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Large-scale Phenotypic Profiling of Gene Deletion Mutants in Candida glabrata
对光滑念珠菌中的基因缺失突变体进行大规模表型分析

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Abstract

Here, we describe a method enabling the phenotypic profiling of genome-scale deletion collections of fungal mutants to detect phenotypes for various stress conditions. These stress conditions include among many others antifungal drug susceptibility, temperature-induced and osmotic as well as heavy metal or oxidative stress. The protocol was extensively used to phenotype a collection of gene deletion mutants in the human fungal pathogen Candida glabrata (C. glabrata) (Schwarzmüller et al., 2014).

Keywords: Phenotyping(表型), Antifungal Drug Susceptiility(抗真菌药物susceptiility), Stress Phenotypes(压力表型), Morphology(形态学), Robotic Screening(机器人筛选)

Materials and Reagents

  1. C. glabrata strain (ATCC2001, clinical isolates)
  2. Sterile water (double distilled)
  3. Bacto™ peptone (BD Biosciences, catalog number: 211820 )
  4. Bacto™ yeast extract (BD Biosciences, catalog number: 212720 )
  5. Bacto™ agar (BD Biosciences, catalog number: 214030 )
  6. Difco™ yeast nitrogen base (YNB) (BD Biosciences, catalog number: 233520 )
  7. Glucose (Merck KGaA, catalog number: 108337 )
  8. Adenine (Sigma-Aldrich, catalog number: A8626 )
  9. L-arginine (Sigma-Aldrich, catalog number: A5006 )
  10. L-tyrosine (Sigma-Aldrich, catalog number: T3754 )
  11. L-isoleucine (Sigma-Aldrich, catalog number: I2752 )
  12. L-phenylalanine (Sigma-Aldrich, catalog number: P2126 )
  13. L-glutamic acid (Sigma-Aldrich, catalog number: G1251 )
  14. L-aspartic acid (Sigma-Aldrich, catalog number: A9256 )
  15. L-threonine (Sigma-Aldrich, catalog number: T8625 )
  16. L-serine (Sigma-Aldrich, catalog number: S4500 )
  17. L-valine (Sigma-Aldrich, catalog number: V0500 )
  18. L-methionine (Sigma-Aldrich, catalog number: M9625 )
  19. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  20. YPD media (see Recipes)
  21. Solid YPD media (see Recipes)
  22. 2x YPD media (see Recipes)
  23. SC media (see Recipes)
  24. Solid SC media (see Recipes)
  25. Amino acid mix (see Recipes)
  26. 15% glycerol solution (see Recipes)

Equipment

  1. Square plates (PlusPlate) (40 ml/plate) (Singer Instruments, catalog number: PLU-001 )
  2. Pin Pad (RePad 96 Long) (Singer Instruments, catalog number: REP-001 )
  3. 96-well plates (200 µl/well) (Starlab, catalog number: CC76827596 )
  4. Petri dishes (92 mm) (SARSTEDT AG, catalog number: 82.1473 )
  5. Culture tubes (Starlab, catalog number: I14850810 )
  6. Yeast replica robot (Singer Instruments, catalog number: RoToR HDA )
  7. Plate reader (Victor3V) (PerkinElmer, catalog number: 1420040 )
  8. 48-well replica stamp (V&P Scientific, catalog number: VP 408H )
  9. 96-well replica stamp (V&P Scientific, catalog number: VP 407 )
  10. Library copier (guide for stamps) (V&P Scientific, model: VP 381 )
  11. Plate mixer (Eppendorf, catalog number: 5353000014 )
  12. Rotary shaker for culture flasks (innova44) (New Brunswick Scientific, catalog number: M1282-0002 )
  13. Incubator (Heraeus Instruments, catalog number: B6120 )

Procedure

  1. Arraying and storage
    1. For arraying deletion strains in the 96-well format see the Note section. Arrayed strains can be suspended in 15% glycerol and stored frozen at minus 70 °C in 96-well plates.
    2. Frozen stocks can be either handled by manual replicating tools or by an automated system such as the RoToR HDA by Singer Instruments. The latter offers a higher reproducibility and reduces the risk of contamination when compared to manual systems. Therefore, this protocol for large-scale screening was optimized for the work with the Singer HDA RoToR replica-plating robot.

  2. Initial large-scale screening (on solid YPD media)
    1. For initial pilot screenings, prepare square plates containing the screening compounds and control plates lacking compounds as shown in Figure 1. The concentration of the compound for the solid screening plate is usually twice the concentration of the IC50. However, the exact concentration should be determined prior to the large-scale screening by plating control strains.
      1. To exclude replica-plating errors, at least two plates should be prepared for each condition (stress-inducing compound, stress-inducing media, temperature, etc.).
      2. Source plates can be either liquid media (96-well microtiter plates) or solid media (square plates).
    2. Dry plates on a clean bench for 10 min, for light-sensitive compounds turn off lights.
      1. Make sure that no droplets of condensed water are on the plates or lids before using them.
      2. Label plates before spotting strains.
        When using a Singer robot use the following settings in the ROTOR HDA software:
      3. For solid source plates activate the “offset” function, to avoid pins taking cells from exactly the same spot.
      4. If liquid source plates are used, enable the “wet mix” option in the software menu (diameter: 1 mm; speed: 3 mm/sec; cycles: 1; Travel: 2D). Alternatively, liquid source plates can be shaken on a MixMate for 15 sec at 750 rpm.
      5. If the so-called RePads are used for more than one target plate (“Recycle Mode”), the “Revisit Source” option should be enabled as well.
    3. After plating, turn plates upside down as soon as spots have dried. In case of solid source plates, the target plates can be turned right away. Incubate plates for 72 h at 30 °C without shaking and inspect colony growth. Pictures should be taken of every plate at 24, 48 and 72 h. Visually compare the growth of each mutant strains and controls under stress and without stress with the growth of wild type strain(s). Important points that should be considered are shown in Figures 2 and 3.

  3. Serial dilution spot assays (solid SC media)
    1. For refined screening, prepare at least two SC media plates for each condition. Prior to their use, dry them on a clean bench. Grow overnight cultures of sensitive and wild type strains from the 96-well screening in SC medium at 30 °C with shaking at 200 rpm (200 rpm are used for growth in culture tubes; a total amount of 5 ml for each culture is sufficient). Dilute cultures into fresh SC medium resulting in an OD600 of ≈ 0.2 and regrow them to an OD600 of ≈ 1.0. Prepare 1:10 serial dilutions of deletions strains in sterile water in a 96-well plate, starting with an OD600 of 0.1.

      Table 1. Serial dilution of cells
      OD600
      Dilution
      Cells/m
      0.1
      1:1
      3 x 106
      0.01
      1:10
      3 x 105
      0.001
      1:100
      3 x 104
      0.0001
      1:1,000
      3 x 103

    2. Spotting uses a 48-well metal pin replicator, also referred to as “frogger” or “hopper”.
      1. Sterilize the tool by submerging the pins in EtOH and brief flaming over a bunsen burner. Repeat this process three times and let the pins cool down.
      2. Stir the serial dilution in the 96-well plate using the replicator and retract the pins from the wells without touching the wall of the wells.
      3. A droplet of cell suspension will remain on each tip of the replicator.
      4.  Place the replicator on a fresh screening plate and apply gentle pressure. Lift the replicator straight up, avoid an angle and repeat the process for other plates.
      5. Incubate the plates as described above at step B3 and takes pictures of every plate after 24, 48 and 72 h of incubation.
    3.  Alternatively, spotting can be performed using a multichannel or a single-channel pipette. When using a pipette, a grid should be placed under the plate to make sure the drops are placed in a straight line.
    4.  After the drops have dried, turn the plates upside down and incubate them at 30 °C for 72 h without shaking. Compare growth of the serial dilutions of each mutant and the wild type control under stress and without stress.
  4. Microdilution of compounds (liquid media)
    1. Grow overnight cultures of deletion and control wild type strains in YPD at 30 °C with shaking at 200 rpm (as described in step C1, 200 rpm are used for culture tubes; 5 ml of each culture are sufficient). Dilute strains in fresh YPD media resulting in an OD600 of ≈ 0.2 and regrow them until an OD600 of ≈ 1.0. Dilute cell suspensions in 2x YPD to a final concentration of 2.5 x 103 cells/ml.
    2. Prepare a series of ten two-fold dilutions of the compound to be tested in sterile water. Fill each well with 100 µl of compound dilution or water. Rows 11 and 12 should only contain sterile water and be used as growth and no-growth control.
    3. Add 100 µl cell suspension in 2x YPD to the wells of the 96-well plate. Seal the plate using a clear breathable foil and incubate the plate at 30 °C without shaking for 48 h. Measure the OD600 on a plate reader after 24 and 48 h. Before reading the plate mix it at 1,000 rpm for 15 sec. Calculate compound concentrations required to obtain 50 % growth inhibition (IC50) for each strain.

  5. Evaluation of results
    1. Since the large-scale screening may result in false-positive hits, it needs independent unbiased inspection of primary results. Therefore, the screenings described in sections C-D should be independently analyzed by at least two persons.


      Figure 1. Screening process-from large scale to single strains. Putative hits (purple, blue, pink) are identified by comparing growth of strains on a control plate (YPD only) with a plate supplemented with screening compounds. In addition, deletion strains must be compared to the corresponding wild types (first two rows). These strains are then used for a refined screening in liquid assays through serial dilution on solid media to confirm primary phenotypes.

    2. To validate the link between a genotype and a phenotype more experiments are necessary. These experiments include the complementation of deletion strains and the deletion of a single gene in different backgrounds.

Notes

  1. Plate layouts
    When designing the layout several aspects should be considered.
    Outer spots grow faster.
    Outer spots are framed red in Figure 2 and show a faster growth when compared to spots in inner rows and columns.


    Figure 2. Faster growth of outer spots

    This phenomenon may hamper comparison of growth phenotypes of deletion strains to the growth of a wild type strain. In the worst case, this may lead to missing relevant phenotype. This problem can be solved by filling all outer rows / columns with a dummy strain (e.g. the wild type). Therefore, all strains that are to be screened are surrounded on all sides and experience the same growth conditions. Of note, this procedure yields only 60 usable spots. Alternatively, a 96-well plate can be spotted in quadruplicate for each deletion in the 384-well format using the Singer HDA RoToR. This method will result in each deletion strain being spotted 4 times on the agar plate as illustrated in Figure 3.


    Figure 3. 96-well to 384-well format. The 384-well format (right panel) offers two distinct advantages when compared to the 96-well format (left panel). Only the outer spots grow faster, so every original spot has at least one spot now that grows normally. In addition, it can be easier to identify growth phenotypes due to the uniform appearance of growth patterns.

    Include empty spots for controls
    To compare results, at least the background strain should be spotted on each plate. Since this strain is usually derived from a wild type strain, the wild type should also be included. Moreover, it is very useful to include empty spots on each plate and to add additional positive and negative control strains for a specific screening. For instance, it can be very helpful to temporarily add a known hypersensitive strain for a specific screening as positive control [for an azole screening a pdr1∆ (CAGL0A00451g) strain is a great control].
  2. Liquid source plates are preferable to solid source plates, since the reproducibility is higher when using liquid sources plates. We experienced that the amount of cells can be inconsistent when using solid source plates. Liquid source plates can be shaken prior to plating, resulting in a very homogenous source cell suspension.
  3. There are various standardized protocols available for the microdilution assays to quantify drug susceptibilities including those of the Clinical and Laboratory Standards Institute (Fothergill et al., 2011) or the European Committee on Antimicrobial Susceptibility Testing (Arendrup et al., 2012).
  4. A video of the Singer RoToR in use is available at:
    https://www.youtube.com/watch?v=1JabqneqXPE.
  5. Stocks for water insoluble compounds can be prepared in the appropriate solvent (e.g. DMSO). However, the dilution should be done in water. Otherwise, the wells would contain up to 50% of solvent.

Recipes

  1. YPD media (yeast extract peptone dextrose)
    25 g/L Bacto™ peptone
    12.5 g/L Bacto™ yeast extract
    2% glucose
  2. Solid YPD media
    25 g/L Bacto™ peptone
    12.5 g/L Bacto™ yeast extract
    2% glucose
    2% agarose
  3. 2x YPD media
    50 g/L Bacto™ peptone
    25 g/L Bacto™ yeast extract
    4% glucose
  4. SC media (synthetic complete)
    3.4 g/L Difco™ YNB
    2.9 g/L amino acid mix
    2 % glucose
  5. Solid SC media
    3.4 g/L Difco™ YNB
    2.9 g/L amino acid mix
    2 % glucose
    2 % agarose
  6. Amino acid mix (g per 11.7 g total mix)
    0.4 g adenine
    0.2 g L-arginine
    0.3 g L-tyrosine
    0.3 g L-isoleucine
    0.5 g L-phenylalanine
    1.0 g L-glutamic acid
    1.0 g L-aspartic acid
    2.0 g L-threonine
    4.0 g L-serine
    1.5 g L-valine
    0.5 g L-methionine
  7. 15% glycerol solution
    150 ml/L glycerol
    850 ml/L water

Acknowledgements

This work was supported by the Austrian Science Foundation FWF through the ERA-Net Pathogenomics project FunPath (FWF-API-0125), and in part by grants from the Christian Doppler Society, the FP7 EC project FUNGITECT, the Marie-Curie ITN ImResFun (MC-ITN-606786) and the FWF Project FWF-P25333 "Chromatin" to KK.

References

  1. Arendrup, M. C., Cuenca-Estrella, M., Lass-Florl, C., Hope, W. and Eucast, A. (2012). EUCAST technical note on the EUCAST definitive document EDef 7.2: method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts EDef 7.2 (EUCAST-AFST). Clin Microbiol Infect 18(7): E246-247.
  2. Fothergill, A. W. (2011). Antifungal susceptibility testing: clinical laboratory and standards institute (CLSI) methods. Interactions of Yeasts, Moulds, and Antifungal Agents 65-74.
  3. Schwarzmuller, T., Ma, B., Hiller, E., Istel, F., Tscherner, M., Brunke, S., Ames, L., Firon, A., Green, B., Cabral, V., Marcet-Houben, M., Jacobsen, I. D., Quintin, J., Seider, K., Frohner, I., Glaser, W., Jungwirth, H., Bachellier-Bassi, S., Chauvel, M., Zeidler, U., Ferrandon, D., Gabaldon, T., Hube, B., d'Enfert, C., Rupp, S., Cormack, B., Haynes, K. and Kuchler, K. (2014). Systematic phenotyping of a large-scale Candida glabrata deletion collection reveals novel antifungal tolerance genes. PLoS Pathog 10(6): e1004211.

简介

在这里,我们描述了一种使表型分析的基因组规模删除集合的真菌突变体检测表型的各种应力条件的方法。 这些胁迫条件包括许多其它抗真菌药物敏感性,温度诱导和渗透以及重金属或氧化应激。 该方案广泛用于表达人真菌病原体光滑假丝酵母( glabrata )中基因缺失突变体的集合(Schwarzmüller等人 >,2014)。

关键字:表型, 抗真菌药物susceptiility, 压力表型, 形态学, 机器人筛选

材料和试剂

  1. C。 glabrata 株(ATCC2001,临床分离株)
  2. 无菌水(双蒸)
  3. Bacto TM蛋白胨(BD Biosciences,目录号:211820)
  4. Bacto TM酵母提取物(BD Biosciences,目录号:212720)
  5. Bacto TM琼脂(BD Biosciences,目录号:214030)
  6. Difco TM酵母氮源(YNB)(BD Biosciences,目录号:233520)
  7. 葡萄糖(Merck KGaA,目录号:108337)
  8. 腺嘌呤(Sigma-Aldrich,目录号:A8626)
  9. L-精氨酸(Sigma-Aldrich,目录号:A5006)
  10. L-酪氨酸(Sigma-Aldrich,目录号:T3754)
  11. L-异亮氨酸(Sigma-Aldrich,目录号:I2752)
  12. L-苯丙氨酸(Sigma-Aldrich,目录号:P2126)
  13. L-谷氨酸(Sigma-Aldrich,目录号:G1251)
  14. L-天冬氨酸(Sigma-Aldrich,目录号:A9256)
  15. L-苏氨酸(Sigma-Aldrich,目录号:T8625)
  16. L-丝氨酸(Sigma-Aldrich,目录号:S4500)
  17. L-缬氨酸(Sigma-Aldrich,目录号:V0500)
  18. L-甲硫氨酸(Sigma-Aldrich,目录号:M9625)
  19. 甘油(Sigma-Aldrich,目录号:G5516)
  20. YPD介质(参见配方)
  21. 固体YPD介质(参见配方)
  22. 2x YPD介质(参见配方)
  23. SC介质(参见配方)
  24. 固体SC介质(参见配方)
  25. 氨基酸混合物(见配方)
  26. 15%甘油溶液(见配方)

设备

  1. 方形板(PlusPlate)(40ml /板)(Singer Instruments,目录号:PLU-001)
  2. Pin Pad(RePad 96 Long)(Singer Instruments,目录号:REP-001)
  3. 96孔板(200μl/孔)(Starlab,目录号:CC76827596)
  4. 培养皿(92mm)(SARSTEDT AG,目录号:82.1473)
  5. 培养管(Starlab,目录号:I14850810)
  6. 酵母复制机器人(Singer Instruments,目录号:RoToR HDA)
  7. 读板器(Victor 3)(PerkinElmer,目录号:1420040)
  8. 48孔复制印模(V& P Scientific,目录号:VP 408H)
  9. 96孔复制印模(V& P Scientific,目录号:VP407)
  10. 图书馆复印机(邮票指南)(V& P Scientific,型号:VP 381)
  11. 板混合器(Eppendorf,目录号:5353000014)
  12. 用于培养瓶的旋转振荡器(innova44)(New Brunswick Scientific,目录号:M1282-0002)
  13. 孵育器(Heraeus Instruments,目录号:B6120)

程序

  1. 阵列和存储
    1. 对于在96孔格式中排列缺失菌株,参见注释 部分。 阵列菌株可以悬浮在15%甘油中并储存 在96孔板中于负70℃冷冻。
    2. 冷冻库存可以 通过手动复制工具或通过自动化系统处理 例如Singer Instruments的RoToR HDA。 后者提供了更高的 重现性,并降低污染的风险 手动系统。 因此,这个大规模筛选的协议 优化了与歌手HDA RoToR复制机器人的工作
  2. 初始大规模筛选(在实体YPD培养基上)
    1. 对于初始试验筛选,制备方形板 筛选化合物和缺少如图所示的化合物的对照板 图1.用于固体筛选的化合物的浓度 板通常是IC 50的浓度的两倍。 但是,确切   浓度应在大规模筛选前确定 通过电镀控制株
      1. 要排除复制电镀错误,请 至少两个板应准备每个条件(应激诱导 化合物,应激诱导介质,温度,等)。
      2. 来源板可以是液体培养基(96孔微量滴定板)或固体培养基(正方形板)。
    2. 干板在干净的工作台上10分钟,对于光敏化合物关闭灯。
      1. 确保在使用之前,板或盖子上没有冷凝水滴。
      2. 标记板前点样菌株。
        使用Singer机器人时,在ROTOR HDA软件中使用以下设置:
      3. 对于固体源板激活"偏移"功能,以避免针从完全相同的斑点接收细胞。
      4. 如果使用液体源板,请启用"湿混合"选项 软件菜单(直径:1mm;速度:3mm /秒;循环:1;行程:2D)。 或者,液体源板可以在MixMate上振荡15分钟 秒,750rpm
      5. 如果所谓的RePads用于多个   目标盘("回收模式"),"再访源"选项应该 启用。
    3. 电镀后,立即将板倒置   斑点已干燥。 在固体源板的情况下,目标板可以   立即转身。 孵育板在30°C下72小时,不摇动   并检查集落生长。 应拍摄每张照片 24,48和72小时。 视觉比较每个突变株的生长和 控制胁迫下和无胁迫与野生型的生长 应变。 应该考虑的重点如下所示 图2和图3。

  3. 连续稀释点测定(固体SC培养基)
    1. 对于精制筛选,为每个至少制备两个SC培养基板 条件。 在使用前,在干净的工作台上干燥。 成长过夜   培养来自96孔筛选的敏感型和野生型菌株 在SC培养基中在30℃下以200rpm振荡(200rpm) 培养管中的生长; 每种培养物的总量为5ml 足够)。 将培养物稀释到新鲜的SC培养基中,导致OD 600约为0.2,并将它们再生至OD≈1.0的600。 准备1:10连续 在96孔板中的无菌水中的缺失菌株的稀释液, 从OD 600 为0.1开始。

      表1.细胞的系列稀释
      OD 600
      稀释
      单元格/m
      0.1
      1:1
      3 x 10 6
      0.01
      1:10
      3 x 10 5
      0.001
      1:100
      3 x 10 4
      0.0001
      1:1,000
      3 x 10 3

    2. 点样使用48孔金属针复制器,也称为"frogger"或"料斗"。
      1. 通过将销浸没在EtOH中和短暂燃烧灭菌工具 在本生灯。 重复这个过程三次,让针 冷却。
      2. 使用在96孔板中连续稀释 复制器并从孔中收回销而不接触 墙的井。
      3. 细胞悬浮液液滴将保留在复制器的每个末端上。
      4.  将复制器放在一个新的筛板上,然后使用温柔 压力。 向上提起复制器,避免角度重复 过程为其他板。
      5. 如上所述在步骤B3孵育平板,并在孵育24,48和72小时后拍摄每个平板的照片。
    3.  或者,可以使用多通道或a 单通道移液器。 使用移液器时,应放置网格 在板下,以确保液滴放置在一条直线上
    4.  滴剂干后,将盘子倒置并孵育 它们在30℃下72小时而不摇动。 比较串行的增长 在胁迫下每个突变体和野生型对照的稀释度 没有压力。
  4. 微量稀释化合物(液体介质)
    1. 生长过夜的缺失培养物并控制野生型菌株 YPD在30℃下以200rpm振动(如步骤C1中所述,200rpm 用于培养管; 每种培养物5ml是足够的)。 稀   菌株在新鲜YPD培养基中,导致OD 600约为0.2并再生长 直到OD <600>≈1.0。 稀释细胞悬浮液在2x YPD中 最终浓度为2.5×10 3个细胞/ml
    2. 准备一系列十   两倍稀释的待测化合物在无菌水中。 填 每孔加入100μl化合物稀释液或水。 第11行和第12行 应仅含无菌水并用作生长和不生长 控制
    3. 添加100微升细胞悬浮液在2x YPD的孔中 96孔板。 使用透明透气箔密封板,孵化   平板在30℃下振荡48小时。 在a上测量OD 600 24和48小时后。 在读板之前,混合1000   rpm,15秒。 计算获得50所需的化合物浓度   %生长抑制(IC 50)。

  5. 结果评价
    1. 由于大规模筛选可能导致假阳性命中,它   需要对主要结果进行独立的无偏见检查。 因此, 在C-D节中描述的筛选应该是独立的 至少由两个人分析

      图1.筛选 过程 - 从大规模到单一菌株。推定命中(紫色,蓝色,   粉红色)通过比较对照板上菌株的生长来鉴定 (仅YPD)与补充筛选化合物的板。 在 此外,缺失菌株必须与相应的野生型比较 类型(前两行)。 然后将这些菌株用于精制 在液体测定中通过在固体培养基上连续稀释进行筛选 确认原发性表型
    2. 验证a之间的链接 基因型和表型更多的实验是必要的。 这些 实验包括缺失菌株的互补和 在不同背景中缺失单个基因。

笔记

  1. 版面布局
    在设计布局时,应考虑几个方面 外点增长更快。
    外部斑点在图2中呈红色,并且与内部行和列中的斑点相比显示更快的生长

    图2.外部点的更快速增长

    这种现象可能妨碍缺失菌株的生长表型与野生型菌株的生长的比较。在最坏的情况下,这可能导致缺失相关表型。这个问题可以通过用虚拟应变(例如。野生型)填充所有外部行/列来解决。因此,所有待筛选的菌株在所有侧面被包围并经历相同的生长条件。值得注意的是,这个过程只产生60个可用点。或者,可以使用Singer HDA RoToR,以384-孔格式每个缺失一式四份地点样96孔板。该方法将导致每个缺失菌株在琼脂平板上点样4次,如图3所示。


    图3. 96孔至384孔格式。 与96孔格式(左图)相比,384孔格式(右图)提供了两个明显的优势。只有外部斑点生长更快,因此每个原始斑点至少有一个斑点现在正常生长。此外,由于生长模式的均匀外观,可以更容易地鉴定生长表型
    包括控制空白点
    为了比较结果,至少背景应变应当在每个板上点样。由于该菌株通常来源于野生型菌株,因此也应包括野生型菌株。此外,在每个平板上包括空白点并添加额外的阳性和阴性对照菌株用于特定筛选是非常有用的。例如,对于特定筛选,作为阳性对照临时添加已知的过敏性菌株可能是非常有帮助的[对于azole筛选,pdr1Δ(CAGL0A00451g)菌株是一个很好的控制]。br/>
  2. 液体源板优于固体源板,因为当使用液体源板时的再现性更高。我们经历,当使用固体源板时,细胞的量可能不一致。液体源板可以在电镀之前摇动,导致非常均匀的源细胞悬浮液
  3. 存在各种用于微量稀释测定以量化药物敏感性的标准化方案,包括临床和实验室标准研究所(Fothergill等人,2011)或欧洲抗菌药物敏感性试验委员会(Arendrup et al。,2012)。
  4. 正在使用的歌手RoToR的视频在以下位置提供:
    https://www.youtube.com/watch?v=1JabqneqXPE
  5. 水不溶性化合物的储备液可以在合适的溶剂(例如 DMSO)中制备。 然而,稀释应在水中进行。 否则,孔将含有高达50%的溶剂。

食谱

  1. YPD培养基(酵母提取物蛋白胨葡萄糖)
    25g/L Bacto TM蛋白胨 12.5g/L Bacto TM酵母提取物
    2%葡萄糖
  2. 实体YPD介质
    25g/L Bacto TM蛋白胨 12.5g/L Bacto TM酵母提取物
    2%葡萄糖 2%琼脂糖
  3. 2x YPD媒体
    50g/L Bacto TM蛋白胨 25g/L Bacto TM酵母提取物
    4%葡萄糖
  4. SC介质(合成完成)
    3.4g/L Difco TM YNB
    2.9g/L氨基酸混合物
    2%葡萄糖
  5. 固体SC介质
    3.4g/L Difco TM YNB
    2.9g/L氨基酸混合物
    2%葡萄糖 2%琼脂糖
  6. 氨基酸混合物(g/11.7g总混合物)
    0.4克腺嘌呤
    0.2克L-精氨酸 0.3克L-酪氨酸 0.3克L-异亮氨酸 0.5克L-苯丙氨酸 1.0g L-谷氨酸 1.0g L-天冬氨酸 2.0克L-苏氨酸 4.0克L-丝氨酸 1.5克L-缬氨酸 0.5克L-甲硫氨酸
  7. 15%甘油溶液
    150ml/L甘油 850 ml/L水

致谢

这项工作得到奥地利科学基金会FWF通过ERA-Net Pathogenomics项目FunPath(FWF-API-0125)的支持,部分得到了基督教多普勒学会,FP7 EC项目FUNGITECT,Marie-Curie ITN ImResFun MC-ITN-606786)和FWF Project FWF-P25333"染色质"

参考文献

  1. Arendrup,M.C.,Cuenca-Estrella,M.,Lass-Florl,C.,Hope,W。和Eucast,A。(2012)。 EUCAST技术说明EUCAST定义文件EDef 7.2: 用于测定酵母菌的抗真菌剂的肉汤稀释最小抑制浓度的方法EDef 7.2(EUCAST-AFST)。临床微生物感染 18(7):E246-247。
  2. Fothergill,A.W。(2011)。 抗真菌药敏试验:临床实验室和标准研究所(CLSI)方法 酵母,霉菌和抗真菌代理 65-74。
  3. Schwarzmuller,T.,Ma,B.,Hiller,E.,Istel,F.,Tscherner,M.,Brunke,S.,Ames,L.,Firon,A.,Green,B.,Cabral, Marcet-Houben,M.,Jacobsen,ID,Quintin,J.,Seider,K.,Frohner,I.,Glaser,W.,Jungwirth,H.,Bachellier-Bassi,S.,Chauvel,M.,Zeidler, U.,Ferrandon,D.,Gabaldon,T.,Hube,B.,d'Enfert,C.,Rupp,S.,Cormack,B.,Haynes,K.and Kuchler,K。 大型的假丝酵母(Candida glabrata)缺失集合的系统表型揭示了新的抗真菌耐受基因。 PLoS Pathog 10(6):e1004211。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Istel, F., Schwarzmüller, T., Tscherner, M. and Kuchler, K. (2015). Large-scale Phenotypic Profiling of Gene Deletion Mutants in Candida glabrata. Bio-protocol 5(14): e1530. DOI: 10.21769/BioProtoc.1530.
  2. Schwarzmuller, T., Ma, B., Hiller, E., Istel, F., Tscherner, M., Brunke, S., Ames, L., Firon, A., Green, B., Cabral, V., Marcet-Houben, M., Jacobsen, I. D., Quintin, J., Seider, K., Frohner, I., Glaser, W., Jungwirth, H., Bachellier-Bassi, S., Chauvel, M., Zeidler, U., Ferrandon, D., Gabaldon, T., Hube, B., d'Enfert, C., Rupp, S., Cormack, B., Haynes, K. and Kuchler, K. (2014). Systematic phenotyping of a large-scale Candida glabrata deletion collection reveals novel antifungal tolerance genes. PLoS Pathog 10(6): e1004211.
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