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Determination of (p)ppGpp Levels During Stringent Response in Streptomyces coelicolor by Thin Layer Chromatography
薄层色谱法测定天蓝色链霉菌在应急反应过程中的(p)ppGpp水平   

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Abstract

The stringent response in bacteria is a stress response that is mediated by the signaling molecules guanosine tetraphosphate and pentaphosphate [(p)ppGpp], alarmones that are also directly related to virulence. Therefore, determination of (p)ppGpp levels is crucial for studying the stringent response. The protocol here outlines in a step-wise manner the detection of (p)ppGpp in the bacterium Streptomyces coelicolor during stringent response (Strauch et al., 1991) by thin layer chromatography (TLC). In the example shown here, stringent response is induced by addition of serine hydroxamate, an inhibitor of seryl tRNA synthetase. This protocol was first published in Molecular Microbiology (Sivapragasam and Grove, 2016).

Background

Thin layer chromatography has been used for analyzing (p)ppGpp levels during stringent response in various bacterial species for a long time, and it is a generally accepted method for this purpose. However, previously published protocols only summarized the main concepts, and it was challenging to identify a comprehensive protocol that comprised every step of the procedure. We present here a detailed protocol that has been optimized for studying stringent response in S. coelicolor. Steps unique to handling of S. coelicolor cultures have been identified, and the protocol can therefore be readily adapted to other bacterial species. The method relies on the use of TLC plates that incorporate polyethyleneimine (PEI), which is a strong basic anion exchanger. PEI is therefore the matrix of choice for separation of ionic compounds such as phosphorylated nucleosides (Calderón-Flores et al., 2005; Mechold et al., 2013; Strauch et al., 1991).

Materials and Reagents

  1. Polyethyleneimine (PEI)-cellulose TLC plates (Sigma-Aldrich, catalog number: Z122882 )
  2. 1.5 ml sterile microcentrifuge tubes with cap
  3. Aluminium foil
  4. Streptomyces coelicolor
  5. 32P-labelled orthophosphate (PerkinElmer, catalog number: NEX053H001MC )
    Note: Use of radioactive materials requires institutional authorization.
  6. Serine hydroxamate (Sigma-Aldrich, catalog number: S4503 )
  7. MilliQ quality water (dH2O)
  8. Formic acid (Thermo Fisher Scientific, Fisher Scientific, catalog number: A118P100 )
  9. 100% ethanol at room temperature
  10. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: 60218 )
  11. 3-morpholinopropane-1-sulfonic acid (MOPS) (AMRESCO, catalog number: 0670 )
  12. Sucrose (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP220-1 )
  13. Magnesium sulfate heptahydrate (AMRESCO, catalog number: 0662 )
  14. Dextrose/glucose (VWR, catalog number: BDH9230 )
  15. BactoTM yeast extract (BD, catalog number: 212750 )
  16. BactoTM peptone (BD, catalog number: 211677 )
  17. BactoTM tryptone (BD, catalog number: 211705 )
  18. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
  19. Casamino acids (BD, catalog number: 228830 )
  20. L-histidine monohydrochloride monohydrate (Sigma-Aldrich, catalog number: H8125 )
  21. L-tryptophan (Sigma-Aldrich, catalog number: T0254 )
  22. L-tyrosine (Sigma-Aldrich, catalog number: T3754 )
  23. L-proline (Sigma-Aldrich, catalog number: P0380 )
  24. Springs (stainless) for dispersing the mycelia (e.g., Grainger, catalog number: 1NCH7 ) (specific to dispersal of S. coelicolor)
  25. Dry ice
  26. Guanosine monophosphate (Sigma-Aldrich, catalog number: G8377 ) (optional)
  27. Guanosine triphosphate (Sigma-Aldrich, catalog number: G8877 ) (optional)
  28. Modified MOPS minimal media (for growing S. coelicolor, see Recipes)
  29. 1.5 M KH2PO4 (pH 3.4) (see Recipes)
  30. 13 M formic acid (see Recipes)
  31. 100 mM serine hydroxamate (see Recipes)
  32. ISP1 medium (for growing S. coelicolor, see Recipes)

Equipment

  1. 50 ml glass Erlenmeyer flasks
  2. TLC chamber with lid (Sigma-Aldrich, catalog number: Z126195 )
  3. Phosphorimager (GE Healthcare)
  4. Phosphor screens with storage cassettes (GE Healthcare)
  5. Refrigerated microcentrifuge with speed up to 16,000 x g and rotor that fits 1.5 ml centrifuge tubes
  6. Shaker-incubator with temperature control

Software

  1. ImageQuant software

Procedure

  1. Setting up the culture (Figure 1)
    1. Grow Streptomyces coelicolor in 5 ml ISP1 media in sterile 50 ml glass flask loosely covered with aluminium foil in shaking incubator (180 rpm) at 28 °C overnight.
      Notes:
      1. The incubation temperature is specific to S. coelicolor and may need to be adjusted for other bacterial species.
      2. Include springs in the growth media to disperse the mycelia and reduce clumping.
    2. Add 150 μl of this culture to 600 μl modified MOPS minimal media in sterile 1.5 ml tubes.
    3. Disperse the mycelia well by vortexing and pipetting and transfer 200 μl to two sterile 1.5 ml tubes.
    4. From this step forward, handle samples behind a radiation shield and follow appropriate practices for handling and disposal of radioactive materials. Only certified personnel authorized by the radiation safety office of their respective institutions should handle the radioactive materials.
    5. To both 200 μl culture samples, add 1.7 μl of 32P-labelled orthophosphate (total 30 µCi) and grow the culture in the shaker incubator for 6 h (OD600 0.4-0.6) at 28 °C.
    6. Add 75 μl of 100 mM serine hydroxamate to one of the tubes and add 75 μl of sterile dH2O to the other tube.
    7. Mix well and incubate at 28 °C in the shaker incubator for 15 min.
    8. Centrifuge the samples at 10,000 x g for 5 min at 4 °C, discard the supernatant, add 25 μl of 13 M formic acid to the pellet and resuspend the pellet by vortexing.
      Note: Use of ice-cold formic acid will stop the metabolic activity rapidly. If possible, estimate the weight of the pellet by subtracting the weight of the empty tube from the weight of the tube with the pellet before adding formic acid and adjust the amount of formic acid used to resuspend the pellets if needed. Discard the supernatant immediately, otherwise the pellet will loosen.
    9. For cell lysis, incubate on dry ice for 1 min and on ethanol bath at room temperature for 1 min.
    10. Repeat step 9 four times.
    11. Incubate the samples on ice for 30 min.
    12. Vortex the samples well and centrifuge at 12,000 x g for 10-15 min at 4 °C.
    13. Store the supernatant in 1.5 ml tubes at -20 °C.


      Figure 1. Setting up the culture

  2. Setting up thin layer chromatography (Figure 2)
    1. Pour 1.5 M KH2PO4 (pH 3.4) buffer into a TLC chamber such that it forms a thin layer (~0.5 cm) at the bottom (50 ml used here) and close the lid in order for the buffer vapors to saturate the chamber (at least 30 min).
      Note: Allow sufficient time for the KH2PO4 buffer vapors to saturate the TLC chamber before inserting the PEI-cellulose plate. This process may be facilitated by first cutting filter papers to a size smaller than that of the PEI-cellulose plates and immersing them in the 1.5 M KH2PO4 buffer. Place the soaked paper against the sides of the TLC chamber to let the vapors diffuse evenly throughout the chamber. Saturating the chamber will prevent the mobile phase from evaporating as it migrates up the plate.
    2. Using a pencil, draw a line approximately 1 cm from the bottom of the PEI-cellulose plate and make fine dots on it approximately 2 cm apart to mark spots for sample loading.
      Notes:
      1. If a smaller TLC chamber is used, cut the PEI-cellulose plates into an appropriate size so that it fits inside the chamber with the lid closed. A covered beaker can substitute for a conventional TLC chamber.
      2. Do not poke the brittle PEI cellulose plates while drawing the baseline and dots. Do not use ink-pen, as the dye may migrate with the mobile phase. Avoid touching the surface of the PEI cellulose plate.
      3. Use an amount of buffer that is just sufficient to immerse the bottom of the PEI-cellulose plate below the drawn baseline; the buffer should not touch the sample application line.
    3. Thaw samples (item 13 above) and spot 1 μl of the sample onto the fine dots on the baseline and air dry for 2-4 min. Note down the sample spotted at each point.
      Note: For less concentrated samples and to get a denser spot of the nucleotide species, add increments of 1 μl to the same spot with intermittent air drying. Allowing each aliquot to dry before applying a second aliquot will prevent the diffusion of the sample that would occur if a greater volume were added at once.
    4. Place PEI-cellulose plate in the chamber, which should be placed on a level surface such that the base of the TLC plate is evenly immersed in the buffer.
    5. Close the lid and allow the mobile phase buffer to migrate until it is near (but not above) the top of the plate (about 3 h).
    6. Take out the plate, mark the top of the buffer front with a pencil to identify the distance migrated by the mobile phase, then air dry the plate for 15 min or longer.
    7. Place the plate in a clean cassette exposing the radioactive surface to a phosphor screen for 24 h or more depending upon the intensity of the 32P signal.
    8. Scan the exposed phosphor screen on a phosphorimager and save the image.


      Figure 2. Setting up TLC

Data analysis

  1. Calculate the relative distance of migration (Retention Factor, Rf) of the different phosphorylated nucleoside species from Rf = (Distance travelled by nucleotide species)/(distance travelled by the mobile phase), with both distances measured relative to the baseline drawn on the TLC plate (origin; Figure 3). Rf values obtained under identical conditions may be compared to previously published results for identification of compounds (Calderón-Flores et al., 2005; Mechold et al., 2013). The more highly phosphorylated species are adsorbed more strongly to the PEI on the TLC plate and will migrate a shorter distance. Repeat the experiment at least twice using independent cultures to verify the data.
    Notes:
    1. The actual distance migrated by the nucleotides depends on the distance of migration of the mobile phase, requiring calculation of the Rf value for comparison purposes.
    2. For verification of sample identity, unlabeled nucleotides may be spotted on the plate and their migration identified by exposure to UV-light; the plate is fluorescent under UV-light, and the nucleotides will absorb the light, causing the appearance of a shadow.
    3. Example data analysis and calculation of Rf:
      Distance traveled by mobile phase = 12.2 cm; distance migrated by GTP = 6.5 cm; distance migrated by ppGpp = 2.9 cm; distance migrated by pppGpp = 1.7 cm.
      For GTP, Rf = 6.5 cm/12.2 cm = 0.53
      For ppGpp, Rf = 2.9 cm/12.2 cm = 0.23
      For pppGpp, Rf = 1.7 cm/12.2 cm = 0.13


      Figure 3. Thin layer chromatography of nucleotides (phosphorylated guanosines) extracted from exponential phase cultures of S. coelicolor treated without (first lane) and with 30 mM serine hydroxamate (second lane) for 15 min. The figure is modified from Figure 10A from Sivapragasam and Grove (2016).

  2. Quantitate the density of spots corresponding to different nucleotide species using ImageQuant software. To compare relative levels of ppGpp and pppGpp between different samples, spot intensity of a nucleotide species may be divided by the combined intensity of both ppGpp and pppGpp after correcting the spot intensity for number of phosphates per nucleotide.
    Example data analysis and calculation of relative levels of ppGpp and pppGpp:
    1. Spot density of ppGpp after correcting for number of phosphates: Density of ppGpp obtained from ImageQuant (volume/area) divided by 4 (number of phosphates) = 131.52/4 = 32.88
    2. Spot density of pppGpp after correcting for number of phosphates: Density of pppGpp obtained from ImageQuant (volume/area)/5 (number of phosphates) = 23.32/5 = 4.66
    3. Total corrected spot density of ppGpp and pppGpp: 32.88 + 4.66
    4. Relative level of ppGpp: Corrected spot density of ppGpp/total corrected spot density of ppGpp and pppGpp = 32.88/(32.88 + 4.66) x 100% = 87.6%
    5. Relative level of pppGpp: Corrected spot density of pppGpp/total corrected spot density of ppGpp and pppGpp = 4.66/(32.88 + 4.66) x 100% = 12.4%

Notes

  1. Since it can be challenging to keep S. coelicolor mycelia dispersed during growth, collecting several samples from each culture condition is advised to increase the chances of collecting a comparable number of cells for each condition.
  2. It should be noted that serine hydroxamate does not induce stringent response in all bacterial species.

Recipes

  1. Modified MOPS minimal media (for growing S. coelicolor)
    100 mM MOPS
    10% sucrose
    1% glucose
    2 mM MgSO4
    0.5% yeast extract
    0.2% peptone
    0.15% casamino acids
    30 mM K2HPO4 (as in complete medium)
    Add all the above ingredients in 800 ml of deionized water. Autoclave the buffer
    50 mg of the amino acids histidine, proline, tyrosine and tryptophan (of each amino acid) is added to 100 ml of deionized water. Mix and slightly heat until all the powder dissolves. Filter the solution and add to the buffer prepared above. Make up the volume to 1 L using autoclaved deionized water.
  2. 1.5 M KH2PO4 (pH 3.4)
    20.4 g of KH2PO4 in 60 ml of dH2O
    Adjust the pH to 3.4 using HCl 
    Make up the volume to 100 ml with dH2O
  3. 13 M formic acid
    Slowly add 2.787 ml of formic acid stock solution (88%) to 1.25 ml of dH2
    Make up the volume to 5 ml using dH2O
    Mix well and freeze the solution at -20 °C
  4. 100 mM serine hydroxamate
    Add 12.11 mg of the powder to 1 ml of dH2O and vortex
    Filter sterilize and store at -20 °C
  5. ISP1 medium (for growing S. coelicolor)
    Add 5 g tryptone and 3 g yeast extract to 1 L of dH2O
    Autoclave 

Acknowledgments

This work was supported by the National Science Foundation (MCB-1515349 to A.G.). We would like to thank Nabanita Bhattacharyya for assisting with the photography and all Grove lab members for their suggestions and helpful discussions. This protocol was derived from (Sivapragasam and Grove, 2016) originally published in Molecular Microbiology.

References

  1. Calderon-Flores, A., Du Pont, G., Huerta-Saquero, A., Merchant-Larios, H., Servin-Gonzalez, L. and Duran, S. (2005). The stringent response is required for amino acid and nitrate utilization, nod factor regulation, nodulation, and nitrogen fixation in Rhizobium etli. J Bacteriol 187(15): 5075-5083.
  2. Mechold, U., Potrykus, K., Murphy, H., Murakami, K. S. and Cashel, M. (2013). Differential regulation by ppGpp versus pppGpp in Escherichia coli. Nucleic Acids Res 41(12): 6175-6189.
  3. Sivapragasam, S. and Grove, A. (2016). Streptomyces coelicolor XdhR is a direct target of (p)ppGpp that controls expression of genes encoding xanthine dehydrogenase to promote purine salvage. Mol Microbiol 100(4): 701-718.
  4. Strauch, E., Takano, E., Baylis, H. A. and Bibb, M. J. (1991). The stringent response in Streptomyces coelicolor A3(2). Mol Microbiol 5(2): 289-298.

简介

细菌中的严格反应是由信号分子鸟苷四磷酸和五磷酸[(p)ppGpp]介导的应激反应,它们也与毒力直接相关。因此,(p)ppGpp水平的确定对于研究严格反应至关重要。这里的方案以分步方式概述在严格反应期间细菌链霉菌(Streptomyces coelicolor)中(p)ppGpp的检测(Strauch等人,1991),通过薄层色谱(TLC)。在本文所示的实施例中,通过添加丝氨酸氧肟酸盐(丝氨酸tRNA合成酶的抑制剂)诱导严格反应。该方案首次发表于Molecular Microbiology(Sivapragasam and Grove,2016)。

[背景] 薄层色谱法用于在严格反应期间分析(p)ppGpp水平在各种细菌菌种中长时间使用,并且它是用于该目的的普遍接受的方法。然而,以前发布的协议仅仅总结了主要概念,并且确定包括该过程的每个步骤的综合协议是具有挑战性的。我们在这里提出已经优化用于研究在严格的反应的详细协议。 coelicolor 。处理 S唯一的步骤。天蓝色文化已经被鉴定,并且因此方案可以容易地适应于其他细菌物种。该方法依赖于使用并入作为强碱性阴离子交换剂的聚乙烯亚胺(PEI)的TLC板。因此,PEI是用于分离离子化合物如磷酸化核苷的选择的基质(Calderón-Flores等人,2005; Mechold等人,2013; Strauch et al。,1991)。

材料和试剂

  1. 聚乙烯亚胺(PEI) - 纤维素TLC板(Sigma-Aldrich,目录号:Z122882)
  2. 1.5 ml无菌微量离心管,带盖
  3. 铝箔
  4. 天蓝链霉菌(Streptomyces coelicolor)
  5. 32 P标记的正磷酸盐(PerkinElmer,目录号:NEX053H001MC)
    注意:使用放射性物质需要机构授权。
  6. 丝氨酸氧肟酸盐(Sigma-Aldrich,目录号:S4503)
  7. MilliQ质量水(dH 2 O)
  8. 甲酸(Thermo Fisher Scientific,Fisher Scientific,目录号:A118P100)
  9. 100%乙醇在室温下洗涤
  10. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:60218)
  11. 3-吗啉代丙烷-1-磺酸(MOPS)(AMRESCO,目录号:0670)
  12. 蔗糖(Thermo Fisher Scientific,Fisher Scientific,目录号:BP220-1)
  13. 硫酸镁七水合物(AMRESCO,目录号:0662)
  14. 葡萄糖/葡萄糖(VWR,目录号:BDH9230)
  15. Bacto TM酵母提取物(BD,目录号:212750)
  16. Bacto TM胨蛋白胨(BD,目录号:211677)
  17. Bacto TM 胰蛋白胨(BD,目录号:211705)
  18. 磷酸氢二钾(K 2 HPO 4)(Sigma-Aldrich,目录号:P3786)
  19. 酪蛋白氨基酸(BD,目录号:228830)
  20. L-组氨酸单盐酸盐一水合物(Sigma-Aldrich,目录号:H8125)
  21. L-色氨酸(Sigma-Aldrich,目录号:T0254)
  22. L-酪氨酸(Sigma-Aldrich,目录号:T3754)
  23. L-脯氨酸(Sigma-Aldrich,目录号:P0380)
  24. 用于分散菌丝体的弹簧(不锈钢)(例如,Grainger,目录号:1NCH7)(专用于分散天蓝色素)
  25. 干冰
  26. 单磷酸鸟苷(Sigma-Aldrich,目录号:G8377)(可选)
  27. 鸟苷三磷酸(Sigma-Aldrich,目录号:G8877)(可选)
  28. 修改MOPS基本媒体(用于生长天蓝色,请参阅配方)
  29. 1.5M KH 2 PO 4(pH 3.4)(参见配方)
  30. 13 M甲酸(见配方)
  31. 100 mM丝氨酸氧肟酸盐(参见配方)
  32. ISP1培养基(用于生长天蓝色,参见Recipes)

设备

  1. 50ml玻璃锥形瓶
  2. 带有盖的TLC室(Sigma-Aldrich,目录号:Z126195)
  3. Phosphorimager(GE Healthcare)
  4. 带有储存盒(GE Healthcare)的荧光屏
  5. 速度高达16,000 x g的冷冻微量离心机和适用于1.5 ml离心管的转子
  6. 带温度控制的振荡培养箱

软件

  1. ImageQuant软件

程序

  1. 建立文化(图1)
    1. 在摇动培养箱(180rpm)中,在28℃下,在5ml的ISP1培养基中在无菌的50ml玻璃烧瓶中生长天蓝色链霉菌(Streptomyces coelicolor)。
      注意:
      1. 孵育温度是特异于天蓝色链霉菌的,并且可能需要针对其他细菌种类进行调整。
      2. 在生长介质中加入弹簧以分散菌丝体,减少结块。
    2. 添加150微升此培养物至600微升修改MOPS基本培养基无菌1.5毫升管
    3. 通过涡旋和移液分散菌丝,并转移200微升到两个无菌1.5毫升管。
    4. 从这一步向前,将样品放在辐射屏幕后面,并遵循放射性材料处理和处置的适当做法。只有各自机构的辐射安全办公室授权的认证人员才能处理放射性物质
    5. 向200μl培养物样品中加入1.7μl32P标记的正磷酸盐(总共30μCi),并在摇床培养箱中培养6小时(OD 600 = 0.4) -0.6)。
    6. 向其中一个管中加入75μl100mM丝氨酸异羟肟酸盐,并向另一个管中加入75μl无菌dH 2 O。
    7. 充分混合并在28℃下在摇床培养箱中孵育15分钟
    8. 在4℃下以10,000×g离心样品5分钟,弃去上清液,向沉淀中加入25μl13M甲酸,并通过涡旋重悬沉淀。
      注意:使用冰冷的甲酸会迅速停止代谢活动。如果可能,通过在加入甲酸之前从含有颗粒的管的重量减去空管的重量并且如果需要调整用于重悬颗粒的甲酸的量来估计颗粒的重量。立即丢弃上清液,否则沉淀会松动。
    9. 对于细胞裂解,在干冰上孵育1分钟,并在乙醇浴中在室温下孵育1分钟。
    10. 重复步骤9四次。
    11. 在冰上孵育样品30分钟。
    12. 涡旋样品,并在4℃下以12,000×g离心10-15分钟。
    13. 将上清液储存在-20℃的1.5ml试管中

      图1.设置文化

  2. 设置薄层色谱(图2)
    1. 将1.5M KH 2 PO 4(pH 3.4)缓冲液倒入TLC室中,使得其在底部形成薄层(?0.5cm)(在此使用50ml) ),并关闭盖子,以使缓冲液蒸汽使室内饱和(至少30分钟) 注意:在插入PEI-纤维素板之前,需要足够的时间使KH sub 2 PO 4缓冲液蒸汽饱和TLC室。可以通过首先将滤纸切割成小于PEI-纤维素板的尺寸并将它们浸入1.5M KH 2 PO 4缓冲液中来促进该过程。将浸泡的纸张靠着TLC室的侧面,让蒸气均匀地扩散到整个室中。饱和腔室将防止流动相随着其在板上迁移而蒸发。
    2. 使用铅笔,从PEI-纤维素板的底部画大约1cm的线,并在其上形成大约2cm的细小的点以标记用于样品加载的点。
      注意:
      1. 如果使用较小的TLC室,将PEI-纤维素板切成适当的尺寸,使其在盖关闭的情况下适合室内。盖式烧杯可代替常规TLC室。
      2. 在绘制基线和点时不要戳脆性PEI纤维素板。不要使用墨水笔,因为染料可能与流动相一起迁移。避免接触PEI纤维素板的表面。
      3. 使用刚好足以将PEI-纤维素板的底部浸入所绘制的基线以下的缓冲剂量;缓冲区不应接触样品应用程序行。
    3. 解冻样品(上面的项目13),将1μl样品点在基线上的细小点上,并风干2-4分钟。记下在每个点上点样的样品。
      注意:对于较低浓度的样品和获得核苷酸物质的更密集的斑点,在间歇性空气干燥的情况下向相同斑点添加1μl的增量。允许每个等分试样在应用第二等分试样之前干燥将防止样品扩散,如果一次添加更大的体积则会发生。
    4. 将PEI-纤维素板放置在室中,其应该放置在水平表面上,使得TLC板的基底均匀地浸没在缓冲液中。
    5. 关闭盖子,让流动相缓冲液迁移,直到它靠近(但不是在板的顶部)(约3小时)的顶部。
    6. 取出板,用铅笔标记缓冲液前部的顶部,以确定流动相迁移的距离,然后将板空气干燥15分钟或更长时间。
    7. 将板放置在干净的盒中,将放射性表面暴露于荧光屏24小时或更长时间,这取决于32 P信号的强度。
    8. 在phosphorimager上扫描曝光的荧光屏,保存图像

      图2.设置TLC

数据分析

  1. 计算不同磷酸化核苷物质的迁移(保留因子,Rf)相对于Rf =(核苷酸物质行进的距离)/(流动相行进的距离)的相对距离,两个距离均相对于TLC上绘制的基线板(来源;图3)。在相同条件下获得的Rf值可以与先前公布的化合物鉴定结果进行比较(Calderón-Flores等人,2005; Mechold等人,2013)。更高度磷酸化的物质更强地吸附到TLC板上的PEI上,并将迁移更短的距离。使用独立培养物重复实验至少两次以验证数据 注意:
    1. 核苷酸迁移的实际距离取决于流动相的迁移距离,为了比较目的,需要计算Rf值。
    2. 为了验证样品的身份,可以将未标记的核苷酸点在板上,并通过暴露于UV光来鉴定它们的迁移;该板在紫外光下是荧光的,并且核苷酸将吸收光,导致阴影的出现。
    3. 示例数据分析和Rf的计算:
      流动相行进距离= 12.2cm;由GTP迁移的距离= 6.5cm;距离由ppGpp = 2.9cm迁移;由pppGpp迁移的距离= 1.7厘米。
      对于GTP,Rf = 6.5cm/12.2cm = 0.53
      对于ppGpp,Rf = 2.9cm/12.2cm = 0.23
      对于pppGpp,Rf = 1.7cm/12.2cm = 0.13


      图3.从S的指数期培养物中提取的核苷酸(磷酸化鸟苷)的薄层色谱。 (第一泳道)和30mM丝氨酸氧肟酸盐(第二泳道)处理15分钟后的天蓝色处理。该图从Sivapragasam和Grove的图10A修改(2016)。
  2. 使用ImageQuant软件定量对应于不同核苷酸物质的斑点的密度。为了比较不同样品之间的ppGpp和pppGpp的相对水平,在校正每个核苷酸的磷酸酯数目的斑点强度之后,核苷酸物质的斑点强度可以除以ppGpp和pppGpp的组合强度。
    示例数据分析和ppGpp和pppGpp的相对水平的计算:
    1. 校正磷酸盐数量后的ppGpp的点密度:从ImageQuant获得的ppGpp的密度(体积/面积)除以4(磷酸盐数)= 131.52/4 = 32.88
    2. 校正磷酸盐数量后的pppGpp的点密度:从ImageQuant获得的pppGpp的密度(体积/面积)/5(磷酸盐数)= 23.32/5 = 4.66
    3. ppGpp和pppGpp的总校正点密度:32.88 + 4.66
    4. ppGpp的相对水平:ppGpp的校正点密度/ppGpp和pppGpp的总校正点密度= 32.88 /(32.88 + 4.66)×100%= 87.6%
    5. pppGpp的相对水平:pppGpp的校正点密度/ppGpp和pppGpp的总校正点密度= 4.66 /(32.88 + 4.66)×100%= 12.4%

笔记

  1. 因为保持 S是有挑战性的。 coelicolor 菌丝体在生长期间分散,从每个培养条件收集几个样品,建议增加收集每个条件相当数量的细胞的机会。
  2. 应当注意,丝氨酸氧肟酸盐不会在所有细菌种类中诱导严格反应

食谱

  1. 修改的MOPS基本媒体(用于生长天蓝色)
    100 mM MOPS
    10%蔗糖 1%葡萄糖 2mM MgSO 4 0.5%酵母提取物
    0.2%蛋白胨 0.15%酪蛋白氨基酸 30mM K 2 HPO 4(如在完全培养基中)
    将所有上述成分加入800ml去离子水中。高压灭菌缓冲液
    将50mg氨基酸组氨酸,脯氨酸,酪氨酸和色氨酸(每种氨基酸)加入到100ml去离子水中。混合并轻微加热,直到所有粉末溶解。过滤溶液并加入到上面制备的缓冲液中。使用高压灭菌的去离子水将体积补至1升。
  2. 1.5M KH 2 PO 4(pH 3.4) 在60ml的dH 2 O中的20.4g的KH 2 PO 4溶液。
    使用HCl<
    将pH调节至3.4 用dH 2 O 2/
    将体积补至100ml
  3. 13 M甲酸
    将2.787ml甲酸储备溶液(88%)缓慢加入到1.25ml dH 2 O 2中
    使用dH 2 O
  4. 100mM丝氨酸氧肟酸酯 将12.11mg粉末加入1ml dH 2 O中并涡旋
    过滤灭菌并储存在-20℃下
  5. ISP1培养基(用于生长天蓝色)
    将5g胰蛋白胨和3g酵母提取物加入1L dH 2 O 2中 高压灭菌器

致谢

这项工作是由国家科学基金会(MCB-1515349到A.G.)支持。我们要感谢Nabanita Bhattacharyya协助摄影和所有Grove实验室成员的建议和有益的讨论。该协议衍生自最初在Molecular Microbiology中发表的(Sivapragasam和Grove,2016)。

参考文献

  1. Calderon-Flores,A.,Du Pont,G.,Huerta-Saquero,A.,Merchant-Larios,H.,Servin-Gonzalez,L.and Duran,S。(2005)。  严格的反应是氨基酸和硝酸盐利用,点头因子调节,结瘤和氮需要的固定在根瘤菌(Rhizobium etli)中。 187(15):5075-5083。
  2. Mechold,U.,Potrykus,K.,Murphy,H.,Murakami,KS和Cashel,M.(2013)。  通过ppGpp与pppGpp在大肠杆菌中的差异调节核酸研究41(12) :6175-6189。
  3. Sivapragasam,S.和Grove,A.(2016)。  < em>天蓝链霉菌 XdhR是(p)ppGpp的直接靶标,其控制编码黄嘌呤脱氢酶的基因的表达以促进嘌呤补救。   100(4):701-718。
  4. Strauch,E.,Takano,E.,Baylis,HA和Bibb,MJ(1991)。  A3(2)中的严格反应。 5(2):289-298。
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引用:Sivapragasam, S. and Grove, A. (2016). Determination of (p)ppGpp Levels During Stringent Response in Streptomyces coelicolor by Thin Layer Chromatography. Bio-protocol 6(21): e1995. DOI: 10.21769/BioProtoc.1995.
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