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Spot Assays for Viability Analysis of Cyanobacteria
蓝藻细菌活性分析的斑点试验   

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Abstract

Cyanobacteria are prokaryotic organisms performing oxygenic photosynthesis. The cyanobacterium Synechocystis sp. PCC 6803 is a model organism for the study of photosynthesis, gene regulation and biotechnological applications because it is easy to manipulate genetically. Moreover, this cyanobacterium can grow photoautotrophically as well as chemoheterotrophically in the dark utilizing glucose. Microbiologists often use optical density measured with a spectrophotometer for the comparison of growth performance of different strains in liquid cultures. Because Synechocystis sp. PCC 6803 (especially motile strains) tend to form aggregates under stress conditions this method might be not suitable for evaluation of different strains under different growth conditions. In addition, many labs are not well equipped with standardized photobioreactors and illumination facilities to ensure reproducibility of growth curves. Here, we describe a highly reproducible spot assay for viability analysis of Cyanobacterial strains.

Keywords: Cyanobacteria(蓝藻), Synechocystis 6803(集胞藻6803), Spot assay(斑点法), Growth assay(生长测定)

Materials and Reagents

  1. Synechocystis sp. PCC 6803 strain (wild type obtained from S. Shestakov, Moscow State University, Russia)
  2. CaCl2.2H2O (Carl Roth, catalog number: 5239.1 )
  3. Citric acid (Carl Roth, catalog number: X863.2 )
  4. NaNO3 (Carl Roth, catalog number: A136.2 )
  5. MgSO4.7H2O (Carl Roth, catalog number: P027.1 )
  6. Na2-EDTA (Carl Roth, catalog number: 8043.2 )
  7. H3BO3 (Carl Roth, catalog number: 6943.1 )
  8. MnCl2.4H2O (Carl Roth, catalog number: T881.3 )
  9. ZnSO4.7H2O (Carl Roth, catalog number: T884.1 )
  10. Na2MoO4.2H2O (Carl Roth, catalog number: 0274.1 )
  11. Co(NO3)2.6H2O (Carl Roth, catalog number: HN16.1 )
  12. CuSO4.5H2O (Carl Roth, catalog number: P024.1 )
  13. Na2CO3 (Carl Roth, catalog number: P028.2 )
  14. Ammonium iron (III) citrate (Carl Roth, catalog number: P027.1)
  15. K2HPO4.3H2O (Merck KGaA, Calbiochem®, catalog number: 105099 )
  16. 2-[(2-Hydroxy-1, 1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES) (Carl Roth, catalog number: 9137.3 )
  17. Bacto Agar (BD Bioscience, catalog number: 214010 )
  18. Syringes (50 ml volume)
  19. Sterile syringe filters (Sarstedt Filtropur S 0.2 µm, catalog number: 83.1826.001 and 0.45 µm, catalog number: 83.1826 )
  20. Sterile 96-well microliter plates (i.e. VWR International, catalog number: 734-2328 )
  21. 100x BG11 medium (see Recipes)
  22. Trace metal mix (see Recipes)
  23. Stock Solutions for BG-11 medium (see Recipes)
  24. 1x BG11 (2x BG11) (see Recipes)
  25. BG11 agar (see Recipes)
  26. Na2S2O3 solution (see Recipes)
  27. 0.25 M Na2-EDTA (see Recipes)

Equipment

  1. Wide neck Erlenmeyer flasks with 100 ml volume (Carl Roth, catalog number: C146.1 )
  2. Light source (18 W/840) (Philips, model: MASTER TL-D Super 80 )
  3. Laboratory shaker (one-dimensional orbital motion) (Heidolph Instruments GmbH, catalog number: 036130180 )
  4. UV/Vis spectrophotometer (Shimadzu, model: UV-2401PC )
  5. Multichannel pipettes (8-channel pipette, 0.5-10 µl and 10-100 µl) (Starlab ErgoOne®, catalog number: S7108-0510 and S7108-1100 )
  6. Petri dishes with square shape (120 x 120 x 17 mm) (Greiner Bio-One GmbH, catalog number: 688102 )
  7. Transmitted light scanner with adjustable cover (i.e. Epson perfection v700 photo, a good camera can be used as an alternative)
  8. Laminar flow hood (i.e. Heraeus HeraSafe HS)
  9. LI-190R Quantum Sensor

Procedure

Every experimental step involving the handling of cyanobacterial cell cultures must be performed under a Laminar flow hood to prevent contamination!

  1. Cyanobacterial liquid cultures were inoculated from agar plates in BG11 medium (Rippka et al., 1979) and incubated with continuous shaking (150 rpm) at 30 °C under constant illumination with white light of 50 µmol photons m-2 s-1 measured at the position of the agar plates for 2-3 days (measured with a LI-190R Quantum Sensor).
    Note: Even though some mutants carry antibiotic resistance gene cassettes inserted in their genome, cultures for the spot assays were always grown in medium lacking antibiotics to exclude effects from growth on selective media. There is no specific volume or OD needed, but the pre-cultures should not reach late stationary phase and their OD750 should be similar.
  2. The well-grown pre-cultures were diluted to OD750 0.2 in a final volume of 20 ml BG11 medium and were incubated further under the above mentioned conditions until they reached logarithmic phase (approximately OD750 0.6-0.7).
    Note: It is important to measure OD at a wavelength where no pigments absorb.
  3. The logarithmically growing cultures were again diluted to OD750 0.2. With the freshly diluted cultures, a dilution series was performed with a multichannel pipette in a sterile microliter plate ranging from 100 to 10-7 (Figure 1).


    Figure 1. Dilution series for viability analysis. Rows B to H were pre-filled with 180 µl BG11 medium. After every dilution step, the cell suspension was thoroughly mixed with the pipette before transferring part of it into the next well.

  1. From each of the dilution steps, 5 µl was spotted in triplicates using the multichannel pipette onto square BG11 agar plates supplemented with 0.3% (w/v) Na2S2O3.
    Note: The exact composition of the agar can be customized. If the experimenter wishes to observe viability under mixotrophic conditions, the BG11 agar can be i.e. supplemented with glucose. It is also possible to exclude iron or nitrogen from the media stock solutions. However do not forget to add Na2S2O3 to the plates. This is important for proper growth of the colonies (Thiel et al., 1989).
  2. The drops of liquid cultures were allowed to dry for approximately 15-20 min (closed plate, at room temperature on the table top). Afterwards, plates were incubated at 30 °C under constant illumination with white light at 50 µmol photons m-2 s-1 for approximately 5 days or until no more colonies appeared in the highest dilution step.
    Note: To prevent phototactic movement of motile strains, the plates should be placed below the light source and lateral illumination from only one side should be avoided. In order to prevent dripping (in case the drops are not completely dry), the plates were incubated with the lid facing upwards for the first day. After the first day, plates were turned upside down to avoid evaporated water dripping on the spots. For investigation of light-dark related phenotypes, constant illumination can also be changed to light-dark-cycles. In our case, we investigated mutants of circadian clock associated genes and therefore chose a 12:12 h light:dark incubation. In this case, the starting OD750 for the dilution series was adjusted to 0.4 instead of 0.2 (step 2).
  3. Record the result (Figure 2) by scanning the plate or by taking a picture. In the best case, it may even be possible to count the single colonies within several spots (you may need a Binocular microscope).


    Figure 2. Spot assay. The dilution series of the cell cultures were spotted in triplicates onto square BG11 agar plates. The plate was incubated until no more colonies appeared in the highest dilution step. Strains have been published by Dörrich et al. (2014).

    Further results generated by using this method were published by Dörrich et al. (2014).

Notes

The spot assays described above are highly reproducible. In contrast to other methods like measuring the optical density of liquid cultures, which is often error-prone due to i.e. the formation of aggregates, we always obtained reliable and reproducible results. Within our group, several researchers have performed this method in two different laboratories and we did not detect any variations in the results. For reproducibility it is highly recommended to use Na2S2O3 in the plates (as described in Recipes) and to record the results after the same time. Composition of agar plates and light conditions can be customized to fit the experimenters’ individual needs and may require optimization for different mutant strains.

Recipes

  1. 100x BG11 medium
    CaCl2.2H2O
    3.6 g/L
    Citric acid
    0.6 g/L
    NaNO3
    149.58 g/L
    MgSO4.7H2O
    7.49 g/L
    0.25 M Na2-EDTA (pH 8)
    0.56 ml/L
    Prepare the solution in ddH2O and sterilize by autoclaving
  2. Trace metal mix
    H3BO3
    2.86 g/L
    MnCl2.4H2O
    1.81 g/L
    ZnSO4.7H2O
    0.222 g/L
    Na2MoO4.2H2O
    0.390 g/L
    Co(NO3)2.6H2O
    0.049 g/L
    Prepare these solutions in ddH2O and sterilize by filtering using a syringe and sterile filter
    Sterile aliquots can be stored at 4 °C
  3. Stock Solutions for BG-11 medium
    Na2CO3
    20 mg/ml
    Ammonium iron (III) citrate
    6 mg/ml
    K2HPO4.3H2O
    30 mg/ml
    TES buffer (pH 8.0 adjust with NaOH)
    1 M
    Prepare these solutions in ddH2O and sterilize by filtering using a syringe and sterile filter
    Sterile aliquots can be stored at 4 °C
  4. 1x BG11 (2x BG11)
    100 x BG11
    10 ml/L (20 ml/L)
    Na2CO3 stock solution
    1 ml/L (2 ml/L)
    K2HPO4.3H2O stock solution
    1 ml/L (2 ml/L)
    Ammonium iron (III) citrate stock solution
    1 ml/L (2 ml/L)
    Trace metal mix stock solution
    1 ml/L (2 ml/L)
    TES buffer (pH 8.0) stock solution
    10 ml/L (20 ml/L)
    Add sterile solutions to ddH2O and adjust the volume to 1 L
    If you use non-sterile solutions and autoclave the medium afterwards there will be slight precipitation of salts. Before using the medium you should mix it thoroughly. This is recommended, when you want to avoid contaminations in your cultures, especially for long-term strain maintenance.
  5. BG11 agar
    Prepare 1.5% bactoagar in ddH2O and autoclave.
    The molten 1.5% bactoagar (approx. 80 °C) is mixed with the same volume of sterile 2x BG11 medium.
  6. Na2S2O3 solution
    30% w/v Na2S2O3
    30 g Na2S2O3
    Add ddH2O to 100 ml
    Filter sterilize (0.2 µm)
    Stored at 4 °C
  7. 0.25 M Na2-EDTA (pH 8)
    9.306 g Na2-EDTA
    Add ddH2O to 100 ml
    Adjust pH to 8.0 with NaOH (use highly concentrated NaOH, Na2-EDTA will not dissolve, until the pH reaches 8.0)
    Stored at room temperature

Acknowledgments

BG11 medium is prepared according to Rippka et al. (1979). This work was supported by DFG grant to A. W. Wi2014/5-1.

References

  1. Dörrich, A. K., Mitschke, J., Siadat, O. and Wilde, A. (2014). Deletion of the Synechocystis sp. PCC 6803 kaiAB1C1 gene cluster causes impaired cell growth under light-dark conditions. Microbiology 160(Pt 11): 2538-2550.
  2. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. and Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of Cyanobacteria. J Gen Microbiol (111): 1-61.
  3. Thiel, T., Bramble, J. and Rogers, S. (1989). Optimum conditions for growth of Cyanobacteria on solid media. FEMS Microbiol Lett 52(1-2): 27-31.

简介

蓝细菌是进行含氧光合作用的原核生物。蓝细菌集胞藻 PCC 6803是一种用于光合作用,基因调控和生物技术应用研究的模式生物,因为它易于基因操作。此外,这种蓝细菌可以在黑暗中利用葡萄糖光化自养地以及化学异养生长。微生物学家经常使用用分光光度计测量的光密度用于比较液体培养物中不同菌株的生长性能。因为 Synechocystis PCC 6803(特别是运动株)倾向于在胁迫条件下形成聚集体,该方法可能不适合在不同生长条件下评估不同的菌株。此外,许多实验室不能很好地配备标准化的光生物反应器和照明设施,以确保生长曲线的可重复性。在这里,我们描述了高度可重复的斑点测定的蓝细菌菌株的生存力分析。

关键字:蓝藻, 集胞藻6803, 斑点法, 生长测定

材料和试剂

  1. 集胞藻 PCC 6803菌株(野生型,从S.Shestakov,Moscow State University,Russia获得)
  2. (Carl Roth,目录号:5239.1)。
  3. 柠檬酸(Carl Roth,目录号:X863.2)
  4. NaNO 3(Carl Roth,目录号:A136.2)
  5. MgSO 4·7H 2 O(Carl Roth,目录号:P027.1)。
  6. Na 2 -EDTA(Carl Roth,目录号:8043.2)

  7. (Carl Roth,目录号:6943.1)

  8. (Carl Roth,目录号:T881.3)
  9. (Carl Roth,目录号:T884.1)
  10. (Carl Roth,目录号:0274.1)。
  11. (Carl Roth,目录号:HN16.1),其中所述氧化还原反应包括以下步骤:(a) br />
  12. (Carl Roth,目录号:P024.1)
  13. (Carl Roth,目录号:P028.2)。
  14. 柠檬酸铁铵(III)(Carl Roth,目录号:P027.1)
  15. (Merck KGaA,Calbiochem , 目录号:105099)
  16. 2 - [(2-羟基-1,1-双(羟甲基)乙基)氨基]乙磺酸(TES)(Carl Roth,目录号:9137.3)
  17. Bacto琼脂(BD Bioscience,目录号:214010)
  18. 注射器(50ml体积)
  19. 无菌注射器过滤器(Sarstedt Filtropur S0.2μm,目录号:83.1826.001和0.45μm,目录号:83.1826)
  20. 无菌96孔微量滴定板( VWR International,目录号:734-2328)
  21. 100x BG11介质(参见配方)
  22. 微量金属混合物(见配方)
  23. BG-11培养基的库存解决方案(参见配方)
  24. 1x BG11(2x BG11)(参见配方)
  25. BG11琼脂(见配方)
  26. 溶液(参见配方)
    (参见配方)。
  27. 0.25 M Na 2 EDTA-EDTA(参见配方)

设备

  1. 具有100ml体积的宽颈锥形瓶(Carl Roth,目录号:C146.1)
  2. 光源(18W/840)(Philips,型号:MASTER TL-D Super 80)
  3. 实验室振动器(一维轨道运动)(Heidolph Instruments GmbH,目录号:036130180)
  4. UV/Vis分光光度计(Shimadzu,型号:UV-2401PC)
  5. 多通道移液管(8通道移液管,0.5-10μl和10-100μl)(Starlab ErgoOne ,目录号:S7108-0510和S7108-1100)
  6. 具有方形形状(120×120×17mm)(Greiner Bio-One GmbH,目录号:688102)的培养皿
  7. 具有可调盖子的透射光扫描器(即爱普生完美v700照片,一个好的相机可以替代)
  8. 层流罩(即 Heraeus HeraSafe HS)
  9. LI-190R量子传感器

程序

每个涉及处理蓝细菌细胞培养物的实验步骤必须在层流罩下进行以防止污染!

  1. 从琼脂平板接种蓝细菌液体培养物   BG11培养基(Rippka等人,1979),并连续振荡温育   (150rpm)在30℃,在50的白光的恒定照明下 在琼脂板的位置测量的2-3μmol/μmol光子m sup -2 s sup-1 天(用LI-190R量子传感器测量)。
    注意:即使 一些突变体携带插入的抗生素抗性基因盒 它们的基因组,用于斑点测定的培养物总是在培养基中生长 缺乏抗生素以排除在选择性培养基上生长的影响。 没有特定的体积或OD需要,但预培养应该 没有达到晚稳定期,它们的OD <750> 应该是相似的。
  2. 将生长良好的预培养物在最终稀释至OD 750±0.2 体积的20ml BG11培养基,并在上述条件下进一步孵育 直到它们达到对数相(约  OD <750> 0.6-0.7)。
    注意:在没有颜料吸收的波长下测量光密度是很重要的。
  3. 将对数生长的培养物再次稀释至OD 750±0.2。 用新鲜稀释的培养物,用a稀释系列  多通道移液管在无菌微量滴定板中,范围从10μL至 10 -7 (图1)。


    图1.活力分析的稀释系列。行B至H用180μlBG11培养基预先填充。 每次稀释步骤后,将细胞悬浮液与移液管充分混合,然后将其一部分转移到下一个孔中

  1. 从每个稀释步骤中,将5μl一式三份点样 使用多通道移液器加入补充的方形BG11琼脂平板上   与0.3%(w/v)Na 2 S 2 O 3 sub的混合物。< br /> 注意:琼脂的确切组成   定制。 如果实验者希望观察下的生存力 混合条件下,BG11琼脂可以补充 葡萄糖。 还可以从介质中排除铁或氮 储备溶液。 然而,不要忘记向板添加Na 2 SubS 2 O 3子。 这对于集落的适当生长是重要的(Thiel等人, 1989)。
  2. 使液体培养物的液滴干燥 约15-20分钟(封闭板,在室温下在桌上 最佳)。然后,将板在30℃恒温孵育 用50μmol光子的白光照射 -2 s -1 约5天或直到最高处没有更多的菌落出现 稀释步骤 注意:防止运动的光线运动 应变,板应放置在光源和侧面下方 应避免仅从一侧照射。为了阻止 滴落(在液滴不完全干燥的情况下),板 与盖子面向上孵育第一天。后第一  天,将板上下颠倒以避免蒸发的水滴 在斑点。对于浅 - 暗相关表型的研究, 恒定照明也可以改变为明暗周期。在我们的 病例,我们调查昼夜节律钟相关基因的突变体 因此选择了12:12小时光照:暗培育。在这种情况下, 将稀释系列的起始OD 750调整为0.4而不是 0.2(第2步)。
  3. 通过扫描记录结果(图2) 板或通过拍照。在最好的情况下,甚至可能 计数几个点(你可能需要一个 双目显微镜)。


    图2.点测定。将细胞培养物的稀释系列一式三份点样到方形BG11琼脂平板上。将板孵育直到在最高稀释步骤中不再出现菌落。菌株已由Dörrich等人公开。 (2014)。

    使用该方法产生的进一步结果由Dörrich等人(2014)出版。

笔记

上述斑点测定法是高度可重复的。与诸如测量液体培养物的光密度的其它方法相反,其通常由于聚集体的形成而易于出现错误,因此我们总是获得可靠和可重复的结果。在我们的小组内,几个研究人员在两个不同的实验室进行了这种方法,我们没有检测到任何结果的变化。为了再现性,强烈推荐 在板中使用Na 2 S 2 Sub 3 O 3(如Recipes中所述),并在同一时间后记录结果。 琼脂板的组成和光条件可以定制以适应实验者的个体需要,并且可能需要优化不同的突变株。

食谱

  1. 100x BG11介质
    CaCl 2 2H O
    3.6 g/L
    柠檬酸
    H3BO3
    2.86 g/L
    MnCl 2 4H O
    1.81 g/L
    ZnSO 4 。 7H O
    0.222 g/L
    Na 2 MoO 4 sub 。 2H O
    0.390g/L
    Co(NO <3>) 2 6H <2> O
    0.049克/升
    在ddH 2 O 2中制备这些溶液,并使用注射器和无菌过滤器过滤灭菌
    无菌等分试样可以在4℃下保存
  2. BG-11培养基的库存解决方案
    Na 2 3
    20 mg/ml
    柠檬酸铁铵(III)
    6 mg/ml
    K 2 HPO 4 3H 2 O
    30 mg/ml
    TES缓冲液(pH8.0用NaOH调节)
    1 M
    准备这些解决方案在ddH2O和灭菌使用注射器和无菌过滤器过滤
    无菌等分试样可以在4℃下保存
  3. 1x BG11(2x BG11)
    100 x BG11
    10ml/L(20ml/L)
    Na 2 CO 2 3储备溶液 1ml/L(2ml/L)
    K 2 HPO 4 3H <2> O储备液
    1ml/L(2ml/L)
    柠檬酸铁铵(III)储液
    1ml/L(2ml/L)
    痕量金属混合原液
    1ml/L(2ml/L)
    TES缓冲液(pH 8.0)储液
    10ml/L(20ml/L)
    将无菌溶液加入ddH 2 O中,并将体积调节至1L
    如果你使用非无菌溶液和高压灭菌媒体后,会有少量盐沉淀。 在使用介质之前,应该彻底混合。 这是建议,当你想避免污染的文化,特别是对长期应变维持。
  4. BG11琼脂
    在ddH 2 O中制备1.5%细菌用的琼脂和高压灭菌器。
    将熔化的1.5%细菌细菌(约80℃)与相同体积的无菌2x BG11培养基混合。
  5. 溶液
    溶液 30%w/v Na 2 S sub 2 O 3 sub / 30g Na 2 2 S 2 O 3 sub
    将ddH 2 O加到100 ml
    过滤灭菌(0.2μm)
    储存在4°C
  6. 0.25M Na 2 -EDTA(pH 8) 9.306g Na 2 2 -EDTA
    将ddH 2 O加到100 ml
    用NaOH调节pH至8.0(使用高浓度NaOH,Na 2 EDTA不会溶解,直到pH达到8.0)
    在室温下储存

致谢

BG11培养基根据Rippka等人制备。 (1979)。这项工作是由DFG授予A.W.Wi2014/5-1支持的。

参考文献

  1. Dörrich,A.K.,Mitschke,J.,Siadat,O。和Wilde,A。(2014)。 删除 Synechocystis sp。在微暗条件下,PCC 6803 kaiAB1C1基因簇引起受损的细胞生长。微生物学 160(Pt 11):2538-2550。
  2. Rippka,R.,Deruelles,J.,Waterbury,J.B.,Herdman,M。和Stanier,R.Y。(1979)。 通用作业,应变历史和纯文化属性> Cyanobacteria 。 J Gen Microbiol (111):1-61。
  3. Thiel,T.,Bramble,J。和Rogers,S。(1989)。 在固体介质上生长蓝藻的最佳条件 。 FEMS Microbiol Lett 52(1-2):27-31。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Dörrich, A. K. and Wilde, A. (2015). Spot Assays for Viability Analysis of Cyanobacteria. Bio-protocol 5(17): e1574. DOI: 10.21769/BioProtoc.1574.
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