对光滑念珠菌中的基因缺失突变体进行大规模表型分析

Fabian Istel; Tobias Schwarzmüller; Michael  Tscherner; Karl Kuchler

doi:10.21769/BioProtoc.1530

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Peer-reviewed

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

FI Fabian Istel ^*

TS Tobias Schwarzmüller ^*

Michael Tscherner

Karl Kuchler email

(*contributed equally to this work) 发布: 2015年07月20日第5卷第14期 DOI: 10.21769/BioProtoc.1530 浏览次数: 10275

评审: Sadri ZnaidiFanglian He

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Cover of PLOS Pathogens, featuring study using the protocol.

Jun 2014

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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

C. glabrata strain (ATCC2001, clinical isolates)
Sterile water (double distilled)
Bacto™ peptone (BD Biosciences, catalog number: 211820 )
Bacto™ yeast extract (BD Biosciences, catalog number: 212720 )
Bacto™ agar (BD Biosciences, catalog number: 214030 )
Difco™ yeast nitrogen base (YNB) (BD Biosciences, catalog number: 233520 )
Glucose (Merck KGaA, catalog number: 108337 )
Adenine (Sigma-Aldrich, catalog number: A8626 )
L-arginine (Sigma-Aldrich, catalog number: A5006 )
L-tyrosine (Sigma-Aldrich, catalog number: T3754 )
L-isoleucine (Sigma-Aldrich, catalog number: I2752 )
L-phenylalanine (Sigma-Aldrich, catalog number: P2126 )
L-glutamic acid(Sigma-Aldrich, catalog number: G1251 )
L-aspartic acid (Sigma-Aldrich, catalog number: A9256 )
L-threonine (Sigma-Aldrich, catalog number: T8625 )
L-serine (Sigma-Aldrich, catalog number: S4500 )
L-valine (Sigma-Aldrich, catalog number: V0500 )
L-methionine (Sigma-Aldrich, catalog number: M9625 )
Glycerol (Sigma-Aldrich, catalog number: G5516 )
YPD media (see Recipes)
Solid YPD media (see Recipes)
2x YPD media (see Recipes)
SC media (see Recipes)
Solid SC media (see Recipes)
Amino acid mix (see Recipes)
15% glycerol solution (see Recipes)

Equipment

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

Procedure

English

中文翻译

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如何引用

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.