml无菌ddH 2 O洗涤沉淀两次。立即将细胞沉淀移至-80°C 5分钟以停止代谢。
    2. 接下来,在连续氮气流下,将细胞沉淀置于-70℃冻干机中过夜。在第二天,在冻干机中干燥〜14小时后,称重干燥的细胞沉淀并记录重量。
    3. 将干燥的细胞沉淀重悬于1ml 30%(w / v)的KOH中(见配方4)。
      完全混合移液(3至5分钟)
    4. 将混合物置于97°C水浴中2小时,用游泳架(提前设置水浴以确保温度正确)。
    5. 将1毫升30%的KOH悬浮样品分成两个单独的Eppendorf管,每个管都有500μl。加入约1.3 ml冰冷的乙醇至最终浓度为70-75%。
      冰上孵育2小时
    6. 在室温下将混合物在17,000×gg下离心2分钟。取出上清液时小心不要打扰管底部的颗粒。用98%乙醇洗涤沉淀三次。
      在Eppendorf管底部看到的颗粒是糖原。
    7. 打开Eppendorf管盖,置于60°C烘箱中10-20分钟(取决于完全干燥需要多长时间)。
    8. 用0.5ml 100mM乙酸钠(pH 4.5)重悬干燥的沉淀(参见配方5)。如果两个试管含有相同的样品(在步骤C5中分成),则它们可以加在一起得到1ml的体积。将固定量的糖原(0.005mg)溶解在1ml 100mM乙酸钠(pH 4.5)中。这是STD glyc 。
    9. 在步骤C8中获得的1ml悬浮液中添加2mg淀粉葡糖苷酶(终浓度2mg / ml)。在60°C水浴中孵育2小时。淀粉葡萄糖苷酶将糖原水解成葡萄糖。
    10. 按照'程序B'中描述的步骤B4至B7测量葡萄糖。

数据分析

  1. 胞外葡萄糖测量
    1. 计算标准(ΔA<540> STD)和测试样品(ΔA<540>样品)的'最终读数'和'初始读数'之间的差异。葡萄糖的量可以使用下面的公式计算:
      Mg葡萄糖=(ΔA540样品)×(0.05ml标准溶液中的葡萄糖量/ΔA540STD)。
    2. 将获得的葡萄糖的量转化为mMol,并通过除以步骤A3中记录的OD 730得到标准化。对于每个样本,我们使用五个生物和三个技术重复。
    3. 打开GraphPad PRISM(版本5.01)软件(软件界面如图2所示)。从'New Graph&amp;'选择'分组'表'列表。从'Sample data'选项中选择'从一个空数据表开始'。从“选择图表”选项中选择“交叉条形,垂直”。从'Y subcolumns for replicates or error bars'选项中,选择5个重复的'Enter'。从'Plot'选项中选择'Mean with SEM',然后点击'创建'。


      图2. GraphPad PRISM软件的界面

    4. 一个页面将在单个标题A(A:Y1,A:Y2,A:Y3,A:Y4,A:Y5)下的顶部打开5个空框。标有1,2,3,4等的标题将出现在左边。将标题“A”的标签替换为“WT”,“B”替换为“D95”和“ C“和”C95“。左侧标题系列标有相应的时间点:0 h,24 h,等。三个技术重复的平均值放在框中,五个生物重复有五个框。同样,D95和C95的数据也放在标题为D95和C95的框中。 D95是其中<! - SIPO - > slr089 基因已被缺失的突变体,导致该突变体与野生型相比对葡萄糖代谢具有不同的响应。 C95是一种内源性基因被删除的菌株,但它与另一种与不同的抗生素抗性标记连锁的野生型版本的 slr089 基因互补,删除菌株D95中的 slr089 基因。 C95具有野生型葡萄糖代谢活性。点击页面顶部的“分析”选项进行分析。点击左侧的“结果”选项即可查看统计信息。点击页面左侧的“图表”选项即可查看图表。图3A可以通过右击并选择复制来复制。


      图3.来自蓝细菌的野生型和不同突变株的细胞外葡萄糖(A)和糖原(B)估计的数据分析和呈现,集胞藻属(Synechocystis sp。) PCC 6803 (详见数据分析A4)。结果发表在科学报告中,关于突变体的更多信息可以在Khan et。(2016)中找到。

    反应的示意图:


  2. 来自蓝细菌的糖原测量
    1. 使用以下公式计算糖原的量:
      测试样品中糖原的量(mg)=(测试样品的葡萄糖量)x(0.005 /从STD glyc获得的葡萄糖的量)。
      计算的糖原量乘以1,000,将单位从mg转换为μg。
    2. 以μg计算的糖原通过除以步骤C2中测量的干重(过程部分)而归一化。为了获得可靠的数据,我们对每个样品使用五种生物和三种技术重复,包括糖原标准(STD glyc )。在步骤C1中使用之前,我们获得从STD glyc复制品获得的平均葡萄糖。
    3. 在数据分析部分的步骤A3至A5之后进行统计分析,样本结果如图3B所示。

食谱

  1. BG-11媒体准备
    1. 步骤1:储备溶液根据表2给出的基于Stainer等报道的溶液制备。 (1971年)。高压灭菌的库存解决方案。用铝箔包装纸3以免光照。
      在室温下储存储备溶液
      表2. BG-11媒体组成


    2. 步骤2:准备1 L培养基,每只股票加1 ml。接下来,加入0.02g / L的Na 2 CO 3和1.5g / L的NaNO 3。
      使用盐酸(HCl)和氢氧化钠(NaOH)将pH调节至约7.5
    3. 第3步:使用液体介质循环将介质倒入锥形瓶和高压灭菌器。
  2. D-葡萄糖
    将1.350克D-葡萄糖溶解在15毫升的dH 2 O中,制成0.5毫升的原液。
    使用0.45μm过滤器消毒
  3. 分析试剂


    添加0.8毫升的茴香胺试剂到含有39.2毫升葡萄糖氧化酶/过氧化物酶试剂的琥珀色瓶中 多次翻转瓶子混合
    尽量减少曝光


    该解决方案在2-8°C时可稳定长达1个月 如果浑浊发展或颜色形式丢弃
  4. 30%(w / v)的KOH制剂
    将15克KOH溶解在50毫升ddH2O中以制备30%(w / v)的KOH溶液
    在室温下储存
  5. 100 mM醋酸钠制剂
    将0.4102克醋酸钠溶于50毫升水中,使终浓度达到100毫摩尔
    使用NaOH或HCl将pH调整至4.5
    在室温下储存

致谢

该协议是Grundel et al。(2012)描述的方法的改进版本。这项工作得到了973计划(2011CBA00803),国家自然科学基金(31671504,81421061)和国家重点科技研发计划(2012BAI01B09)的支持。
作者声明不存在任何利益冲突或利益冲突。

参考

  1. Anderson,S.L。和McIntosh,L。(1991)。 蓝藻集胞藻的光激活异养生长 sp。应变PCC 6803:需要蓝光的过程。 J Bacteriol 173(9):2761-2767。
  2. Bloye,S.A.,Silman,N.J.,Mann,N.H。和Carr,N.G。(1992)。 集胞藻的碳酸氢盐浓度 PCC6803:调节蛋白质磷酸化和无机碳运输通过葡萄糖。植物生理学 99(2):601-606。
  3. Grundel,M.,Scheunemann,R.,Lockau,W。和Zilliges,Y。(2012)。 受损的糖原合成引起代谢溢流反应并影响蓝藻集胞的应激反应 sp。 PCC 6803. 微生物学 158(Pt 12):3032-3043。
  4. Kaplan,A。和Reinhold,L。(1999)。 Co2在光合微生物中的浓缩机制 Annu Rev Plant Physiol Plant Mol Biol 50:539-570。
  5. Khan,R. I.,Wang,Y. S.,Afrin,S.,Wang,B.,Liu,Y.,Zhang,X.Q.,Chen,L.,Zhang,W.W.,He,L.and Ma,G.(2016)。 转录调节因子PrqR在葡萄糖代谢和氧化应激适应<集胞蓝细菌 sp。中扮演负面角色。 PCC 6803. Sci Rep 6:32507.
  6. Lee,S.,Ryu,J.Y.,Kim,S.Y.,Jeon,J.H.,Song,J.Y。,Cho,H.T.,Choi,S.B。,Choi,D.,de Marsac,N.T.and Park,Y.I。(2007)。 蓝藻集胞藻呼吸基因的转录调控 sp。 PCC 6803在对葡萄糖喂养的早期反应中。植物生理学 145(3):1018-1030。
  7. Nagarajan,S.,Srivastava,S.和Sherman,L. A.(2014)。 质粒hik31操纵子在调控集胞藻黑暗中的中央代谢中的重要作用 EM> SP。 PCC 6803. Mol Microbiol 91(1):79-97。
  8. Narainsamy,K.,Cassier-Chauvat,C.,Junot,C.和Chauvat,F.(2013)。 通过与LTQ-Orbitrap质量耦合的液相色谱对蓝藻代谢进行高效分析光谱仪:证明葡萄糖重新编程整个碳代谢并触发氧化应激。代谢组学 9(1):21-32。
  9. Ryu,J.Y.,Song,J.Y.,Lee,J.M.,Jeong,S.W.,Chow,W.S.,Choi,S.B。,Pogson,B.J.and Park,Y.I。(2004)。 葡萄糖诱导的黑素中类胡萝卜素生物合成基因的表达是由蓝细菌中的细胞质pH介导的> Synechocystis sp。 PCC 6803. Biol Chem 279(24):25320-25325。
  10. Takahashi,H.,Uchimiya,H。和Hihara,Y。(2008)。 集胞藻的光合自养和光混合营养培养物代谢物水平的差异 PCC 6803通过毛细管电泳电喷雾电离质谱测定。 J Exp Bot 59(11):3009-3018。
  11. Stanier,R.Y.,Kunisawa,R.,Mandel,M.和Cohen-Bazire,G.(1971)。 单细胞蓝藻的纯化和特性(订购Chroococcales)。 Bacteriol Rev 35(2):171-205。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Khan, M. I., Wang, Y., Afrin, S., He, L. and Ma, G. (2018). Glycogen and Extracellular Glucose Estimation from Cyanobacteria Synechocystis sp. PCC 6803. Bio-protocol 8(9): e2826. DOI: 10.21769/BioProtoc.2826.
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