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Genetic Transformation of Candida glabrata by Electroporation
采用电穿孔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 by electroporation of the human fungal pathogen Candida glabrata (C. glabrata). The protocol can be used for transformations in single well or in 96-well microtiter plates. It has been extensively used to generate a genome-scale gene deletion library using the C. glabrata background recipient strain ATCC2001 (Schwarzmüller et al., 2014).

Keywords: Candida glabrata(光滑念珠菌), Transformation(转型), High-Throughput(高吞吐量), Gene deletion(基因缺失), 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 (highly purified)
  3. Electroporation cuvette (Life Technologies, InvitrogenTM, catalog number: P51050 )
    This product has been discontinued.
  4. Deep well plate (96-well) (Nunc®, catalog number: 7322662 )
  5. Reservoir (autoclavable) (VWR International, catalog number: 6130466 )
  6. Multichannel pipette (200 µl) (BrandTech Scientific)
  7. Sterile water (double-distilled)
  8. Bacto™ peptone (BD Biosciences, catalog number: 211820 )
  9. Bacto™ yeast extract (BD Biosciences, catalog number: 212720 )
  10. Bacto™ agar (BD Biosciences, catalog number: 214030 )
  11. Glucose (Merck KGaA, catalog number: 108337)
  12. Tris base (Sigma-Aldrich, catalog number: T1503 )
  13. EDTA (Sigma-Aldrich, catalog number: E6758 )
  14. Lithium acetate dihydrate (Sigma-Aldrich, catalog number: L6883 )
  15. DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  16. Nourseothricin-dihydrogen sulfate (Werner BioAgents, catalog number: 5.0 )
  17. TE buffer (see Recipes)
  18. DTT solution (see Recipes)
  19. YPD media (see Recipes)
  20. Solid selective media (see Recipes)

Equipment

  1. 96-wel electroporation plate (2 mm gap) (Harvard Apparatus, catalog number: HTP962450450 )
  2. Electroporation cuvettes (1 mm gap) (Life Technologies, InvitrogenTM, catalog number: P51050 )
    This product (catalog P51050 ) has been discontinued.
  3. Culture flasks with baffles
  4. Centrifuge for tubes (50 ml) (Eppendorf, catalog number: 5702R )
  5. Centrifuge for tubes (1.5 ml) (Eppendorf, catalog number: 5417R )
  6. Rotary shaker for culture flasks (New Brunswick Scientific, catalog number: innova44 )
  7. Stratagene Stratalinker 2400 UV Crosslinker (Artisan Technology Group®, catalog number: 532741 )
  8. Single-well scaleTransfection apparatus consisting of capacitance extender, pulse controller and gene pulser (Bio-Rad Laboratories, AbD Serotec®, catalog numbers: 165-2087 ; 165-2098 ; 165-2077 )
  9. 96-well microtiter plates
    1. 96-well electroporation device (BTX The Electroporation Experts, catalog number: ECM63 or Harvard Apparatus, catalog number: 450422 )
    2. Plate handler (BTX The Electroporation Experts, catalog number: HT-100 or Harvard Apparatus, catalog number: 450400 )

Procedure

  1. Culture preparation
    1. Grow the recipient strain overnight in YPD media at 30 °C with shaking at 160 rpm.
    2. Dilute the culture in fresh YPD media to an OD600 of about 0.3, and continue culture until an OD600 of 1.5 is reached (50 ml of culture are sufficient for 12 transformations).
    3. Harvest the culture in 50 ml tubes by centrifugation at 1,000 x g for 5 min. Wash the cell pellet(s) once with 25 ml of sterile water at room temperature.
  2. Treatment of cells
    1. Each cell pellet derived from 50 ml culture is very gently resuspended by slow pipetting in 8 ml sterile water, 1 ml 10x TE buffer and 1 ml 1 M LiAc.
    2. Incubate the cell suspension(s) for 30 min at 30 °C and 130 rpm.
    3. Add 250 µl of DTT and incubate the suspension(s) for 60 min at 30 °C and 130 rpm.
    4. Add 40 ml of sterile pre-cooled water and centrifuge the suspension(s) at 1,000 x g for 5 min and 4 °C.
    5. Carefully resuspend cell pellets by slow pipetting in 25 ml of sterile water (4 °C) and repeat the centrifugation step.
    6. Carefully resuspend cell pellets in 5 ml pre-cooled 1 M sorbitol and repeat the centrifuging step. Finally, resuspend the pellets of electrocompetent cells in 550 µl of pre-cooled 1 M sorbitol.
  3. Electroporation
    Single-well scale
    1. Transfer 45 µl aliquots of electrocompetent cell suspensions into sterile precooled tubes.
    2. Add 5 to 10 µl of highly purified transforming DNA (1 to 3 µg), mix gently and thoroughly and incubate tubes on ice for 10 min.
    3. Apply electro pulses (200 Ω, 1.5 kV, 25 µF), the time constant should be at about 4.6 Ω. µF.
    4. Add 950 µl of YPD media to each cuvette and transfer the suspension to a tube.

    96-well scale
    1. Transfer 5 to 10 µl aliquots of the highly purified transforming DNA (1 to 3 µg) into each well of the electroporation plate.
    2. Add 45 µl of electrocompetent cell suspensions and incubate the plate for 10 min on ice. Make sure that the DNA samples and the cell suspension are at the bottom of the wells.
    3. Cover the plate with a sterile lid or a seal it with a foil for the incubation.
    4. After the incubation place the plate in the plate handler and pulse each column individually (200 Ω, 1.5 kV, 25 µF).
    5. Transfer the cell suspension into a 96-well plate with 950 µl of YPD media.
    6. The media in the plate can be used to flush the wells of the electroporation plate.
  4. Regeneration of cells
    1. Incubate cell suspensions in tubes or in a plate for 1 to 4 h (depending on the marker) at 30 °C without shaking.
    2. Afterwards, centrifuge the tubes or the plate 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 media and incubate plates for 1 to 2 days at 30 °C until colonies are visible.

Notes

  1. The protocol was optimized for C. glabrata ATCC2001 and all derivative strains.
  2. Speed of rotary shaker depends on the type of culture flasks used. Flasks without baffles require higher shaking speeds about 220 rpm.
  3. Handle cells as gently as possible after adding LiAc (no vortexing!) and keep them on ice. Add LiAc AFTER adding sterile water and the TE buffer (this automatically dilutes the LiAc to the appropriate concentration).
  4. In section “Procedure” part 2 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. 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 electroporation cuvettes and plates can be reused. They should be cleaned after use with a mild detergent and then thoroughly rinsed with distilled water. After drying, they can be sterilized using a Crosslinker (3 times “Auto-Crosslink” - equals an exposure of 3 times 120 mJoules).

Recipes

  1. TE buffer
    10 mM Tris base (pH 8)
    1 mM EDTA
  2. DTT solution
    Dissolve 1 M dithiothreitol in 0.01 M NaAc (pH 5.2)
  3. YPD media (yeast extract peptone dextrose)
    25 g/L Bacto™ peptone
    12.5 g/L Bacto™ yeast extract
    2 % glucose
  4. Solid selective media (nourseothricin)
    25 g/L Bacto™ peptone
    12.5 g/L Bacto™ yeast extract
    2 % glucose
    2 % agarose
    0.2 g/ml nourseothricin

Acknowledgments

This protocol was adapted from a previously published method (Reuss et al., 2004). This work was supported by the Austrian Science Fund 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. Reuss, O., Vik, A., Kolter, R. and Morschhauser, J. (2004). The SAT1 flipper, an optimized tool for gene disruption in Candida albicans. Gene 341: 119-127.
  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.

简介

在这里,我们报道了通过电穿孔转化人类真菌病原体(光滑念珠菌)的方法( C.glabrata )。 该方案可用于在单孔或96孔微量滴定板中的转化。 它已广泛用于使用C产生基因组规模的基因缺失文库。 glabrata 背景受体菌株ATCC2001(Schwarzmüller等人,2014)。

关键字:光滑念珠菌, 转型, 高吞吐量, 基因缺失, 优势选择

材料和试剂

  1. 受体菌株[ATCC2001,HTL或临床分离株(Schwarzmuller等人,2014)]
  2. DNA缺失构建体/转化DNA(高度纯化)
  3. 电穿孔杯(Life Technologies,Invitrogen TM ,目录号:P51050)
    此产品已停产。
  4. 深孔板(96孔)(Nunc ,目录号:7322662)
  5. 水库(高压灭菌)(VWR International,目录号:6130466)
  6. 多通道移液器(200μl)(BrandTech Scientific)
  7. 无菌水(双蒸)
  8. Bacto TM蛋白胨(BD Biosciences,目录号:211820)
  9. Bacto TM酵母提取物(BD Biosciences,目录号:212720)
  10. Bacto TM琼脂(BD Biosciences,目录号:214030)
  11. 葡萄糖(Merck KGaA,目录号:108337)
  12. Tris碱(Sigma-Aldrich,目录号:T1503)
  13. EDTA(Sigma-Aldrich,目录号:E6758)
  14. 乙酸锂二水合物(Sigma-Aldrich,目录号:L6883)
  15. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  16. Nourseothricin-dihydrogen sulfate(Werner BioAgents,目录号:5.0)
  17. TE缓冲区(参见配方)
  18. DTT解决方案(参见配方)
  19. YPD介质(参见配方)
  20. 固体选择性培养基(参见配方)

设备

  1. 96孔电穿孔板(2mm间隙)(Harvard Apparatus,目录号:HTP962450450)
  2. 电穿孔杯(1mm间隙)(Life Technologies,Invitrogen TM ,目录号:P51050)
    此产品(目录P51050)已停产。
  3. 带挡板的培养瓶
  4. 离心管(50ml)(Eppendorf,目录号:5702R)
  5. 离心管(1.5ml)(Eppendorf,目录号:5417R)
  6. 用于培养瓶的旋转振荡器(New Brunswick Scientific,目录号:innova44)
  7. Stratagene Stratalinker 2400 UV交联剂(Artisan Technology Group ,目录号:532741)
  8. 由电容延长器,脉冲控制器和基因脉冲器(Bio-Rad Laboratories,AbD Serotec ,目录号:165-2087; 165-2098; 165-2077)组成的单孔转移装置
  9. 96孔微量滴定板
    1. 96孔电穿孔装置(BTX The Electroporation Experts,目录号:ECM63或Harvard Apparatus,目录号:450422)
    2. 板处理器(BTX The Electroporation Experts,目录号:HT-100或Harvard Apparatus,目录号:450400)

程序

  1. 文化准备
    1. 在YPD培养基中在30℃下以160rpm振荡使受体菌株生长过夜。
    2. 将新鲜YPD培养基中的培养物稀释至约0.3的OD 600, 继续培养直至达到1.5的OD 600(50ml培养物 足以进行12次转化)。
    3. 收获在50毫升的文化 通过在1,000×g离心5分钟。 洗涤细胞沉淀 在室温下用25ml无菌水洗涤一次。
  2. 细胞治疗
    1. 来自50ml培养物的每个细胞沉淀非常温和 通过缓慢移液在8ml无菌水,1ml 10×TE缓冲液中重悬 和1ml 1M LiAc。
    2. 在30℃和130rpm下孵育细胞悬浮液30分钟。
    3. 加入250μlDTT,并在30℃和130rpm下孵育悬浮液60分钟。
    4. 加入40ml无菌预冷却的水,并将悬浮液以1,000×g离心5分钟和4℃。
    5. 通过缓慢移液在25 ml无菌水(4°C)中小心地重悬细胞沉淀,并重复离心步骤。
    6. 小心地重悬细胞沉淀在5ml预冷却的1M山梨醇和 重复离心步骤。 最后,重悬颗粒 电感受态细胞在550μl预冷却的1M山梨醇中。
  3. 电穿孔
    单井规模
    1. 将45μl等分的电感受态细胞悬浮液转移到无菌预冷管中。
    2. 加入5到10微升高度纯化的转化DNA(1到3微克),轻轻地彻底混合,并在冰上孵育管10分钟。
    3. 施加电脉冲(200Ω,1.5 kV,25μF),时间常数应为约4.6Ω。 μF。
    4. 添加950微升的YPD介质到每个比色杯,并将悬浮液转移到管。

    96孔规模
    1. 转移5到10微升的高纯度转化DNA(1-3微克)到电穿孔板的每个孔的等分试样。
    2. 加入45微升电感受态细胞悬液,并在冰上孵育板10分钟。 确保DNA样品和细胞悬浮液在孔底部。
    3. 用无菌盖子盖住平板或用箔片密封以进行孵育。
    4. 孵育后,将板置于板处理器中,并单独脉冲每个柱(200Ω,1.5kV,25μF)。
    5. 转移细胞悬液到96孔板与950微升的YPD媒体。
    6. 板中的介质可用于冲洗电穿孔板的孔。
  4. 细胞再生
    1. 孵育细胞悬浮液在管或板中1至4小时(取决于标记)在30°C,无振荡。
    2. 然后,将管或板在1,000×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. 将板细胞悬浮在选择性培养基上并在30℃孵育平板1至2天,直到可见菌落

笔记

  1. 该协议针对 C进行了优化。 glabrata ATCC2001和所有衍生菌株
  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220转/分
  3. 处理细胞后尽可能轻轻地加入LiAc(不涡旋!),并保持在冰上。 添加LiAc后,加入无菌水和TE缓冲液(这自动将LiAc稀释至适当的浓度) />
  4. 在"程序"第2部分中,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. 再生时间取决于选择标记。 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. 电穿孔比色皿和板可以重复使用。 使用后应用温和的清洁剂清洁,然后用蒸馏水彻底冲洗。 干燥后,可以使用交联剂(3次"Auto-Crosslink" - 等于3次120mJoules的暴露量)对其进行灭菌。

食谱

  1. TE缓冲区
    10mM Tris碱(pH8)
    1mM EDTA
  2. DTT解决方案
    将1M二硫苏糖醇溶解在0.01M NaAc(pH5.2)中
  3. 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
  4. 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%琼脂糖 0.2 g/ml诺尔丝菌素

致谢

此协议改编自先前公布的方法(Reuss等人,2004)。 这项工作得到奥地利科学基金会FWF的支持,通过ERA-Net Pathogenomics项目FunPath(FWF-API-0125),部分由基督教多普勒学会,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. Reuss,O.,Vik,A.,Kolter,R。和Morschhauser,J。(2004)。 SAT1鳍状物,一种用于在白色念珠菌中进行基因破坏的优化工具。 341:119-127。
  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。
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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 Electroporation. Bio-protocol 5(14): e1528. DOI: 10.21769/BioProtoc.1528.
  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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