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Pseudomonas aeruginosa is a Gram negative bacterium. Separating the cell envelope compartments enables proteins to be localized to confirm where in the cell they function. Cell fractionation can also provide a first step in a protein purification strategy (Williams et al., 1998). This protocol has been designed to obtain the different fractions of P. aeruginosa, namely the inner membrane, outer membrane, cytoplasmic and periplasmic compartments. Specific detection of the arginine specific autotransporter (AaaA) (Luckett et al., 2012) in the outer membrane of P. aeruginosa has been performed using this protocol.

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Cell Fractionation of Pseudomonas aeruginosa
绿脓杆菌的细胞分级分离

微生物学 > 微生物细胞生物学 > 细胞器分离
作者: Esteban Paredes-Osses
Esteban Paredes-OssesAffiliation: Centre for Biomolecular Sciences, University of Nottingham, Nottingham, UK
For correspondence: mrxeap@nottingham.ac.uk
Bio-protocol author page: a872
 and Kim R. Hardie
Kim R. HardieAffiliation: Centre for Biomolecular Sciences, University of Nottingham, Nottingham, UK
For correspondence: kim.hardie@nottingham.ac.uk
Bio-protocol author page: a873
Vol 3, Iss 19, 10/5/2013, 3778 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.922

[Abstract] Pseudomonas aeruginosa is a Gram negative bacterium. Separating the cell envelope compartments enables proteins to be localized to confirm where in the cell they function. Cell fractionation can also provide a first step in a protein purification strategy (Williams et al., 1998). This protocol has been designed to obtain the different fractions of P. aeruginosa, namely the inner membrane, outer membrane, cytoplasmic and periplasmic compartments. Specific detection of the arginine specific autotransporter (AaaA) (Luckett et al., 2012) in the outer membrane of P. aeruginosa has been performed using this protocol.

[Abstract]

Materials and Reagents

  1. P. aeruginosa strain (In this experiment, AaaA deficient mutant strains are used). The strain was bearing the pME6032 shuttle expression vector (Heeb et al., 2002). This vector is IPTG-inducible and has very good stability in Pseudomonas.
  2. Polyclonal antibody anti-AaaA (rabbit) (not commercial available) (Luckett et al., 2012)
  3. Anti-Rabbit IgG (whole molecule)–Peroxidase antibody produced in goat (Sigma-Aldrich, catalog number: A6154 )
  4. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, catalog number: I5502 )
  5. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS )
  6. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  7. Luria Bertani (LB) (Difco, catalog number: 244620 )
  8. Autoclaved sterilized Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417 )
  9. Sucrose (Sigma-Aldrich, catalog number: S7903 )
  10. Sodium Lauryl Sarcoscine (SLS) (Sigma-Aldrich, catalog number: L5125 )
  11. Magnesium Sulphate (Sigma-Aldrich, catalog number: 63139 )
  12. MgCl2 (Sigma-Aldrich, catalog number: M2393 )
  13. DNAase I (Sigma-Aldrich, catalog number: D4527 )
  14. RNAase A (Sigma-Aldrich, catalog number: R5503 )
  15. 100% Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T6399 )
  16. Acetone (Sigma-Aldrich, catalog number: 179124 )
  17. NaOH (Sigma-Aldrich, catalog number: 221465 )
  18. Tris(hydroxymethyl) aminomethane (Sigma-Aldrich, catalog number: 252859 )
  19. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779 )
  20. Sodium Dodecyl Sulphate (SDS) (Sigma-Aldrich, catalog number: L4509 )
  21. Bromophenol Blue (Sigma-Aldrich, catalog number: B0126 )
  22. Glycerol (Sigma-Aldrich, catalog number: G8773 )
  23. 4x Loading buffer (see Recipes)

Equipment

  1. French Pressure cell (Manufactured by Amicon and supplied by Thermo Fisher Scientific)
  2. 50 ml Falcon tube (BD Biosciences, Falcon®, catalog number: 352070 ) (Supplied by Scientific Laboratory Supplies)
  3. Eppendorf® Safe-Lock micro test tubes (Eppendorf, catalog number: 0030121023 ) (Supplied by Scientific Laboratory Supplies)
  4. Microfuge, larger volume centrifuge and high speed centrifuge (Beckman Coulter, model: Allegra X-22R Centrifuge )
  5. 1 ml pipettman
  6. W380 sonicator (Ultrasonics)

Procedure

  1. Set up overnight cultures for P. aeruginosa strains of interest from a single colony. Conditions: 10 ml LB broth in 50 ml Falcon tube at 37 °C shaking at 200 rpm.
  2. Next day, set up fresh cultures from the overnight cultures in new LB broth using a 1:100 ratio dilution, and grow at 37 °C shaking at 200 rpm until the exponential growth phase (0.4 - 0.5 OD600 nm).
  3. When the exponential phase is reached, add IPTG (1 mM final concentration) for 1 h to induce the production of the protein of interest if required.
  4. After the hour of induction, centrifuge the culture at 3,000 x g for 5 min at 4 °C. Discard the supernatant and gently resuspend the pellet in 10 ml of room temperature PBS using a 1 ml pipettman.
  5. Repeat step 4 twice so that the cells are washed three times in total.
  6. Resuspend the final pellet in 10 ml PBS solution.
  7. Check OD600 nm and dilute to an OD600 nm of 1.0 with PBS solution. This will be referred to as the ‘main solution’.
  8. Take 1 ml of the main solution to prepare the whole cell fraction. Centrifuge at 3,700 x g for 5 min in an Eppendorf tube and resuspend in 200 μl of 1x loading buffer, then sonicate for 10 seconds in W380 sonicator using the following settings: duty cycle, 40%; output control, 41/2 position and cycle time, continuous and boil at 100 °C for 10 minutes. The supernatant from the centrifugation can be kept on ice as a sample from the supernatant if secreted proteins are also to be analyzed.
  9. Take another 1 ml of the main solution to prepare the periplasmic and cytoplasmic fractions. Centrifuge at 3,700 x g for 2 min at room temperature in an Eppendorf tube.
  10. Wash with 300 μl of 25 mM Tris pH 7.4 three times by centrifuging at 3,700 x g for 5 min (room temperature) and gently resuspending the pellet with a pipettman.
  11. After the last centrifugation, the pellet needs to be resuspended in 50 μl of 25 mM Tris pH 7.4, 1 μl of 0.1 M EDTA and 50 μl of 40% w/w sucrose in 25 mM Tris pH 7.4.
  12. Mix the sample gently at room temperature for 10 min.
  13. Centrifuge at 3,700 x g for 5 min (room temperature), discard supernatant and resuspend pellet in 100 μl of ice cold 0.5 mM Magnesium Sulphate.
  14. Incubate on ice for 10 min, gently inverting occasionally to mix.
  15. Centrifuge for 5 min at 10,300 x g in microfuge (4 °C).
  16. The supernatant of the sample has the periplasmic fraction, and should be stored on ice.
  17. The pellet needs to be resuspended in 600 μl of 10 mM Tris pH 7.4 containing 20 μg/ml PMSF.
  18. The sample from step 17 should be frozen and thawed three times on dry ice.
  19. Add 19.9 μl MgCl2 (1 M) and 1.2 μl DNase I (1 mg/ml) to the sample from step 18. Mix by inversion.
  20. Incubate at 37 °C for 15 min.
  21. Centrifuge for 15 min at 10,300 x g, and the supernatant contains the cytoplasmic fraction, which should be stored on ice.
  22. The rest of main solution (approximately 5 ml) is used to get the membrane fractions.
  23. Centrifuge at 3,000 x g for 10 min at 4 °C, discard supernatant and resuspend pellet in 3 ml 20 mM Tris pH 7.4 containing 0.1 mg/ml DNase and 0.1 mg/ml RNase.
  24. Pass through the French Press three times at 16,000 lb/in, on ice.
  25. Centrifuge 3,000 x g for 20 min at 4 °C to remove unlysed cells.
  26. The resultant supernatant is transferred to a fresh tube, and centrifuged at 30,000 x g for 40 minutes at 4 °C.
  27. Discard the supernatant and resuspend the pellet in 200 μl 20 mM Tris pH 7.4 containing 0.7% (w/v) SLS.
  28. Incubate at 4 °C for 25 minutes and centrifuge at 30,000 x g for 40 minutes at 4 °C.
  29. The supernatant contains the inner membrane fraction, and should be stored in a fresh tube on ice.
  30. The pellet needs to be resuspended in 200 μl 20 mM Tris pH 7.4 and contains the outer membrane.
    Note: The samples (periplasmic, inner membrane, cytoplasmic outer membrane fractions and culture supernatant) need to be subjected to TCA precipitation (Cooksley et al., 2003) to concentrate the proteins and remove residual detergents and salts. To TCA precipitate the proteins add TCA (from a 100% stock solution kept at 4 °C in the dark) to a final concentration of 10% v/v. Incubate the samples on ice for 30 minutes and centrifuge for 15 minutes at top speed in a microfuge at room temperature or 4 °C. Discard the supernatant carefully without disturbing the small and fragile pellet. Add 500 μl of ice cold acetone to the pellet, and centrifuge for 5 minutes in the microfuge. The supernatant is discarded again and the pellet air dried for 15 min. Finally, the pellet is resuspended in 20 μl of 50 mM NaOH plus 180 μl loading buffer 1x, sonicated for 10 sec, and boiled for 10 min.
  31. The proteins can then be visualized by SDS-PAGE or Immunoblotting (Cooksley et al., 2003) as seen in Figure 1. It is good practice to have available control antibodies against proteins known to be localized to each of the cell fractions analyzed to verify that there is no cross contamination.


    Figure 1. AaaA is found in the outer membrane fraction of P. aeruginosa. Cell fractionation was performed as described, followed by immunoblotting with specific anti-AaaA sera as described previously (Luckett et al., 2012). Whole cell (W), cytoplasmic (C), Inner Membrane (IM), Perplasmic (P) and outer membrane (OM) fractions are shown, and the expected position of AaaA is indicated by the arrow. The P. aeruginosa strains analyzed were the AaaA deficient mutant containing the plasmid pME6032 [PA01 delta aaaA (pME6032)], or a derivative of pME6032 with an insert encoding wild type AaaA [PA01 delta aaaA (pME6032::aaaA)] or a site directed mutant of AaaA replacing alanine with the residue at either position G89 [PA01 delta aaaA (pME6032:: aaaA G89A)] or E149 [PA01 delta aaaA (pME6032::aaaA E149A)]. To verify that the fractionation has occurred correctly, it is important to strip the blots and reprobe with antibodies specific for proteins that are only found in the fractions of interest as was shown in Luckett et al. (2012).

Recipes

  1. 4x Loading buffer
    200 mM Tris(hydroxymethyl) aminomethane (pH 6.8)
    800 mM Dithiothreitol (DTT)
    8% Sodium Dodecyl Sulphate (SDS)
    0.4% w/v Bromophenol Blue
    40% v/v Glycerol

Acknowledgments

We acknowledge the use of this protocol in Luckett et al. (2012). We would like to thank the Chilean Government for financially supporting Esteban Paredes. We also thank Prof Miguel Camara for critical analysis of our work and everyone else in the Bacteriology Laboratories of the Centre of Biomolecular Sciences, University of Nottingham for helpful discussion about our work.

References

  1. Cooksley, C., Jenks, P. J., Green, A., Cockayne, A., Logan, R. P. and Hardie, K. R. (2003). NapA protects Helicobacter pylori from oxidative stress damage, and its production is influenced by the ferric uptake regulator. J Med Microbiol 52(Pt 6): 461-469.
  2. Heeb, S., Blumer, C. and Hass, D. (2002). Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescent CHA0. J Bacteriol 184(4):1046-56. 
  3. Methods in Microbiology: Bacterial Pathogenesis, Academic Press. (1998). Eds: Paul Williams, Peter Williams, George Salmond, Julian Ketley Chapter 6.1: p185-191.
  4. Luckett, J. C., Darch, O., Watters, C., Abuoun, M., Wright, V., Paredes-Osses, E., Ward, J., Goto, H., Heeb, S., Pommier, S., Rumbaugh, K. P., Camara, M. and Hardie, K. R. (2012). A novel virulence strategy for Pseudomonas aeruginosa mediated by an autotransporter with arginine-specific aminopeptidase activity. PLoS Pathog 8(8): e1002854. 

材料和试剂

  1. p。 铜绿假单胞菌菌株(在本实验中,使用了AaaA 缺陷型突变菌株)。 菌株携带pME6032穿梭表达载体(Heeb等人,2002)。 该载体是IPTG诱导的,并且在假单胞菌中具有非常好的稳定性。
  2. 多克隆抗体抗AaaA(兔)(不可商购)(Luckett等人,2012)
  3. 山羊中生产的抗兔IgG(完整分子) - 过氧化物酶抗体(Sigma-Aldrich,目录号:A6154)
  4. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Sigma-Aldrich,目录号:I5502)
  5. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:EDS)
  6. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  7. Luria Bertani(LB)(Difco,目录号:244620)
  8. 高压灭菌的磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:P4417)
  9. 蔗糖(Sigma-Aldrich,目录号:S7903)
  10. 月桂基Sarcoscine(SLS)(Sigma-Aldrich,目录号:L5125)
  11. 硫酸镁(Sigma-Aldrich,目录号:63139)
  12. MgCl 2(Sigma-Aldrich,目录号:M2393)
  13. DNA酶I(Sigma-Aldrich,目录号:D4527)
  14. RNA酶A(Sigma-Aldrich,目录号:R5503)
  15. 100%三氯乙酸(TCA)(Sigma-Aldrich,目录号:T6399)
  16. 丙酮(Sigma-Aldrich,目录号:179124)
  17. NaOH(Sigma-Aldrich,目录号:221465)
  18. 三(羟甲基)氨基甲烷(Sigma-Aldrich,目录号:252859)
  19. 二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D9779)
  20. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L4509)
  21. 溴酚蓝(Sigma-Aldrich,目录号:B0126)
  22. 甘油(Sigma-Aldrich,目录号:G8773)
  23. 4x加载缓冲区(参见配方)

设备

  1. French压力室(由Amicon制造,由Thermo Fisher Scientific提供)
  2. 50ml Falcon管(BD Biosciences,Falcon ,目录号:352070)(由Scientific Laboratory Supplies提供)
  3. Eppendorf Safe-Lock微量试管(Eppendorf,目录号:0030121023)(由Scientific Laboratory Supplies提供)
  4. Microfuge,更大体积的离心机和高速离心机(Beckman Coulter,型号:Allegra X-22R离心机)
  5. 1 ml pipettman
  6. W380超声波仪(超声波)

程序

  1. 对来自单个菌落的感兴趣的绿脓杆菌菌株建立过夜培养物。 条件:10ml LB肉汤在50ml Falcon管中,37℃,200rpm振荡
  2. 第二天,使用1:100比例稀释,在新LB肉汤中设置来自过夜培养物的新鲜培养物,并在37℃下以200rpm振荡生长,直到指数生长期(0.4-0.5OD 600/> nm)。
  3. 当达到指数期时,如果需要,加入IPTG(1mM终浓度)1小时以诱导感兴趣的蛋白质的产生。
  4. 诱导1小时后,在4℃下以3,000xg离心培养物5分钟。 弃去上清液,并使用1ml pipettman轻轻地将沉淀重悬于10ml室温PBS中。
  5. 重复步骤4两次,使细胞总共洗涤三次。
  6. 将最终沉淀重悬于10ml PBS溶液中。
  7. 检查OD 600nm并用PBS溶液稀释至OD 600nm为1.0。这将被称为"主要解决方案"。
  8. 取1ml的主溶液以制备全细胞级分。在Eppendorf管中以3,700×g离心5分钟,并重悬于200μl1x加样缓冲液中,然后在W380超声波仪中使用以下设置超声处理10秒:占空比,40%;输出控制,41/2位置和循环时间,连续并在100℃煮沸10分钟。如果还要分析分泌的蛋白质,则来自离心的上清液可以保存在冰上作为来自上清液的样品
  9. 取另一个1ml的主要溶液来制备周质和细胞质部分。在Eppendorf管中在室温下以3,700×g离心2分钟。
  10. 通过在3,700×g离心5分钟(室温),用300μl25mM Tris pH 7.4洗涤三次,并用移液管轻轻地重悬沉淀。
  11. 在最后一次离心后,沉淀需要重悬于50μl25mM Tris pH7.4,1μl0.1M EDTA和50μl40%w/w蔗糖的25mM Tris pH 7.4中。
  12. 在室温下轻轻混合样品10分钟。
  13. 以3,700×g离心5分钟(室温),弃去上清液并将沉淀重悬在100μl冰冷的0.5mM硫酸镁中。
  14. 在冰上孵育10分钟,轻轻颠倒偶尔混合。
  15. 在微量离心机(4℃)中以10,300×g离心5分钟。
  16. 样品的上清液具有周质级分,并且应该储存在冰上。
  17. 沉淀需要重悬在600μl含有20μg/ml PMSF的10mM Tris pH7.4中
  18. 来自步骤17的样品应该在干冰上冷冻和解冻三次。
  19. 向步骤18的样品中加入19.9μlMgCl 2(1M)和1.2μlDNA酶I(1mg/ml)。通过倒置混合。
  20. 在37℃孵育15分钟。
  21. 在10,300×g离心15分钟,上清液含有细胞质级分,其应储存在冰上。
  22. 剩余的主溶液(约5ml)用于得到膜级分
  23. 在4℃下以3,000xg离心10分钟,弃去上清液并在含有0.1mg/ml DNA酶和0.1mg/ml核糖核酸酶的3ml 20mM Tris pH7.4中重悬沉淀。
  24. 在冰上以16,000磅/英寸的速度通过法国报纸三次。
  25. 在4℃下离心3,000xg,20分钟以除去未裂解的细胞。
  26. 将所得上清液转移至新鲜试管中,并在4℃以30,000xg离心40分钟。
  27. 弃去上清液并将沉淀物重悬在200μl含有0.7%(w/v)SLS的20mM Tris pH 7.4中。
  28. 在4℃下孵育25分钟,在4℃下以30,000×g离心40分钟。
  29. 上清液含有内膜级分,并应储存在冰上的新鲜管中
  30. 沉淀需要重悬在200μl20mM Tris pH7.4中,并含有外膜。
    注意:样品(周质,内膜,胞质外膜级分和培养物上清液)需要进行TCA沉淀(Cooksley等人,2003)以浓缩蛋白质并除去残留的去污剂和盐。为了TCA沉淀,将TCA(来自在黑暗中保持在4℃下的100%储备溶液)加至10%v/v的终浓度。在冰上孵育样品30分钟,并在微量离心机中在室温或4℃下以最高速度离心15分钟。小心弃去上清液,不要打扰小而脆的颗粒。加入500微升冰冷的丙酮到沉淀,并在微型离心机离心5分钟。再次弃去上清液,将沉淀物空气干燥15分钟。最后,将沉淀重悬于20μl50mM NaOH加180μl上样缓冲液1x中,超声处理10秒,煮沸10分钟。
  31. 然后可以通过SDS-PAGE或免疫印迹(Cooksley等人,2003)使蛋白质显色,如图1所示。优良实践是具有针对已知位于每个的分析的细胞级分以验证没有交叉污染。


    图1. AaaA存在于 P的外膜部分。铜绿假单胞菌 。。如所述进行细胞分级分离,然后如前所述用特异性抗AaaA血清进行免疫印迹(Luckett等,2012 )。显示了全细胞(W),细胞质(C),内膜(IM),全细胞(P)和外膜(OM)级分,AaaA的预期位置由箭头指示。 p。分析的铜绿假单胞菌菌株是含有质粒pME6032 [PA01 delta aaaA(pME6032)]或含有编码野生型插入片段的插入片段的pME6032的衍生物的AaaA缺陷型突变体AaaA [PA01δ aaaA (pME6032 :: aaaA )]或AaaA的位点定向突变体替换丙氨酸与位置G89 [PA01δ(pME6032 :: aaaA G89A)]或E149 [PA01 delta aaaA (pME6032 :: emaA E149A)]。为了验证分馏是否正确发生,重要的是剥离印迹并用对仅在感兴趣的级分中发现的蛋白质特异性的抗体进行再次探测,如Luckett等人(2012)所示。 。

食谱

  1. 4x加载缓冲区
    200mM三(羟甲基)氨基甲烷(pH6.8) 800mM二硫苏糖醇(DTT) 8%十二烷基硫酸钠(SDS)
    0.4%w/v溴酚蓝
    40%v/v甘油

致谢

我们承认这个协议在Luckett等人(2012)中的使用。我们要感谢智利政府对Esteban Paredes的资助。我们还感谢Miguel Camara教授对我们的工作和诺丁汉大学生物分子科学中心细菌学实验室的所有其他人进行批判性分析,对我们的工作进行了有益的讨论。

参考文献

  1. Cooksley,C.,Jenks,P.J.,Green,A.,Cockayne,A.,Logan,R.P.and Hardie,K.R。(2003)。 NapA保护幽门螺杆菌免受氧化应激损伤,其生产受铁摄取调节剂的影响。/a> J Med Microbiol 52(Pt 6):461-469。
  2. Heeb,S.,Blumer,C。和Hass,D。(2002)。 (2002).Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescent CHA0.J Bacteriol 184(4) 1046-56。靶向作用于在假单胞菌荧光CHA0中的外部产物形成的GacA/RsmA依赖性全局控制中作为介质的调节性RNA。细菌 184(4) :1046-56。 
  3. 微生物方法 :细菌病理学,Academic Press。 (1998)。 Eds:Paul Williams,Peter Williams,George Salmond,Julian Ketley第6.1章:p185-191。
  4. Luckett,JC,Darch,O.,Watters,C.,Abuoun,M.,Wright,V.,Paredes-Osses,E.,Ward,J.,Goto,H.,Heeb,S.,Pommier, ,Rumbaugh,KP,Camara,M。和Hardie,KR(2012)。 一种针对绿脓杆菌的新型毒力策略 由精氨酸 - 具体的氨基肽酶活性。

    8(8):e1002854。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Paredes-Osses, E. and Hardie, K. R. (2013). Cell Fractionation of Pseudomonas aeruginosa. Bio-protocol 3(19): e922. DOI: 10.21769/BioProtoc.922; Full Text
  2. Luckett, J. C., Darch, O., Watters, C., Abuoun, M., Wright, V., Paredes-Osses, E., Ward, J., Goto, H., Heeb, S., Pommier, S., Rumbaugh, K. P., Camara, M. and Hardie, K. R. (2012). A novel virulence strategy for Pseudomonas aeruginosa mediated by an autotransporter with arginine-specific aminopeptidase activity. PLoS Pathog 8(8): e1002854.




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    我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

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


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