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Genetic Transformation of Candida glabrata by Heat Shock
采用热激法对光滑念珠菌进行遗传操作   

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Abstract

Here, we report a method for the transformation of Candida glabrata using a heat shock method. The protocol can be used for transformations in single well or in 96-well scale. It has been employed as an alternative method to the electroporation protocol to construct a genome-scale gene deletion collection in the human fungal pathogen Candida glabrata ATCC2001 and related strains. Furthermore, the protocol can be used to generate gene deletions in clinical isolates of Candida glabrata (C. glabrata).

Keywords: Candida glabrata(光滑念珠菌), Transformation(转型), Heat Shock(热休克), Knock-Out(敲), Dominant Selection(优势选择)

Materials and Reagents

  1. Recipient strain [ATCC2001, HTL or clinical isolates (Schwarzmuller et al., 2014)]
  2. DNA deletion construct/transforming DNA
  3. Sterile water (double distilled)
  4. Bacto™ peptone (BD Biosciences, catalog number: 211820 )
  5. Bacto™ yeast extract (BD Biosciences, catalog number: 212720 )
  6. Bacto™ agar (BD Biosciences, catalog number: 214030 )
  7. Glucose (Merck KGaA, catalog number: 108337 )
  8. Lithium acetate dehydrate (LiAc) (Sigma-Aldrich, catalog number: L6883 )
  9. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 472301 )
  10. Polyethylene glycol (PEG 3350) (Sigma-Aldrich, catalog number: P4338 )
  11. Nourseothricin-dihydrogen sulfate (Werner BioAgents, catalog number: 5.0 )
  12. ssDNA (Sigma-Aldrich, catalog number: D1626 )
  13. YPD media (see Recipes)
  14. Solid selective media (see Recipes)

Equipment

  1. Deep well plate (96-well) (Nunc®, catalog number: 732-2662 )
  2. Reservoir (autoclavable) (VWR International, catalog number: 6130466 )
  3. Multichannel pipette (200 µl) (Brandt Transferpette, WU2160016)
  4. Culture flasks with baffles
  5. Centrifuge (50 ml tubes) (Eppendorf, catalog number: 5702R )
  6. Centrifuge (1.5 ml tubes) (Eppendorf, catalog number: 5417R )
  7. Rotary shaker for culture flasks (New Brunswick Scientific, catalog number: innova44 )

Procedure

  1. Culture preparation
    1. Grow background strains overnight in YPD media at 30 °C with shaking in flasks at 160 rpm.
    2.  Dilute the culture into fresh YPD medium to an OD600 of about 0.3, and regrow until an OD600 of 1.5 is reached (for 96-well heat shock transformations, 600 ml of culture are required).
    3. Harvest cultures in 50 ml Falcon tubes by centrifugation at 1,000 x g for 5 min.
  2. Treatment of cells
    1. Wash cell pellets twice with 25 ml of sterile water, centrifuge and discard supernatants. Resuspend cell pellets gently in 1 ml of 100 mM LiAc.
    2. Combine all cell suspensions from one strain in two 50 ml tubes.
    3.  After centrifugation at 1,000 x g for 30 sec, add the following sterile solution for each 50 ml of cells grown in step 1 in the order they are listed:
      1,920 µl 50 % PEG-3350
      400 µl ssDNA (10 mg/ml; heat-denatured)
      288 µl of 1 M LiAc
    4. Gently resuspend cells by aspirating with a pipette.
  3. Heat-shock

    96-well scale
    1. Prior to preparing competent cells, place 50 µl of transformation DNA constructs at the bottom of wells of a deep well plate.
    2. Add 326 µl of cell suspension to each well and mix gently by aspiration with a multi-channel or single-channel pipette. Seal the plate using a breathable adhesive foil.
    3. Incubate plates for 30 min at 30 °C in an incubator without shaking.
    4. Add 45 µl of DMSO and mix immediately by aspirating with a pipette.
    5. Incubate plates at 42 °C in an incubator without shaking for exactly 15 min.
    6. Centrifuge plates at 1,000 x g for 5 min and remove supernatants.
    7. Add 950 µl of YPD media and gently resuspend fungal cells by aspirating with a pipette.

    Single-well scale
    1. Add 326 µl of the cell suspension to a 1.5 ml tube containing 50 µl transformation DNA constructs and gently mix by aspirating.
    2. Incubate tubes at 30 °C for 30 min. Add 45 µl of DMSO and mix immediately.
    3. Incubate tubes at 42 °C for exactly 15 min without agitation.
    4. Sediment cells by centrifugation at 1,000 x g for 5 min and remove supernatants by aspiration.
    5. Add 950 µl of YPD medium and gently resuspend cells.
  4. Regeneration of cells
    1. Incubate microtiter plates or tubes at 30 °C for 1 to 4 h at 30 °C without shaking.
    2. Afterwards, centrifuge tubes or plates at 1,000 x g for 5 min.
    3. Discard supernatants and resuspend cell pellets in 100 µl of sterile water.
    4. Plate cell suspensions on selective medium and incubate the plates at 30 °C for a few days until colonies become visible.

Notes

  1. This transformation protocol was optimized for C. glabrata ATCC2001 and all derived strains, as well as for clinical isolates of C. glabrata.
  2. The speed of rotary shaker depends on the type of culture flasks used. Flasks without baffles require higher shaking speeds around about 220 rpm for good oxygenation.
  3. Handle cells VERY gently after adding LiAc (no vortex-mixing!) and keep them on ice. Add the LiAc after adding sterile water and TE buffer (this automatically dilutes the LiAc to the appropriate concentration).
  4. In section “Procedure” we mention that cell pellets should be resuspended carefully by pipetting. For this step we recommend a manual 1,000 µl pipette. By slow aspiration and release the cell pellet can be gently resuspended.
  5. The required regeneration time depends on the selective marker. We experienced that transformants with a HIS3 marker can be plated after 1 h, while those with a NAT1 marker may require up to 4 h of regeneration.
  6. The ssDNA solution is prepared according to the manual described in Molecular cloning (Sambrook and Russell, 2001). Each aliquot is heated to 95 °C for 5 min and immediately cooled on ice before use.

Recipes

  1. YPD media (yeast extract peptone dextrose)
    25 g/L Bacto ™ peptone
    12.5 g/L Bacto ™ yeast extract
    2 % glucose
  2. Solid selective media (nourseothricin)
    25 g/L Bacto™ peptone
    12.5 g/L Bacto™ yeast extract
    2 % glucose
    2 % agar
    0.2 g/L nourseothricin

Acknowledgments

We thank Suzanne Noble and Alexander Johnson for their advice in setting up the heat-shock deletion workflow for gene deletion in the 96-well format (Noble and Johnson, 2005). 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. Noble, S. M. and Johnson, A. D. (2005). Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans. Eukaryot Cell 4(2): 298-309.
  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.
  3. Sambrook, J. and Russell, D. W. (2001). Molecular cloning: A laboratory manual. 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA

简介

在这里,我们报告使用热休克方法转化假丝酵母的方法。 该方案可用于单孔或96孔规模的转化。 它已被用作电穿孔方案的替代方法,以在人真菌病原体光滑假丝酵母ATCC2001和相关菌株中构建基因组规模的基因缺失集合。 此外,该方案可以用于在光滑念珠菌(Emicum Candida glabrata)( C.glabrata )的临床分离株中产生基因缺失。

关键字:光滑念珠菌, 转型, 热休克, 敲, 优势选择

材料和试剂

  1. 受体菌株[ATCC2001,HTL或临床分离株(Schwarzmuller et al。,2014)]
  2. DNA缺失构建体/转化DNA
  3. 无菌水(双蒸)
  4. Bacto TM蛋白胨(BD Biosciences,目录号:211820)
  5. Bacto TM酵母提取物(BD Biosciences,目录号:212720)
  6. Bacto TM琼脂(BD Biosciences,目录号:214030)
  7. 葡萄糖(Merck KGaA,目录号:108337)
  8. 乙酸酐二水合物(LiAc)(Sigma-Aldrich,目录号:L6883)
  9. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:472301)
  10. 聚乙二醇(PEG 3350)(Sigma-Aldrich,目录号:P4338)
  11. Nourseothricin-dihydrogen sulfate(Werner BioAgents,目录号:5.0)
  12. ssDNA(Sigma-Aldrich,目录号:D1626)
  13. YPD介质(参见配方)
  14. 固体选择性培养基(参见配方)

设备

  1. 深孔板(96孔)(Nunc ,目录号:732-2662)
  2. 水库(高压灭菌)(VWR International,目录号:6130466)
  3. 多通道移液管(200μl)(Brandt Transferpette,WU2160016)
  4. 带挡板的培养瓶
  5. 离心(50ml管)(Eppendorf,目录号:5702R)
  6. 离心(1.5ml管)(Eppendorf,目录号:5417R)
  7. 用于培养瓶的旋转振荡器(New Brunswick Scientific,目录号:innova44)

程序

  1. 文化准备
    1. 在30℃下在YPD培养基中在160rpm下在烧瓶中振荡培养背景菌株过夜。
    2. 将培养物稀释到新鲜的YPD培养基中至OD大约为0.3,并且 再生直到达到1.5的OD 600(对于96孔热休克) 转化,需要600ml培养物)。
    3. 通过在1,000×g离心5分钟,在50ml Falcon管中收获培养物。
  2. 细胞治疗
    1. 用25 ml无菌水洗涤细胞沉淀两次,离心和 丢弃上清液。 重悬细胞沉淀在1毫升的100毫米 LiAc。
    2. 将来自一个菌株的所有细胞悬浮液混合在两个50ml管中。
    3.  在1,000 x g离心30秒后,加入以下无菌   溶液用于在步骤1中按照它们的顺序生长的每50ml细胞 列出:
      1,920μl50%PEG-3350
      400μlssDNA(10mg/ml;热变性)
      288μl的1M LiAc
    4. 轻轻地用移液器吸取细胞重悬。
  3. 热冲击

    96井规模
    1. 在准备感受态细胞前,放置50μl转化DNA   构建体在深孔板的孔的底部。
    2. 添加326 μl的细胞悬浮液加入到每个孔中并通过抽吸轻轻混合 多通道或单通道移液器。 使用a。密封板 透气的粘性箔。
    3. 孵育板30分钟,在30°C在孵箱中,不摇动。
    4. 加入45微升的DMSO,并立即用移液器吸取混合。
    5. 孵育板在42℃下在孵育器中,没有振荡精确15分钟。
    6. 离心板在1,000×g离心5分钟,并去除上清液。
    7. 加入950微升YPD媒体,轻轻地重悬浮真菌细胞通过用吸管吸气。

    单井规模
    1. 添加326微升的细胞悬浮液到一个1.5毫升管,含有50微升 转化DNA构建体并通过抽吸轻轻混合。
    2. 在30℃下孵育30分钟。 加入45μl的DMSO,立即混合。
    3. 孵育管在42°C精确15分钟,无需搅拌。
    4. 通过在1,000×g离心5分钟沉淀细胞并通过抽吸除去上清液。
    5. 加入950微升YPD培养基,轻轻地重悬细胞
  4. 细胞再生
    1. 孵育微量滴定板或管在30°C下1至4小时,在30°C,不摇动。
    2. 然后,将离心管或平板以1000×g离心5分钟。
    3. 弃去上清液并将细胞沉淀重悬于100μl无菌水中。
    4. 将板细胞悬浮液在选择性培养基上并在30℃下孵育几天直到菌落变得可见

笔记

  1. 该转化方案针对C进行优化。 (glabrata)ATCC2001和所有衍生的菌株,以及C的临床分离株。 glabrata 。
  2. 旋转振荡器的速度取决于所使用的培养瓶的类型。无挡板的烧瓶需要更高的振动速度,约220rpm左右,以获得良好的氧合作用
  3. 处理细胞非常轻轻地加入LiAc(无涡混合!)并保持在冰上。加入无菌水和TE缓冲液(这自动稀释LiAc到适当的浓度)后添加LiAc
  4. 在"程序"一节中,我们提到细胞沉淀应通过移液小心地重悬浮。对于这一步,我们建议手动1000微升移液器。通过缓慢吸出和释放,可以轻轻地重悬细胞沉淀
  5. 所需的再生时间取决于选择性标记。我们经历了具有 HIS3 标记的转化体可以在1小时后铺板,而具有 NAT1 标记的转化体可能需要长达4小时的再生。
  6. ssDNA溶液根据Molecular cloning(Sambrook和Russell,2001)中描述的手册制备。 将每个等分试样加热至95℃5分钟,并在使用前立即在冰上冷却

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. 固体选择性培养基(诺尔丝菌素)
    25g/L Bacto TM蛋白胨 12.5g/L Bacto TM酵母提取物
    2%葡萄糖 2%琼脂
    0.2 g/L诺尔丝菌素

致谢

我们感谢Suzanne Noble和Alexander Johnson在96孔模式中建立基因缺失的热休克删除工作流程(Noble和Johnson,2005)。 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"至KK。

参考文献

  1. Noble,S.M.and Johnson,A.D。(2005)。 用于大规模基因缺失研究的菌株和策略二倍体人类真菌病原体白色念珠菌 。 Eukaryot Cell 4(2):298-309。
  2. 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。
  3. Sambrook,J。和Russell,D.W。(2001)。 分子克隆:实验室手册。 第3版,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,USA
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
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). Genetic Transformation of Candida glabrata by Heat Shock. Bio-protocol 5(14): e1529. DOI: 10.21769/BioProtoc.1529.
  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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