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Intracellular Glycogen Assays
胞内糖原分析   

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Abstract

Glycogen, a soluble multi-branched glucose homopolysaccharide, is composed of chains of α-1,4-linked glucose residues interconnected by α-1,6-linked branches. The classical biosynthetic pathway involves phosphoglucomutase (Pgm), glucose-1-phosphate adenylyltransferase (GlgC or GlgCD), glycogen synthase (GlgA) and branching enzyme (GlgB). Phosphoglucomutase converts glucose-6-phosphate into glucose-1-phosphate, which serves as a substrate for ADP-glucose synthesis catalyzed by GlgC or GlgCD. Then, GlgA catalyzes the transfer of glucosyl units from ADP-glucose to the elongating chain of linear α-1,4-glucan. GlgB subsequently cleaves off portions of the glucan and links it to internal glucose molecules in existing chains via α-1,6 glycosidic bonds to form the glycogen structure. Glycogen breakdown is mediated by glycogen phosphorylase (GlgP) and debranching enzyme (GlgX), which catalyze the sequential phosphorolysis of α-1,4-glucosyl linkages in the glucan chain from the non-reducing ends and debranching of the limit dextrins generated by GlgP, respectively. An increasing number of studies have revealed the involvement of glycogen metabolism in a multitude of physiological functions in some prokaryotes beyond the function of synthesizing energy storage compounds. Lactobacillus acidophilus NCFM was the first probiotic lactic acid bacterium demonstrated to possess a functional glycogen biosynthesis pathway that is involved in its growth, bile tolerance and complex carbohydrate metabolism (Goh and Klaenhammer, 2013). The following qualitative (for detection of intracellular glycogen) and quantitative (for measurement of intracellular glycogen content) intracellular glycogen assay protocols for Lactobacillus acidophilus (L. acidophilus) were modified from previous works (Govons et al., 1969; Law et al., 1995; Parrou and Francois, 1997) and should be applicable to other lactic acid bacteria as well as most microorganisms.

Part I. Qualitative detection of intracellular glycogen with iodine-staining method

Materials and Reagents

  1. L. acidophilus strains, or desired bacterial strains
  2. MRS broth (Difco), or other liquid growth medium of choice (stored at 4 °C)
  3. Iodine solution (see Recipes)
  4. Solid semi-defined medium (SDM) with 2% (w/v) carbohydrate (see Recipes)

Equipment

  1. 37 °C incubator
  2. 37 °C anaerobic chamber incubator
  3. Pipettor

Procedure

  1. Inoculate L. acidophilus strains of interest in 5 ml of MRS broth and grow overnight at 37 °C under anaerobic or ambient atmospheric condition.
  2. Transfer and spot 5 to 8 µl of each overnight culture onto solid agar growth medium (we use solid SDM supplemented with 2% trehalose or glucose). Include positive and/or negative control strains, if possible.
    Optional: The spotted plates can be prepared in multiple replicates to provide several iodine staining trials (see steps 4-6).
  3. Allow the spotted cultures to absorb into the agar medium, and incubate the plate at 37 °C anaerobically for 36-48 h until colonies are visible.
  4. Carefully transfer 5 ml of freshly prepared iodine solution (0.01 M I2, 0.03 M KI) onto the agar plate with the grown colonies.
  5. Incubate the cells with the iodine solution at room temperature for 30 sec to 1 min.
  6. Immediately remove the iodine solution from the plate with a pipettor. Cells containing intracellular glycogen will stain brown; whereas cells that lack intracellular glycogen will appear yellow or colorless.
    Optional: To enhance color development for capturing image of the stained colonies, the stained plates (after removal of iodine solution) can be incubated for an additional 10 min in the dark.
    Note: The stain from iodine solution is not permanent and will gradually disappear after about 30 min to 1 h.

Representative data

  1. Example: Spotted cultures of L. acidophilus NCFM and glycogen metabolic mutants stained with iodine solution.


    Figure 1. Iodine staining of L. acidophilus parent strain (NCK1909) and isogenic glycogen metabolic mutants grown on solid SDM containing 2% trehalose. Both deletion mutants of the glycogen synthesis pathway, ΔglgA and ΔglgB, appeared as yellow/colorless (-) indicative of glycogen-deficient phenotype. Like the parent strain, the ΔglgP and Δamy mutants of the glycogen degradation pathway were stained brown (+), indicating that their ability to synthesize intracellular glycogen was unaffected. glgA, glycogen synthase gene; glgB, branching enzyme gene; glgP, glycogen phosphorylase gene, and amy, putative α-amylase/debranching enzyme gene. Figure from Goh and Klaenhammer (2013).

Recipes

  1. Iodine solution
    0.01 M I2
    0.03 M KI
    Prepare fresh prior to each experiment
    Keep in dark
  2. Semi-defined medium (SDM; modified from Reference 3)
    Tween 80 (1 g/L)
    Ammonium citrate (2 g/L)
    Sodium acetate (5 g/L)
    MgSO4.7H2O (0.1 g/L)
    MnSO4 (0.05 g/L)
    K2HPO4 (2 g/L)
    Yeast nitrogen base (5 g/L)
    Casitone (10 g/L)
    Carbohydrate substrate, e.g. glucose or trehalose, 2% (or desired) final concentration
    Note: For trehalose or any heat-labile sugars, prepare a 25-40% (w/v) stock solution, filter-sterilize, and add appropriate volume to autoclaved SDM to a final concentration of 2%. For carbohydrates with poor solubility e.g. raffinose, add 2% (w/v) of the sugar directly into SDM (sterile or non-sterile), stir to solubilize, and filter-sterilized through 0.45 µm filter.
    For solid medium, add agar, 15 g/L
    Sterilize by autoclaving or filter-sterilize with 0.45 µm filter (see above; for broth only)
    Stored at 4 °C

Part II. Quantitative measurement of intracellular glycogen

Materials and Reagents

  1. L. acidophilus strains (or other bacterial strains)
  2. MRS broth (stored at 4 °C)
  3. Phosphate-buffered saline (PBS, pH 7.4) (e. g. Life Technologies, catalog number: 10010-023 )
  4. 0.25 M Na2CO3 solution (filter-sterilized with 0.45 µm filter)
  5. 1 M acetic acid
  6. 0.2 M sodium acetate (pH 5.2) (filter-sterilized with 0.45 µm filter)
  7. Sterile distilled water
  8. Amyloglucosidase (10 mg/ml ≈ 0.14 U/ µl) (Roche Diagnostics, catalog number: 10102857001 ) (stored at 4 °C)
  9. Glucose assay kit (Glucose assay reagent) (Sigma-Aldrich, catalog number: G3293 ) (stored at 4 °C)
  10. Glycogen (5-20 mg/ml solution) (e. g. Life Technologies, catalog number AM9510 ) (stored at -20 °C)
  11. SDM broth with 2% (w/v) carbohydrate substrate (see Recipes) (or liquid growth medium of choice)

Equipment

  1. 37 °C incubator
  2. Centrifuge (fits 15-ml and 50-ml tubes; 1,717 x g)
  3. Microcentrifuge (14,549 x g)
  4. Pipettor
  5. Disposable conical centrifuge tubes (15-ml and 50-ml)
  6. 1.5-ml screw-capped tubes (sterile) (e. g. Thermo Fisher Scientific, catalog numbers: 02-681-343 and 02-681-368 ) (autoclave tubes with caps on prior to use)
  7. 1.5-ml microcentrifuge tubes (sterile)
  8. Analytical balance
  9. Vortex
  10. Hot plate
  11. Large glass beaker (e. g. 1,000-ml pyrex glass beaker)
  12. Microcentrifuge tube floating rack (round, to fit into large glass beaker)
  13. Thermometer
  14. Incubator at 57 °C equipped with agitator (or hybridization oven)
  15. Spectrophotometer or microtiter plate reader (capable of measuring absorbance at 340 nm)

Procedure

  1. Cultivate L. acidophilus cells in SDM broth containing 2% (w/v) carbohydrate substrate (we supplemented with carbohydrate substrate that is known to induce glycogen biosynthesis) at 37 °C to desired growth phase and harvest by centrifugation (1,717 x g, 10 min, room temperature). Cell pellets can be processed right after centrifugation or stored at -80 °C.
    Notes:
    1. In general, for early log (OD0.3 to 0.4), mid-log (OD0.6), stationary (OD1.0 to 1.6) and late stationary phase cultures (OD ≥ 2.0), we harvest aliquots of ca. 50 ml, 30 ml, 20 ml and 15 ml, respectively, in disposable conical centrifuge tubes, to yield ca. 0.04 to 0.06 g of cell wet weight.  
    2. We prepare cultures and cell pellets representing at least two biological replicates in order to demonstrate reproducibility of the experimental results.
  2. Pre-label, pre-weigh (with cap on), and designate each 1.5-ml screw-capped tube for each sample.
  3. Resuspend each cell pellet in 1 ml of PBS and transfer to designated pre-weighed 1.5 ml screw-capped tube.
  4. Centrifuge (14,549 x g, 1 min, room temperature) and remove all supernatant with a pipettor.
  5. Repeat step 4 centrifugation for at least twice to remove all the supernatant in order to obtain an accurate cell wet weight.
  6. Weigh the tubes (with caps on) with the cell pellets. Subtract the previous weights of the empty tubes (with cap; from step 2 above) from the weight values to obtain net cell wet weight.
  7. Add 0.25 ml of 0.25 M Na2CO3 solution to the cell pellet. Resuspend by gentle vortexing.
    Note: Do not resuspend the cell pellet with a pipettor since any amount of cells adhering to the pipet tip will change the net cell wet weight.
    For controls: Include 2 additional tubes (no pre-weighing necessary). In each tube, add ~400 µg (e. g. 20 µl of 20 mg/ml) of glycogen to 0.25 ml of 0.25 M Na2CO3 solution. Follow steps 8-12.
  8. Incubate the cell suspensions at 95 to 98 °C for 4 h (We place the tubes securely in a microcentrifuge tube floating rack and incubate in a large beaker of water heated on a hot plate.).  Gently vortex to mix the cell suspension 1 to 2 times during the 4-h incubation.
  9. Add 0.15 ml of 1 M acetic acid and 0.6 ml of 0.2 M sodium acetate (pH 5.2) to the cell suspension to bring the pH to ~5.2 and the final volume to 1 ml.
  10. Add 10 µl of amyloglucosidase (0.14 U/µl) to the cell suspension (1.4 U/ml final concentration of amyloglucosidase).
    For controls: Into the two control tubes containing 400 µg of glycogen (see step 7), add 10 µl of amyloglucosidase to one tube (labeled as positive control) and 10 µl of sterile distilled water to the remaining tube (labeled as negative control).
  11. Incubate the tubes at 57 °C overnight with constant agitation (We seal the tubes with parafilm and incubate the tubes in a hybridization oven.).
  12. The next day, centrifuge the cell suspensions (5,000 x g, 3 min, room temperature) to pellet the cell debris.
  13. Measure the glucose released from the intracellular glycogen using a hexokinase/glucose-6-phosphate dehydrogenase-based glucose assay kit according to manufacturer’s instructions.  
  14. The resulting glucose concentration (mg of glucose in 1 ml of the sample from step 9) is used to calculate the intracellular glycogen content per g of cell wet weight (expressed as mg of glucose per g of cell wet weight).

Representative data

  1. Example: Quantitative determination of intracellular glycogen contents in L. acidophilus parent strain (NCK1909) and isogenic glycogen metabolic mutants.


    Figure 2. Intracellular glycogen contents of mid-log phase cells grown in SDM containing 2% trehalose. In this quantitative assay, glycogen was not detected from both ΔglgA and ΔglgB mutants. This confirms the results from the qualitative detection assay above using iodine-staining method (Figure 1). Data shown represent the mean ± standard deviation for two independent biological replicates. Figure adapted from Goh et al. (2003).

Recipes

  1. Semi-defined medium [(SDM; modified from Kimmel and Roberts (1998)]
    Tween 80 (1 g/L)
    Ammonium citrate (2 g/L)
    Sodium acetate (5 g/L)
    MgSO4.7H2O (0.1 g/L)
    MnSO4 (0.05 g/L)
    K2HPO4 (2 g/L)
    Yeast nitrogen base (5 g/L)
    Casitone (10 g/L)
    Carbohydrate substrate, e.g. glucose or trehalose, 2% (or desired) final concentration
    Note: For trehalose or any heat-labile sugars, prepare a 25-40% (w/v) stock solution, filter-sterilize, and add appropriate volume to autoclaved SDM to a final concentration of 2%. For carbohydrates with poor solubility e.g. raffinose, add 2% (w/v) of the sugar directly into SDM (sterile or non-sterile), stir to solubilize, and filter-sterilized through 0.45 µm filter.
    For solid medium, add agar, 15 g/L
    Sterilize by autoclaving or filter-sterilize with 0.45 µm filter (see above; for broth only)
    Stored at 4 °C

Acknowledgments

The protocols were published in Goh and Klaenhammer (2013), and were developed based on modifications from the previous works of Govons et al. (1969), Kimmel and Roberts (1998), Law et al. (1995), and Parrou and Francois (1997). The funding sources for this work included Danisco/DuPont Nutrition and Health and the North Carolina Agricultural Foundation.

References

  1. Goh, Y. J. and Klaenhammer, T. R. (2013). A functional glycogen biosynthesis pathway in Lactobacillus acidophilus: expression and analysis of the glg operon. Mol Microbiol 89(6): 1187-1200.
  2. Govons, S., Vinopal, R., Ingraham, J. and Preiss, J. (1969). Isolation of mutants of Escherichia coli B altered in their ability to synthesize glycogen. J Bacteriol 97(2): 970-972.
  3. Kimmel, S. A. and Roberts, R. F. (1998). Development of a growth medium suitable for exopolysaccharide production by Lactobacillus delbrueckii ssp. bulgaricus RR. Int J Food Microbiol 40(1-2): 87-92.
  4. Law, J., Buist, G., Haandrikman, A., Kok, J., Venema, G. and Leenhouts, K. (1995). A system to generate chromosomal mutations in Lactococcus lactis which allows fast analysis of targeted genes. J Bacteriol 177(24): 7011-7018.
  5. Parrou, J. L. and Francois, J. (1997). A simplified procedure for a rapid and reliable assay of both glycogen and trehalose in whole yeast cells. Anal Biochem 248(1): 186-188.

简介

糖原,一种可溶性多分支葡萄糖同多糖,由通过α-1,6连接的分支相互连接的α-1,4-连接的葡萄糖残基的链组成。经典的生物合成途径包括磷酸葡萄糖变位酶(Pgm),葡萄糖-1-磷酸腺苷酰转移酶(GlgC或GlgCD),糖原合酶(GlgA)和分支酶(GlgB)。磷酸葡萄糖变位酶将葡萄糖-6-磷酸转化为葡萄糖-1-磷酸,其作为由GlgC或GlgCD催化的ADP-葡萄糖合成的底物。然后,GlgA催化葡萄糖基单位从ADP-葡萄糖向线性α-1,4-葡聚糖的延长链的转移。 GlgB随后切割葡聚糖的部分并且通过α-1,6糖苷键将其连接到现有链中的内部葡萄糖分子以形成糖原结构。糖原分解由糖原磷酸化酶(GlgP)和脱支酶(GlgX)介导,其催化来自非还原末端的葡聚糖链中的α-1,4-葡萄糖基键的顺序磷酸解和由GlgP产生的极限糊精的脱支, 分别。越来越多的研究已经揭示糖原代谢在一些原核生物的多种生理功能中的参与超过合成能量储存化合物的功能。嗜酸乳杆菌 NCFM是第一种证明具有功能性糖原生物合成途径的益生菌乳酸菌,其参与其生长,胆汁耐受性和复杂的碳水化合物代谢(Goh和Klaenhammer,2013)。用于嗜酸乳杆菌(嗜酸乳杆菌)的以下定性(用于检测细胞内糖原)和定量(用于测量细胞内糖原含量)细胞内糖原测定方案从之前的工作(Govons等人,1969; Law等人,1995; Parrou和Francois,1997),并且应当适用于其它乳酸菌以及大多数微生物。

第I部分。用碘染色法定性检测细胞内糖原

材料和试剂

  1. L。 嗜酸菌菌株或所需的细菌菌株
  2. MRS肉汤(Difco)或其他选择的液体生长培养基(在4℃下保存)
  3. 碘溶液(见配方)
  4. 含有2%(w/v)碳水化合物的固体半定义培养基(SDM)(参见配方)

设备

  1. 37℃孵育器
  2. 37℃厌氧培养箱
  3. Pipettor

程序

  1. 接种 L。嗜酸乳杆菌菌株在5ml MRS肉汤中感兴趣并在37℃下在无氧或环境大气条件下生长过夜。
  2. 转移和点5到8微升的每个过夜培养物在固体琼脂生长培养基(我们使用固体SDM补充2%海藻糖或葡萄糖)。如果可能,包括阳性和/或阴性对照菌株 可选:斑点板可以多次重复制备,以提供多次碘染色试验(见步骤4-6)。
  3. 让点样的培养物吸收到琼脂培养基中,并在37℃厌氧培养板36-48小时,直到菌落可见。
  4. 小心地将5ml新鲜制备的碘溶液(0.01M I 2,0.03M KI)转移到具有生长的集落的琼脂板上。
  5. 在室温下用碘溶液孵育细胞30秒至1分钟
  6. 立即用移液器从板中除去碘溶液。含有细胞内糖原的细胞会染成褐色;而缺乏胞内糖原的细胞将呈现黄色或无色 任选:为了增强用于捕获染色菌落图像的显色,可以将染色板(在除去碘溶液后)在黑暗中孵育另外10分钟。
    注意:来自碘溶液的染色剂不是永久性的,并且在约30分钟至1小时后将逐渐消失。

代表数据

  1. 实施例:嗜酸性粒细胞 NCFM和用碘溶液染色的糖原代谢突变体

    图1. l的碘染色。嗜酸性亲本菌株(NCK1909)和在含有2%海藻糖的固体SDM上生长的同基因糖原代谢突变体。 糖原合成途径的缺失突变体Δg1ggA和Δg1ggβ显示为黄色/无色( - ),表明糖原缺乏表型。与亲本菌株一样,糖原降解途径的Δemggp和Δamy突变体染色为棕色(+), 表明它们合成细胞内糖原的能力 不受影响。 glgA ,糖原合酶基因; glgB ,分支酶基因; glgP ,糖原磷酸化酶基因和amy ,假定的α-淀粉酶/脱支酶基因。 Figure from Goh and Klaenhammer(2013) size:10.0pt; line-height:115%; font-family:"">。

食谱

  1. 碘溶液
    0.01 M I <2>
    0.03 M KI
    在每次实验前准备新鲜的
    保持阴暗
  2. 半定义介质(SDM;修改自参考文献3)
    吐温80(1g/L) 柠檬酸铵(2g/L)
    醋酸钠(5g/L)
    MgSO 4·7H 2 O(0.1g/L)。< br /> MnSO 4(0.05g/L)
    HPO <4>(2g/L) 酵母氮基(5g/L)
    中腹(10克/升)
    碳水化合物底物,例如葡萄糖或海藻糖,2%(或期望的)最终浓度。
    注意:对于海藻糖或任何热不稳定的糖,准备25-40%(w/v)储备溶液,过滤灭菌,并向高压灭菌的SDM添加适当体积至最终浓度为2%。 对于溶解性差的碳水化合物。 棉子糖,将2%(w/v)的糖直接加入SDM(无菌或未灭菌)中,搅拌以溶解,并通过0.45μm过滤器过滤灭菌。 对于固体培养基,加入琼脂,15 g/L
    通过高压灭菌或用0.45μm过滤器过滤灭菌(见上文;仅用于肉汤)
    储存在4°C

第二部分。 细胞内糖原的定量测量

材料和试剂

  1. L。 嗜酸杆菌菌株(或其他细菌菌株)
  2. MRS肉汤(储存在4℃)
  3. 磷酸盐缓冲盐水(PBS,pH 7.4)(例如Life Technologies,目录号:10010-023)
  4. 0.25M Na 2 CO 3溶液(用0.45μm过滤器过滤灭菌)
  5. 1M乙酸
  6. 0.2M乙酸钠(pH5.2)(用0.45μm过滤器过滤灭菌)
  7. 无菌蒸馏水
  8. 淀粉葡糖苷酶(10mg/ml,≈0.14U/μl)(Roche Diagnostics,目录号:10102857001)(保存在4℃)
  9. 葡萄糖测定试剂盒(葡萄糖测定试剂)(Sigma-Aldrich,目录号:G3293)(保存在4℃)
  10. 糖原(5-20mg/ml溶液)(例如Life Technologies,目录号AM9510)(储存在-20℃)
  11. 具有2%(w/v)碳水化合物底物(参见Recipes)(或选择的液体生长培养基)的SDM肉汤

设备

  1. 37℃孵育器
  2. 离心(适用于15ml和50ml管; 1,717×g )
  3. 微量离心机(14,549 x g )
  4. Pipettor
  5. 一次性锥形离心管(15 ml和50 ml)
  6. 1.5ml螺旋盖管(无菌)(例如Thermo Fisher Scientific,目录号:02-681-343和02-681-368)(在使用前具有盖的高压灭菌管)
  7. 1.5 ml微量离心管(无菌)
  8. 分析天平
  9. 涡流
  10. 热板
  11. 大玻璃烧杯(例如1000ml Pyrex玻璃烧杯)
  12. 微型离心管浮动架(圆形,适合大型玻璃烧杯)
  13. 温度计
  14. 在配有搅拌器(或杂交炉)的57℃的孵育器
  15. 分光光度计或微量滴定板读数器(能够测量340nm处的吸光度)

程序

  1. 培养 L。 在含有2%(w/v)碳水化合物底物(我们补充了已知诱导糖原生物合成的碳水化合物底物)的SDM肉汤中在37℃下培养至期望的生长期的嗜酸乳杆菌细胞, xg,10分钟,室温)。 细胞沉淀可以在离心后立即加工或储存在-80℃ 注意:
    1. 一般来说,对于早期记录(OD 0.3到0.4),中期日志(OD 〜 0.6),静止 (OD 〜 <1.0> 1.6)和后期稳定期培养物(OD 〜 <2.0) 收获等分试样。 50ml,30ml,20ml和15ml 一次性锥形离心管, 0.04〜0.06g的细胞   湿重。  
    2. 我们准备培养物和细胞沉淀 代表至少两个生物学重复以证明 实验结果的重现性。
  2. 预标签,预称重(带盖),并为每个样品指定每个1.5 ml螺旋盖管。
  3. 将每个细胞沉淀重悬于1ml PBS中,并转移至指定的预称重的1.5ml螺旋管
  4. 离心(14,549×g,1分钟,室温),用移液器除去所有上清液。
  5. 重复步骤4离心至少两次以去除所有上清液,以获得准确的细胞湿重
  6. 用细胞团称重管(带盖)。从重量值中减去空管(有盖;来自上述步骤2)的先前重量,以获得净细胞湿重。
  7. 向细胞沉淀中加入0.25ml的0.25M Na 2 CO 3溶液。通过温和涡旋重悬。
    注意:不要使用移液器重悬细胞沉淀,因为任何数量的细胞粘附在移液管尖端会改变净细胞湿重。
    对于控制:包括2个额外的管(无需预先称重)。在每个管中,加入约400μg(例如20μl的20mg/ml)糖原至0.25ml的0.25M Na 2 CO 3 sub >解决方案。按照步骤8-12。
  8. 孵育细胞悬浮液在95至98℃下4小时(我们将管安全地放置在微量离心管浮动架,并在一个大的烧杯水在热板上加热孵化。在4小时孵育期间轻轻涡旋混合细胞悬液1至2次
  9. 向细胞悬浮液中加入0.15ml 1M乙酸和0.6ml 0.2M乙酸钠(pH5.2),使pH为〜5.2,最终体积为1ml。
  10. 向细胞悬浮液(1.4 U/ml的淀粉葡糖苷酶终浓度)中加入10μl淀粉葡糖苷酶(0.14 U /μl)。
    对于对照:向含有400μg糖原的两个对照管(参见步骤7)中,向其中一管(标记为阳性对照)和10μl无菌蒸馏水中加入10μl淀粉葡糖苷酶(标记为阴性对照) 。
  11. 孵育管在57°C过夜,持续搅拌(我们用石蜡膜密封管,并在杂交炉中孵育管)。
  12. 第二天,离心细胞悬浮液(5,000x g,3分钟,室温)以沉淀细胞碎片。
  13. 使用己糖激酶/基于葡萄糖-6-磷酸脱氢酶的葡萄糖测定试剂盒根据制造商的说明书测量从细胞内糖原释放的葡萄糖。  
  14. 使用所得葡萄糖浓度(1ml来自步骤9的样品中的葡萄糖的mg)来计算每g细胞湿重的细胞内糖原含量(表示为每g细胞湿重的mg葡萄糖)。

代表数据

  1. 实施例:定量测定细胞内糖原含量。 嗜酸性亲本菌株(NCK1909)和同基因糖原代谢突变体

    图2.在含有2%海藻糖的SDM中生长的对数期中期细胞的细胞内糖原含量。在该定量测定中,从ΔggAΔ和Δ< em> glgB 突变体。 这证实了定性检测的结果 测定方法(图1)。 数据显示 代表两个独立生物学的平均值±标准偏差 复制。 图改编自Goh等人。 (2003)。

食谱

  1. 半定义培养基[(SDM;从Kimmel和Roberts(1998)修改]
    吐温80(1g/L) 柠檬酸铵(2g/L)
    醋酸钠(5g/L)
    MgSO 4·7H 2 O(0.1g/L)。< br /> MnSO 4(0.05g/L)
    HPO 4(2g/L) 酵母氮基(5g/L)
    中腹(10克/升)
    碳水化合物底物,例如葡萄糖或海藻糖,2%(或期望的)最终浓度。
    注意:对于海藻糖或任何热不稳定的糖,准备25-40%(w/v)储备溶液,过滤灭菌,并向高压灭菌的SDM添加适当体积至最终浓度为2%。对于溶解性差的碳水化合物。棉子糖,将2%(w/v)的糖直接加入SDM(无菌或未灭菌)中,搅拌以溶解,并通过0.45μm过滤器过滤灭菌。 对于固体培养基,加入琼脂,15 g/L
    通过高压灭菌或用0.45μm过滤器过滤灭菌(见上文;仅用于肉汤)
    储存在4°C

致谢

所述方案在Goh和Klaenhammer(2013)中公开,并且基于来自Govons等人的先前作品(1969),Kimmel和Roberts(1998),Law& al。(1995),和Parrou and Francois(1997)。这项工作的资金来源包括丹尼斯克/杜邦营养与健康和北卡罗来纳农业基金会。

参考文献

  1. Goh,Y. J.和Klaenhammer,T. R.(2013)。 嗜酸乳杆菌中的功能性糖原生物合成途径:表达和分析 glg 操纵子。 Mol Microbiol 89(6):1187-1200。
  2. Govons,S.,Vinopal,R.,Ingraham,J。和Preiss,J.(1969)。 大肠杆菌突变体的分离 b在合成糖原的能力上有所改变。


    97(2):970-972。
  3. Kimmel,S.A。和Roberts,R.F。(1998)。 开发适合于德氏乳杆菌(Lactobacillus delbrueckii)ssp的胞外多糖生产的生长培养基。 bulgaricus RR。 Int J Food Microbiol 40(1-2):87-92。
  4. Law,J.,Buist,G.,Haandrikman,A.,Kok,J.,Venema,G。和Leenhouts,K。(1995)。 在乳酸乳球菌中产生染色体突变的系统,它允许快速分析 靶向基因。细菌 177(24):7011-7018。
  5. Parrou,J.L.and Francois,J。(1997)。 在整个酵母细胞中快速可靠地测定糖原和海藻糖的简化程序。 a> Anal Biochem 248(1):186-188
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引用:Goh, Y. J. and Klaenhammer, T. R. (2014). Intracellular Glycogen Assays. Bio-protocol 4(11): e1148. DOI: 10.21769/BioProtoc.1148.
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