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Heat Shock Treatment of Chlamydomonas reinhardtii and Chlorella Cells
热击处理莱茵衣藻和小球藻细胞

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Abstract

The protocol is very reliable and simple for inducing heat shock in unicellular green algae cells. The main purpose was to compare cellular response of three Chlorella species, isolated from different habitats: Chlorella vulgaris 8/1- thermophilic, Chlorella kesslery- mesophilic and C. vulgaris- extremophilic. Species were isolated from different habitats and differ in their temperature preferences and tolerance. Temperature induced stress response was measured as cell survival, induction of chloroplast HSP70B and DSBs induction and rejoining.

Materials and Reagents

  1. Species:
    Three Chlorella species were used: C. vulgaris, isolated from soil samples of Livingston Island, the South Shetland Archipelago, Antarctic; C. vulgaris strain 8/1, isolated in 1968 from thermal springs in the region of Rupite, Bulgaria, and cultivated in our laboratory since 1975 and Chlorella kesslery a mesophile, from the Trebon collection.
    Cultivation: Chlorella species were cultivated on Tris Acetate Phosphate (TAP) medium under continuous light of 60 μmol/m2/s and a temperature of 23 °C ± 0.1 °C in a Phytotron GC 400 growth chamber. The species were cultivated at this temperature because it is well known, that eurythermal algae, could be grown at a wide range of temperatures.
  2. TAP medium (see Recipes)
  3. Sager–Granick medium (see Recipes)

Equipment

  1. Phytotron GC 400 growth chamber (NUVE Ankara/Turkey 2009)
  2. Microscope
  3. Bürker chamber
  4. Circulating water bath

Procedure

  1. Cultivation
    1. Cultivate for 4-5 days algae strains or species on TAP medium (Harris, 1989) under continuous light of 60 μmol/m2/s and a temperature of 23 °C ± 0.1 °C in a Phytotron GC 400 growth chamber to the end of exponential and early stationary phase of growth.
    2. Check under the microscope whether cell culture is not contaminated.
    3. Count cell number under the microscope using Bürker chamber.
    4. Centrifuge certain volume (depending on the cell density, counted in step A-3) of cell suspension at 1,200 x g by RT, resuspend in Sager-Granick medium or other appropriate medium, so to get 10 ml of cell suspension with a density of 1 x 106 cells/ml for every sample.

  2. Temperature treatment
    1. Keep 10 ml cell culture with a density 1 x 106 cells/ml in an incubator under continuous shaking, at different temperatures:
      t = 39 °C for 30 min
      t = 42 °С for 5 min
      t = 45 °С for 5 min
      Notes:
      1. The same heating procedure could be done in a circulating water bath at the same three temperatures: t = 39 °С for 30 min, t = 42 °С for 5 min, and t = 45 °С for 5 min.
      2. Use 50 ml flasks to comply with the requirement that volume of the cell culture must be not more than 1/3 of the volume of the flask.
    2. Place on ice to stop the heating process.
    3. For HSP70B analysis keep cells for 2 and 4 h after the step II-2 at t = 23 °C ± 0.1 °C to allow cells to recover.
    4. Centrifuge 10 ml cell suspension at 1,200 x g for 5 min.

Recipes

  1. TAP medium
    Stock solution for 1 L of TAP media
    1 M Tris base
    20 ml
    Phosphate Buffer II (see 1-a)
    1.0 ml
    Solution A (see 1-b)
    10.0 ml
    Hutner's trace elements (see 1-c)
    1.0 ml
    Glacial acetic acid (pH to 7.0)
    1.0 ml
    1. Phosphate buffer II (Stock solution)
      Component (For 100 ml)
      K2HPO4
      10.8 g
      KH2PO4
      5.6 g
    2. Solution A
      Component (For 500 ml)
      NH4Cl
      20 g
      MgSO4.7H2O
      5.0 g
      CaCl2.2H2O
      2.5 g
    3. Hutner's trace elements
      1. Dissolve 50 g of acid free EDTA in 250 ml of deionized. Heat to dissolve.
      2. Dissolve the following one by one in order. Heating to approximately 100 °C in 500 ml deionized H2O.
        Component
        Quantity
        H3BO3
        11.4 g
        ZnSO4.7H2O
        22.0 g
        MnCl2.4H2O
        5.06 g
        FeSO4.7H2O
        4.99 g
        CoCl2.6H2O
        1.61 g
        CuSO4.5H2O
        1.57 g
        Mo7O24(NH4)6.4H2O
        1.1 g
      3. Mix the two solutions together. The resulting solution should be blue-green.
      4. Heat to 100 °C. Cool slightly, but don't let the temperature drop below 80 °C – 90 °C.
      5. Adjust pH to 6.5–6.8 with 20% KOH (approximately 83 ml).
  2. Sager and Granick medium (adjust pH 6.8-7.0)
    Component
    In 1 L stock
    For 1 L media
    Trace elements*
    --
    1 ml
    NaCitrate.2H2O
    100 g
    5 ml
    FeCl3.6H2O
    10 g
    1 ml
    CaCl2.6H2O
    58 g
    1 ml
    MgSO4.7H2O
    100 g
    3 ml
    NH4NO3
    100 g
    3 ml
    KH2PO4
    100 g
    1 ml
    K2HPO4
    100 g
    1 ml
    *Trace Elements
    Component
    In 1 L stock
    H3BO3
    1.0 g
    ZnSO4.7H2O
    1.0 g
    MnSO4.H2O
    0.303 g
    CoCl2.6H2O
    0.2 g
    Na2MoO4.2H2O
    0.2 g
    CuSO4.5H2O
    0.063 g

References

  1. Chankova, S. G., Yurina, N. P., Dimova, E. G., Ermohina, O. V., Oleskina, Y. P., Dimitrova, M. T. and Bryant, P. E. (2009). Pretreatment with heat does not affect double-strand breaks DNA rejoining in Chlamydomonas reinhardtii. J Ther Biol 34(7): 332-336.
  2. Chankova, S., Mitrovska, Z., Miteva, D., Oleskina, Y. P. and Yurina, N. P. (2013). Heat shock protein HSP70B as a marker for genotype resistance to environmental stress in Chlorella species from contrasting habitats. Gene 516(1): 184-189.

简介

该协议是非常可靠和简单的诱导单细胞绿藻细胞热休克。 主要目的是比较从不同栖息地分离的三种小球藻物种的细胞反应:小球藻(Chlorella vulgaris)8/1-嗜热的,小球藻乳酪 - < 嗜温和。 vulgaris - 嗜极的。 从不同的栖息地分离物种,其温度偏好和耐受性不同。 温度诱导的应激反应测量为细胞存活,叶绿体HSP70B的诱导和DSB诱导和重新接合。

材料和试剂

  1. 物种:
    使用三种小球藻:C。 寻常的,从利文斯顿岛,南设得兰群岛,南极的土壤样品分离; C。 vulgaris 菌株8/1,于1968年从保加利亚Rupite地区的温泉泉中分离,并自1975年以来在我们的实验室中栽培,并且从Trebon集合中孵化出嗜中性粒细胞。
    培养:小球藻属物种在Tris乙酸磷酸盐(TAP)培养基上在60μmol/m 2 s/s的连续光下和23℃±0.1℃的温度下培养 在Phytotron GC 400生长室中。 该种在该温度下培养,因为众所周知,热的藻类可以在宽的温度范围内生长。
  2. TAP介质(参见配方)
  3. Sager-Granick培养基(见配方)

设备

  1. Phytotron GC 400生长室(NUVE Ankara/Turkey 2009)
  2. 显微镜
  3. Bürker房间
  4. 循环水浴

程序

  1. 培养
    1. 在60μmol/m 2/s的连续光下和在Phytotron中的23℃±0.1℃的温度下在TAP培养基(Harris,1989)上培养4-5天藻类菌株或物种。 GC 400生长室至指数期和早期稳定期的生长末期
    2. 在显微镜下检查细胞培养是否没有被污染
    3. 使用Bürker腔室在显微镜下计数细胞数目
    4. 通过RT将在1,200×g下的细胞悬浮液的一定体积(取决于细胞密度,在步骤A-3中计数)离心,重悬于Sager-Granick培养基或其它合适的培养基中,从而得到10ml 细胞悬浮液,每个样品的密度为1×10 6个细胞/ml
  2. 温度处理
    1. 在连续振荡下,在不同温度下在培养箱中保持10ml细胞培养物,密度为1×10 6个细胞/ml:
      t = 39℃30分钟
      t = 42°С,5分钟
      t = 45°С,5分钟
      注意:
      1. 可以在相同的三个温度下在循环水浴中进行相同的加热程序:t = 39℃30分钟,t = 42℃5分钟,t = 45℃5分钟。/em>
      2. 使用50ml烧瓶以符合细胞培养物的体积必须不大于烧瓶体积的1/3的要求。
    2. 放在冰上停止加热过程。
    3. 对于HSP70B分析,在t = 23℃±0.1℃下,在步骤II-2后保持细胞2和4小时以允许细胞恢复。
    4. 在1,200×g离心10分钟细胞悬浮液5分钟

食谱

  1. TAP媒体
    1 L的TAP培养基的库存溶液
    1 M Tris碱
    20ml
    磷酸盐缓冲液II(见1-a)
    1.0 ml
    解决方案A(见1-b)
    10.0 ml
    Hutner的微量元素(见1-c)
    1.0 ml
    冰醋酸(pH至7.0)
    1.0 ml
    1. 磷酸盐缓冲液II(储存溶液)
      组分(对于100ml)
      K 2 HPO 4
      10.8克
      KH 2 PO 4
      5.6 g
    2. 解决方案A
      组分(对于500ml)
      NH 4 Cl
      20克
      MgSO 4·7H 2 O 2·h/v 5.0克
      CaCl 2 2·2H 2 O·dm/2 2.5克
    3. Hutner的微量元素
      1. 将50g无酸的EDTA溶于250ml去离子水中。 加热溶解。
      2. 依次溶解以下物品。 在500ml去离子H 2 O中加热至约100℃
        组件
        数量
        H 3 BO 3
        11.4克
        ZnSO 4·7H 2 O·m/2 22.0克
        MnCl 2 2·4H 2 O·m/2 5.06克
        FeSO 4 47H 2 O
        4.99克
        CoCl <2> .6H 2 O 1.61克
        CuSO 4 H 5 H 2 O·m/2 1.57克
        Mo 7 SubO 4(NH 4)6 Si 4 H 2 O b /> 1.1克
      3. 将两种溶液混合在一起。 所得溶液应为蓝绿色。
      4. 加热至100℃。 稍微冷却,但不要让温度降低到80°C - 90°C
      5. 用20%KOH(约83ml)调节pH至6.5-6.8
  2. Sager和Granick培养基(调节pH 6.8-7.0)
    组件
    在1升股票
    对于1 L媒体
    微量元素*
    -
    1 ml
    柠檬酸盐.H 2 O 2 100克
    5 ml
    FeCl 3·6H 2 O 10克
    1 ml
    CaCl 2 2·6H 2 O·dm/2 58克
    1 ml
    MgSO 4·7H 2 O 2·h/v 100克
    3 ml
    NH 4 3
    100克
    3 ml
    KH 2 PO 4
    100克
    1 ml
    K 2 HPO 4
    100克
    1 ml
    *跟踪元素
    组件
    在1升股票
    H 3 BO 3
    1.0 g
    ZnSO 4·7H 2 O·m/2 1.0 g
    MnSO 4·H 2 O·m/2 0.303克
    CoCl <2> .6H 2 O 0.2 g
    Na 2 MoO 4 ... 2H H 2 O
    0.2 g
    CuSO 4 H 5 H 2 O·m/2 0.063克

参考文献

  1. Chankova,S.G.,Yurina,N.P.,Dimova,E.G.,Ermohina,O.V.,Oleskina,Y.P.,Dimitrova,M.T.and Bryant,P.E。(2009)。 热预处理不会影响双链断裂重新结合于莱茵衣藻中的DNA 。 J Ther Biol 34(7):332-336。
  2. Chankova,S.,Mitrovska,Z.,Miteva,D.,Oleskina,Y.P.和Yurina,N.P。(2013)。 热休克蛋白HSP70B作为基因型对环境压力的标记,在小球藻中 >物种来自对比的栖息地。基因 516(1):184-189。
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引用:Chankova, S., Mitrovska, Z. and Yurina, N. (2013). Heat Shock Treatment of Chlamydomonas reinhardtii and Chlorella Cells. Bio-protocol 3(15): e849. DOI: 10.21769/BioProtoc.849.
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