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Induction, Isolation and Counting of Akinetes in Aphanizomenon ovalisporum
束丝藻属中原垣孢子的诱导、分离和计数

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Abstract

Akinetes are spore-like resting (dormant) cells formed by strains of filamentous cyanobacteria for surviving long periods of unfavorable conditions. During deprivation for potassium, vegetative photosynthetic cells along the filaments of the cyanobacterium Aphanizomenon ovalisporum (A. ovlisporum) (strain ILC-164) differentiate into akinetes. Akinetes are larger than vegetative cell, have a thick wall, accumulate storage compounds (cyanophycine, glycogen, lipids) and excess of DNA (Sukenik et al., 2015; Sukenik et al., 2007; Maldener et al., 2014). Differences in structure and composition between akinetes and vegetative cells allow separation and isolation of akinetes. Akinetes isolated by the described protocol can be utilized for protein analysis, measurements of metabolic activities, fluorescence in situ hybridization (FISH) studies and more.

Materials and Reagents

  1. Erlenmeyer flasks of appropriate volume
  2. 50 ml tubes (SARSTEDT AG & Co, catalog number: 62.547.004 )
  3. 250 ml centrifuge bottle (TermoFisher SCIENTIFIC, catalog number: 3141-0250 )
  4. Aphanizomenon ovalisporum, strain ILC-164 [isolated from Lake Kinneret, Israel (Banker et al., 1997)]
  5. BG11 growth medium (Stanier et al., 1971)
  6. BG/-K akinete induction medium
    Note: BG11 medium in which the K2HPO4 component was substituted with Na2HPO4.
  7. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  8. Ethylenediaminetetraacetic acid disodium salt dehydrate (Na-EDTA) (Sigma-Aldrich, catalog number: E5134 )
  9. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  10. Lysozyme from chicken egg white (Sigma-Aldrich, catalog number: L6876 )
    Note: It is also named as “Mucopeptide N-acetylmuramoylhydrolase” or “muramidase”.
  11. Lugol’s solution (Sigma-Aldrich, catalog number: L6146 )
  12. SYTOX (Invitrogen, catalog number: S7020 )
    Note: Currently, it is “SYTOX® Green Nucleic Acid Stain-5 mM Solution in DMSO” (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: S7020).
  13. Coomassie brilliant blue R-250 staining solution (BIO RAD catalog number: 1610436 ).
  14. Molecular weight markers [All blue pre-stained protein standards (M. W. 10-250 kD)] (Bio-Rad Laboratories, catalog number: 1610373 )
  15. Tris (Sigma-Aldrich, catalog number: 252859 )
    Note: It is also named as “Tris (hydroxymethyl) aminomethane” on Sigma-Aldrich website.
  16. Tris buffer(see Recipes)
  17. Tris-EDTA-Mg buffer (see Recipes)
  18. Tris-Mg buffer (see Recipes)
  19. TE buffer (see Recipes)
  20. 0.5 M EDTA stock solution (see Recipes)
  21. 1 M MgCl2 stock solute (see Recipes)
  22. 1 M NaCl (see Recipes)

Equipment

  1. Spectrophotometer (such as Uvikon XS SECOMAM)
  2. Sorvall centrifuge RC 6 Plus and appropriate rotor for the centrifuge tubes or bottles
  3. Vortex
  4. Sonicator [e.g. Sonifier 450 (Branson Ultrasonics)]
  5. Orbital shaker (e.g. MaxQTM 2000 and 3000 benchtop orbital shaker, TermoFisher SCIENTIFIC, model number: SHKA2000 )
  6. Incubator (regulated temperature and light)
  7. Bright field/fluorescent inverted microscope (e.g. Zeiss Axioobserver Z1)
    Note: Use the following filter sets: for chlorophyll (EX-425-443 nm; BS-452 nm; EM-496 nm LP), for Phycobilins (EX-510-550 nm; BS-565 nm; EM-582 nm LP) and for SYTOX (EX-445-495 nm; BS-500 nm; EM-505-555 nm).
  8. Utermohl sedimentation chamber (Aquatic Research Instruments, http://www.aquaticresearch.com/sedimentation_chamber.htm)
  9. Thermostatic water bath

Procedure

  1. Cultivation and akinete induction
    1. In order to obtained sufficient biomass for akinete induction, cultivate A. ovlisporum in Erlenmeyer flasks using 0.5-1.0 liters BG11 medium. Incubate cultures at 28 ℃ under continuous light (PAR) at intensity of 30 µmol photon m-2 s-1. Culture can be aerated at 0.5 liter/min using aquarium air pump.
    2. Toward the end of the exponential growth phase (after 7-10 days, OD750 should be around 1.0. In case this OD has not reach consider restart this step with a new inoculum), remove the culture to a lower temperature incubator (20-24 ℃). Allow 3-4 days of acclimation under continuous light (PAR) at intensity of 30 µmol photon m-2 s-1 and aeration. Then harvest the culture by centrifugation.
    3. Centrifuge the culture at 5,000 x g for 5-10 min at room temperature, discard the supernatant. If available, use a centrifuge with the rotor fitting 500 ml tubes or distribute the culture into 50 ml Falcon tubes and perform several rounds of centrifugations. At the end of centrifugation collect the biomass in one 50 ml falcon tube and discard as much of growth medium as possible.
    4. Wash the pellet in BG/-K medium 3 times by re-suspending the pellet in the potassium depleted medium by gently vortexing, followed by centrifugation at 5,000 x g at room temperature. Finally re-suspend the pellet in 0.5 liters BG/-K medium using a 1-liter Erlenmeyer flask. Incubate the K deprived culture at a controlled temperature between 20 and 24 ℃ under continuous light (PAR) at intensity of 70-80 µmol photon m-2 s-1.
    5. Sample 3-5 ml from the induced culture to follow the progress of the akinete differentiation process. Collect samples at days 4, 7, 10, 14, 21 and 28-post induction. Young akinetes attached to filaments can be observed since day 5 or 7. Later on, more attached akinetes can be observed together with free akinetes whose diameter is larger than the young attached akinetes (Figure 1).
      Note: Akinete differentiation is a non-synchronized process.

  2. Isolation of akinete [modified from Razquin et al. (1996)]
    1. Centrifuge a 21 (or 28) days old akinete-induced culture at 5,000 x g at room temperature for 5-10 min, discard the supernatant.
    2. Re-suspend the pellet and wash few times in Tris-EDTA-Mg buffer. Finally re-suspend the washed pellet in 20-50 ml Tris-Mg buffer and add lysozyme to a final concentration of 1 mg ml-1. Use freshly prepared lysozyme stock solution.
    3. Incubate the suspension for 2 h at 37 ℃ to allow the disruption of vegetative cells, followed by low speed (3,000 x g) centrifugation for 3 min. During the incubation period, sample the suspension for microscopic observation of cell disruption. If necessary, extend the incubation with lysozyme for an additional hour.
    4. Re-suspend the akinete-enriched pellet in TE buffer and wash several times in the same buffer to remove cellular residues and the soluble lysozyme.
    5. Finally re-suspend the akinete enriched pellet in Tris buffer keep on ice, and briefly sonicate it using 30% of the full intensity at 20 sec intervals (Sonifier 450). Repeat the sonication steps 3-5 times but monitor the akinete integrity under microscope. The aim of this sonication step is to disrupt intact vegetative cells that remained in the suspension. Be sure that the sonication is not too aggressive and akinetes survive this step.
    6. Centrifuge the akinete suspension i at low speed (3,000 x g) wash it a few more times in sterile distilled water and finally re-suspend the pellet in a small volume of sterile distilled water. Keep the suspension at 15 ℃ and dark conditions. An akinetes’ suspension can be kept under those conditions for at least 6 months.
    7. At this stage, sample an aliquot from the akinete suspension for microscopic observation and enumeration.

  3. Counting akinetes
    1. Collect aliquots (1-3 ml) from cultures at various stages of akinete differentiation or during the akinete isolation process. Collect samples after vigorous shaking of the culture to ensure homogeneous suspension of filaments and akinetes.
    2. Preserve the samples in Lugol’s solution (add 1 drop-50 µl to 3 ml sample). Lugol-preserved samples can be stored at room temperature under dark until counting. Preserved samples can be stored for 4-6 months.
    3. Filaments, vegetative cells and akinetes are counted in an Utermohl sedimentation chamber using an inverted microscope (Utermohl, 1958). Two categories of akinetes can be defined; filament-attached akinetes (Figure 1. D7 and D14) and mature free akinetes (Figure 1. D21 and IA). The size of filament-attached akinetes increased as their differentiation proceeded and they exceeded a biomass of 0.4 ng (Sukenik et al., 2013). During their differentiation, akinetes are characterized by a thickened the cell wall and the accumulation of apparent cyanophycin granules (Figure 1).


      Figure 1. Light micrographs of filaments and akinetes from akinete induced culture of Aphanizomenon ovalisporum. D0, A filament with vegetative cells prior the induction (transfer to a BG/-K medium); D7, A filament 7 days post induction. Note the presence of young akinetes (arrowhead); D14, A filament 14 days post induction. A well-developed attached akinetes is marked with an arrowhead; D21, A short filament with attached akinetes and free akinetes 21 days post induction; IA, Isolated akinetes. Gray arrowheads in D7 and D14 indicate cyanophycine granule. Scale bar=10 µm

  4. Quality control of isolated akinetes
    1. Residual lysozyme on isolated akinetes
      Although the isolated akinetes suspension is vigorously washed to remove excess lysozyme, we found residual lysozyme in the akinete fraction, presumably associated with cell walls. These residues may interfere later on with akinete germination. The removal of the enzyme from the isolated akinetes is carried out by several washes with TE Buffer containing 100 mM NaCl as assessed by SDS-12% PAGE (Figure 2).
      Note: Three washes with TE + 100 mM NaCl are sufficient to remove residual lysozyme from lysozyme treated isolated akinetes.
    2. Assessment of akinete vitality
      In order to assess the percentage of vital akinetes use SYTOX solution at final concentration of 1 µM. SYTOX is a high affinity nucleic acid stain that can enter the cell only through damaged plasma membranes, such as those of dead cells. Thus it is possible to discriminate between dead and live akinetes. As a confirmation of vital akinetes, it is recommended to use SYTOX staining in addition to auto-fluorescence of the phycobilisome antenna and Chlorophyll a in viable akinetes (Figure 3).


      Figure 2. The effect of salt titration on lysozyme dissociation from akinetes. Proteins were separated on Laemmmli SDS-PAGE system (12% acrylamide) and stained by Coomassie blue. M, Molecular Weight Markers (All Blue pre-stained protein standards, M. W. 10-250 kD); AK, crude soluble protein extracted from isolated akinetes; Lanes 0 to 420 are soluble proteins after isolated akinete were washed with TE Buffer + NaCl at different concentrations (0-420 mM NaCl, respectively). Lyzozyme peptide (MW 14.4 kD) released by that treatment is indicated by an arrow. Notice the higher the salt concentration more lysozyme dissociated from isolated akinetes.


      Figure 3. Akinete induced Aphanizomenon ovalisporum samples before and after isolation of akinetes. Upper panel, culture at day 17 of induction; Lower panel, Isolated akinetes. Samples were stained with SYTOX and observed using fluorescence microscope with filters’ cubes set for fluoresce of chlorophyll (EX- 425-443 nm; BS-452 nm; EM-496 nm LP), Phycobilins (EX-510-550 nm; BS-565 nm; EM-582 nm LP) and SYTOX (EX-445-495 nm; BS-500 nm; EM-505-555 nm). Notice the four dead (SYTOX positive) akinetes prior to isolation (yellow arrows) and one dead akinete post isolation. Scale bar=50 µm

Notes

  1. Notes about reproducibility and variability:
    1. Optimal conditions for akinete induction are potassium deficiency, medium light intensity (75 µmol photon m-2 s-1) and temperature of 20-24 ℃ as described by Sukenik et al. (2013).
    2. Follow the differentiation process every 3-4 days as some variation may occur in the development schedule.
  2. Other notes and technical tips:
    1. The final concentration of lysozyme and the incubation time can be slightly adjusted as you gain experience with this protocol.
    2. Gentle sonication is required at step 5B of the isolation procedure. Follow the effectivity of the sonication in between cycles by microscopic observation.

Recipes

  1. Tris buffer
    50 mM Tris
    pH 7.5
  2. Tris-EDTA-Mg buffer
    50 mM Tris (pH 7.5) containing 10 mM EDTA and 1 mM MgCl2
  3. Tris-Mg buffer
    50 mM Tris (pH 7.5) containing 1 mM MgCl2
  4. TE buffer
    50 mM Tris (pH 7.5) with 10 mM EDTA
  5. 0.5 M EDTA stock solution
    Na-EDTA (186.12 g/L)
  6. 1 M MgCl2 stock solute (95.21 g/L)
  7. 1 M NaCl (58.44 g/L)

Acknowledgements

The protocol was modified from Razquin et al. (1996). The work was supported by research grants from the Israel Science Foundation (ISF grant No. 319/12), a Joint German-Israeli-Project (FKZ 02WT0985, WR803) from German Ministry of Research and Technology (BMBF) and Israel Ministry of Science and Technology (MOST). R. N. K-L was supported by Levy Eshkol Fellowship (no. 3-9476) Israel Ministry of Science and Technology (MOST).

References

  1. Banker, R., Carmeli, S., Hadas, O., Teltsch, B., Porat, R. and Sukenik, A. (1997). Identification of cylindrospermopsin in Aphanizomenon ovalisporum (Cyanophyceae) isolated from Lake Kinneret, Israel. Journal of Phycology 33(4): 613-616.
  2. Maldener, I., Summers, M. L. and Sukenik, A. (2014). Cellular differentiation in Filamentous Cyanobacteria. In: Flores, E. and Herrero, A. (eds). The cell biology of cyanobacteria. Caister Academic Press: 263-290.
  3. Razquin, P., Fillat, M. F., Schmitz, S., Stricker, O., Bohme, H., Gomez-Moreno, C. and Peleato, M. L. (1996). Expression of ferredoxin-NADP+ reductase in heterocysts from Anabaena sp. Biochem J 316 (Pt 1): 157-160.
  4. 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.
  5. Sukenik, A., Kaplan-Levy, R. N., Viner-Mozzini, Y., Quesada, A. and Hadas, O. (2013). Potassium deficiency triggers the development of dormant cells (akinetes) in Aphanizomenon ovalisporum (Nostocales, Cyanoprokaryota)(1). J Phycol 49(3): 580-587.
  6. Sukenik, A., Maldener, I., Delhaye, T., Viner-Mozzini, Y., Sela, D. and Bormans, M. (2015). Carbon assimilation and accumulation of cyanophycin during the development of dormant cells (akinetes) in the cyanobacterium Aphanizomenon ovalisporum. Front Microbiol 6: 1067.
  7. Sukenik, A., Beardall, J. and Hadas, O. (2007). Photosynthetic characterization of developing and mature akinetes of Aphanizomenon ovalisporum (Cyanoprokaryota). Journal of Phycology 43(4): 780-788.
  8. Utermohl, H. (1958). Zur Vervollkommnung der quantitativen Phytoplankton-Methodik. Inter Ver Theor Ange Limnol 9:1-38.

简介

Akinetes是由丝状蓝细菌菌株形成的孢子样休眠(休眠)细胞,用于在长期不利条件下存活。 在剥夺钾期间,沿着蓝细菌Aphanizomenon ovalisporum( ovlisporum )(菌株ILC-164)的细丝的营养光合细胞分化成动力基因。 Akinetes大于营养细胞,具有厚壁,积累储存化合物(氰基霉素,糖原,脂质)和过量的DNA(Sukenik等人,2015; Sukenik等人, em>,2007; Maldener等人,,2014)。 动力学和营养细胞之间的结构和组成的差异允许动力蛋白的分离和分离。 通过所述方案分离的Akinet可以用于蛋白质分析,代谢活性测量,荧光原位杂交(FISH)研究等。

材料和试剂

  1. 合适体积的锥形瓶
  2. 50ml管(SARSTEDT AG& Co,目录号:62.547.004)
  3. 250ml离心瓶(TermoFisher SCIENTIFIC,目录号:3141-0250)
  4. Aphanizomenon ovalisporum ,菌株ILC-164 [从以色列的Kinneret湖分离(Banker等人,1997)]
  5. BG11生长培养基(Stanman等人,1971)
  6. BG/-K动力感应培养基
    注意:其中K 2 HPO 4亚组分被Na 2 HPO 4亚组分代替的BG11培养基, 。
  7. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  8. 无水乙二胺四乙酸二钠盐(Na-EDTA)(Sigma-Aldrich,目录号:E5134)
  9. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  10. 来自鸡蛋清的溶菌酶(Sigma-Aldrich,目录号:L6876)
    注意:它也称为"Mucopeptide N-乙酰胞壁酰水解酶"或"muramidase"。
  11. Lugol溶液(Sigma-Aldrich,目录号:L6146)
  12. SYTOX(Invitrogen,目录号:S7020) 注意:目前,它是"SYTOX Green Nucleic Acid Stain-5mM Solution in DMSO"(Thermo Fisher Scientific,Molecular Probes TM ,目录号:S7020 )。
  13. 考马斯亮蓝R-250染色溶液(BIO RAD目录号:1610436)
  14. 分子量标记[所有蓝色预染色的蛋白质标准(M.W.10-250kD)](Bio-Rad Laboratories,目录号:1610373)
  15. Tris(Sigma-Aldrich,目录号:252859) 注意:在Sigma-Aldrich网站上也称为"三(羟甲基)氨基甲烷"。
  16. Tris缓冲液(见配方)
  17. Tris-EDTA-Mg缓冲液(见配方)
  18. Tris-Mg缓冲液(见配方)
  19. TE缓冲区(参见配方)
  20. 0.5 M EDTA储备溶液(见配方)
  21. 1 MgCl 2 2储备溶质(参见配方)
  22. 1 M NaCl(见配方)

设备

  1. 分光光度计(如Uvikon XS SECOMAM)
  2. Sorvall离心机RC 6 Plus和适当的转子用于离心管或瓶子
  3. 涡流
  4. 超声波发生器[例如 Sonifier 450(Branson Ultrasonics)]
  5. 轨道摇床(例如MaxQ TM sup/2000和3000台式轨道摇床,TermoFisher SCIENTIFIC,型号:SHKA2000)
  6. 孵化器(调节温度和光)
  7. 明场/荧光倒置显微镜(例如Zeiss Axioobserver Z1)
    注意:对于藻胆蛋白(EX-510-550nm; BS-565nm; EM-496nm LP)使用以下滤光片组:对于叶绿素(EX-425-443nm; 582nm LP)和SYTOX(EX-445-495nm; BS-500nm; EM-505-555nm)。
  8. Utermohl沉淀室(Aquatic Research Instruments, http://www.aquaticresearch.com/sedimentation_chamber.htm
  9. 恒温水浴

程序

  1. 种植和动力诱导
    1. 为了获得足够的生物量用于动力诱导,培养  在Erlenmeyer烧瓶中使用0.5-1.0升BG11培养基。 孵育培养物在28℃下连续光照(PAR)强度 30μmol光子m -1 s -1 。培养物可以0.5升/分钟通气 水族馆空气泵。
    2. 接近指数增长的结束 相(在7-10天后,OD <750>应该在1.0左右) 没有达到考虑重新启动这一步与一个新的接种),删除 培养至低温培养箱(20-24℃)。允许3-4天 在连续光(PAR)下以30μmol光子的强度驯化 m <-200 s -1 和通气。然后通过离心收获培养物
    3. 在室温下以5,000xg离心培养物5-10分钟, 丢弃上清液。如果可用,使用离心机与转子装配500ml管或将培养物分配到50ml Falcon管中 并进行几轮离心。 在......的最后 离心收集生物质在一个50ml falcon管中并丢弃 尽可能多的生长培养基
    4. 在BG/-K中洗涤沉淀 培养基3次,通过将沉淀物重悬在钾中 通过轻轻涡旋,随后在5,000×g离心5分钟 室内温度。 最后将沉淀重悬在0.5升BG/-K中 使用1升锥形瓶。 孵育K剥夺的文化   在控制温度在20和24℃之间在连续光   (PAR),强度为70-80μmol光子m -1 -2 -1
    5. 样品3-5ml 从诱导文化跟随机器的进步 分化过程。 在第4,7,10,14,21和4天收集样品 28后诱导。 可以观察到附着到细丝的年轻动子自5天或7天后,可以观察到更多的附着动力 以及直径大于年轻人的自由曲柄 附件akinetes(图1)。
      注意:Akinete微分是一个非同步过程。

  2. 动力学的分离[从Razquin等人修改]。 (1996)]
    1. 在室温下以5,000xg离心21(或28)天的动物诱导培养物5-10分钟,弃去上清液。
    2. 重悬浮沉淀并在Tris-EDTA-Mg缓冲液中洗涤几次。 最后将洗过的沉淀重悬在20-50ml Tris-Mg缓冲液中,加入 溶菌酶至终浓度为1mg ml -1 -1。 使用新鲜制备的溶菌酶原液
    3. 在37℃下孵育悬浮液2小时,以允许营养细胞的破坏,然后低速(3,000×g )  离心3分钟。在孵化期间,取样 悬浮液用于细胞破碎的显微观察。如果需要,  将与溶菌酶的孵育时间再延长1小时
    4. 重新悬浮富含骨髓的颗粒在TE缓冲液中,并洗几个 次在相同的缓冲液中以除去细胞残余物和可溶性 溶菌酶。
    5. 最后将富含akinete的沉淀物重悬在Tris中  缓冲液保持在冰上,并简单地超声波使用30%的满 强度(20秒间隔)(Sonifier 450)。重复超声处理 步骤3-5次,但在显微镜下监测动力学完整性。的 这个超声处理步骤的目的是破坏完整的营养细胞 保留在悬浮液中。确保超声不是太 侵略性和动力生存这一步。
    6. 在低速(3,000xg)下离心机动搅拌器悬浮液i   在无菌蒸馏水中洗几次,最后 将沉淀重悬于少量无菌蒸馏水中。 保持   在15℃和黑暗条件下悬浮。 akinetes的悬浮液可以在这些条件下保存至少6个月。
    7. 在此阶段,从动力学悬浮液中取样,用于显微镜观察和计数。

  3. 计算动力
    1. 从动物的各个阶段的培养物中收集等分试样(1-3ml) 分化或在动力分离过程中。 收集样品之后剧烈振荡培养物以确保均匀悬浮 的丝和动力学。
    2. 保存在Lugol的样品 溶液(向3ml样品中加入1滴-50μl)。 Lugol保存的样品可以 在室温下避光保存直至计数。保存 样品可以储存4-6个月。
    3. 细丝,营养细胞  并在Utermohl沉降室中使用a计算动力学 倒置显微镜(Utermohl,1958)。两类akinetes可以 定义;细丝附着的动力蛋白(图1D1和D14)和成熟的 自由动力学(图1D 21和IA)。细丝附着的尺寸 随着它们的分化进行,动量增加,它们超过a  生物量0.4ng(Sukenik等人,2013)。在它们的分化期间,  动脉瘤的特征在于细胞壁增厚 表观藻青素颗粒的积累(图1)。


      图1。   来自动力学诱导的细丝和动量的光学显微照片 D ,A filament with vegetative (转移到BG/-K培养基); 细丝7   诱导后天数。 注意年轻akinetes的存在(箭头); D 14,诱导后14天的细丝。 一个发达的附件 akinetes标有箭头; D 21,短细丝 附着动力学和自由动力学诱导21天后; IA,隔绝   akinetes。 D sub 7和D 14中的灰色箭头指示氰霉素颗粒。 比例尺=10μm

  4. 孤立动力学的质量控制
    1. 孤立动力蛋白上的残留溶菌酶
      虽然孤立 强烈洗涤动力蛋白悬浮液以除去过量的溶菌酶,我们 发现残余溶菌酶在动力学部分,推测是相关的 与细胞壁。这些残基可能在后来干扰动力 发芽。从分离的动力学中去除酶 通过用含有100mM NaCl的TE缓冲液洗涤几次进行 通过SDS-12%PAGE评估(图2)。
      注意:使用TE + 100mM NaCl的三次洗涤足以从溶菌酶处理的分离的动力学中除去残留的溶菌酶。
    2. 动机生命力的评估
      为了评估重要骨骼的百分比使用SYTOX解决方案 最终浓度为1μM。 SYTOX是高亲和力核酸 仅可通过损伤的质膜进入细胞的染色, 例如死细胞的那些。因此可以区分  死和活的动力。作为重要动力的确认,它是 建议使用SYTOX染色以及自动荧光藻类天线和叶绿素a在活的动力学(图 3)。


      图2.盐滴定对溶菌酶解离的影响  从akinetes。 蛋白在Laemmmli SDS-PAGE系统(12%  丙烯酰胺)并用考马斯蓝染色。 M,分子量标记 (所有蓝色预染色的蛋白质标准,M.W.10-250kD); AK,粗品 从孤立动力学提取的可溶性蛋白;泳道0至420为 用TE Buffer +洗涤分离的动物后的可溶性蛋白 NaCl(分别为0-420mM NaCl)。溶菌酶  通过该处理释放的肽(MW 14.4kD)由a表示 箭头。注意,盐浓度越高,溶菌酶越高 从孤立的动力学中解离

      图3.在分离之前和之后Akinete诱导的Aphanizomenon ovalisporum 样品 akinetes。 上图,培养在诱导的第17天;下面板, 孤立的akinetes。样品用SYTOX染色并使用观察荧光显微镜与滤波器的立方体设置为荧光 叶绿素(EX-425-443nm; BS-452nm; EM-496nm LP),藻胆蛋白 (EX-510-550nm; BS-565nm; EM-582nm LP)和SYTOX(EX-445-495nm; BS-500nm; EM-505-555nm)。 注意四个死(SYTOX正) 分离前的动力学(黄色箭头)和一个死的动力学 隔离。 比例尺=50μm

笔记

  1. 有关再现性和变异性的说明:
    1. 动机诱导的最佳条件是钾缺乏,中等光强度(75μmol光子m -2 -2s -1 s -1)和温度20-24℃,如Sukenik等人所述。 (2013)。
    2. 每3-4天遵循分化过程,因为开发计划表中可能会发生一些变化。
  2. 其他说明和技巧提示:
    1. 当您获得该方案的经验时,可以稍微调整溶菌酶的最终浓度和孵育时间
    2. 在分离程序的步骤5B需要温和的超声处理。   按照循环之间的超声处理的有效性 显微镜观察。

食谱

  1. Tris缓冲液
    50 mM Tris
    pH 7.5
  2. Tris-EDTA-Mg缓冲液
    含有10mM EDTA和1mM MgCl 2·h/v的50mM Tris(pH7.5)
  3. Tris-Mg缓冲液
    含有1mM MgCl 2·h/v的50mM Tris(pH7.5)
  4. TE缓冲区
    50mM Tris(pH7.5)和10mM EDTA
  5. 0.5 M EDTA储备液
    Na-EDTA(186.12g/L)
  6. 1 MgCl 2 2储备溶质(95.21g/L)
  7. 1M NaCl(58.44g/L)

致谢

The protocol was modified from Razquin et al. (1996). The work was supported by research grants from the Israel Science Foundation (ISF grant No. 319/12), a Joint German-Israeli-Project (FKZ 02WT0985, WR803) from German Ministry of Research and Technology (BMBF) and Israel Ministry of Science and Technology (MOST). R. N. K-L was supported by Levy Eshkol Fellowship (no. 3-9476) Israel Ministry of Science and Technology (MOST).

References

  1. Banker, R., Carmeli, S., Hadas, O., Teltsch, B., Porat, R. and Sukenik, A. (1997). Identification of cylindrospermopsin in Aphanizomenon ovalisporum (Cyanophyceae) isolated from Lake Kinneret, Israel. Journal of Phycology 33(4): 613-616.
  2. Maldener, I., Summers, M. L. and Sukenik, A. (2014). Cellular differentiation in Filamentous Cyanobacteria. In: Flores, E. and Herrero, A. (eds). The cell biology of cyanobacteria. Caister Academic Press: 263-290.
  3. Razquin, P., Fillat, M. F., Schmitz, S., Stricker, O., Bohme, H., Gomez-Moreno, C. and Peleato, M. L. (1996). Expression of ferredoxin-NADP+ reductase 在来自鱼腥藻的异囊中。生物化学316(Pt 1):157-160。
  4. Stanier,R.Y.,Kunisawa,R.,Mandel,M。和Cohen-Bazire,G。(1971)。 单细胞蓝绿藻的纯化和性质(Chroococcales订单) Bacteriol Rev 35(2):171-205。
  5. Sukenik,A.,Kaplan-Levy,R.N.,Viner-Mozzini,Y.,Quesada,A.and Hadas,O.(2013)。 钾缺乏会引发Aphanizomenon ovalisporum中的休眠细胞(akinetes)的发展。 (Nostocales,Cyanoprokaryota)(1)。

    49(3):580-587。
  6. Sukenik,A.,Maldener,I.,Delhaye,T.,Viner-Mozzini,Y.,Sela,D。和Bormans,M。 藻蓝蛋白在蓝细菌中休眠细胞(akinetes)发育期间的碳同化和积累Aphanizomenon ovalisporum 。 前微型生物 6:1067.
  7. Sukenik,A.,Beardall,J。和Hadas,O。(2007)。 光合作用的发育和成熟机能的表征 (Cyanoprokaryota)。 43(4):780-788。
  8. Utermohl,H.(1958)。 Zur Vervollkommnung der quantitativen Phytoplankton -Methodik。 Inter Ver Theor Ange Limnol 9:1-38。
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引用:Sukenik, A., Kaplan-Levi, R. N., Viner-Mozzini, Y., Lupu, A. and Sela, D. (2016). Induction, Isolation and Counting of Akinetes in Aphanizomenon ovalisporum. Bio-protocol 6(10): e1808. DOI: 10.21769/BioProtoc.1808.
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