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CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in Saccharomyces cerevisiae

Featured protocol,  Authors: Yu Sasano
Yu SasanoAffiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a4563
 and Satoshi Harashima
Satoshi HarashimaAffiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto City, Japan
For correspondence: harashima@bio.sojo-u.ac.jp
Bio-protocol author page: a4564
date: 5/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2306.

Brief version appeared in Sci Rep, Aug 2016
Chromosome engineering is an important technology with applications in basic biology and biotechnology. Chromosome splitting technology called PCS (PCR-mediated Chromosome Splitting) has already been developed as a fundamental chromosome engineering technology in the budding yeast. However, the splitting efficiency of PCS technology is not high enough to achieve multiple splitting at a time. This protocol describes a procedure for achieving simultaneous and multiple chromosome splits in the budding yeast Saccharomyces cerevisiae by a new technology called CRISPR-PCS. At least four independent sites in the genome can be split by one transformation. Total time and labor for obtaining a multiple split yeast strain is drastically reduced when compared with conventional PCS technology.

Pathogenicity Assay of Penicillium expansum on Apple Fruits

Featured protocol,  Authors: Yong Chen
Yong ChenAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4471
Boqiang Li
Boqiang LiAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a1847
Zhanquan Zhang
Zhanquan ZhangAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a1845
 and Shiping Tian
Shiping TianAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Beijing, China
For correspondence: tsp@ibcas.ac.cn
Bio-protocol author page: a1848
date: 5/5/2017, 167 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2264.

Brief version appeared in Mol Plant Microbe Interact, Jun 2015
Penicillium expansum, a widespread filamentous fungus, is a major causative agent of fruit decay and leads to huge economic losses during postharvest storage and shipping. Furthermore, it produces mycotoxin on the infected fruits that may cause harmful effects to human health. This pathogenicity assay involves a stab inoculation procedure of P. expansum on apple fruit, an important experimental technique to study fungal pathogenesis. This assay can be applied to analyze the virulence of postharvest pathogen on other fruits such as orange, pear and kiwifruit.

CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in Saccharomyces cerevisiae

Authors: Yu Sasano
Yu SasanoAffiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a4563
 and Satoshi Harashima
Satoshi HarashimaAffiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto City, Japan
For correspondence: harashima@bio.sojo-u.ac.jp
Bio-protocol author page: a4564
date: 5/20/2017, 93 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2306.

[Abstract] Chromosome engineering is an important technology with applications in basic biology and biotechnology. Chromosome splitting technology called PCS (PCR-mediated Chromosome Splitting) has already been developed as a fundamental chromosome engineering technology in the budding yeast. However, the splitting ...

Pathogenicity Assay of Penicillium expansum on Apple Fruits

Authors: Yong Chen
Yong ChenAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4471
Boqiang Li
Boqiang LiAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a1847
Zhanquan Zhang
Zhanquan ZhangAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a1845
 and Shiping Tian
Shiping TianAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Beijing, China
For correspondence: tsp@ibcas.ac.cn
Bio-protocol author page: a1848
date: 5/5/2017, 167 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2264.

[Abstract] Penicillium expansum, a widespread filamentous fungus, is a major causative agent of fruit decay and leads to huge economic losses during postharvest storage and shipping. Furthermore, it produces mycotoxin on the infected fruits that may cause harmful effects to human health. This pathogenicity assay ...

In vitro Detection of Neutrophil Traps and Post-attack Cell Wall Changes in Candida Hyphae

Authors: Alex Hopke
Alex HopkeAffiliation: Molecular & Biomedical Sciences, University of Maine, Orono, Maine, USA
Bio-protocol author page: a4321
 and Robert T. Wheeler
Robert T. WheelerAffiliation 1: Molecular & Biomedical Sciences, University of Maine, Orono, Maine, USA
Affiliation 2: Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, Maine, USA
For correspondence: robert.wheeler1@maine.edu
Bio-protocol author page: a4322
date: 4/5/2017, 297 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2213.

[Abstract] In this protocol we describe how to visualize neutrophil extracellular traps (NETs) and fungal cell wall changes in the context of the coculture of mouse neutrophils with fungal hyphae of Candida albicans. These protocols are easily adjusted to test a wide array of hypotheses related to the impact of ...

[Bio101] Phenotypic Profiling of Candida glabrata in Liquid Media

Authors: Fabian Istel
Fabian IstelAffiliation: Medical University Vienna, Max F. Perutz Laboratories, Department of Medical Biochemistry, A-1030 Vienna, Austria
Bio-protocol author page: a2372
Miha Tome
Miha TomeAffiliation: Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, 1000, Ljubljana, Slovenia
Bio-protocol author page: a4219
Sabrina Jenull
Sabrina JenullAffiliation: Medical University Vienna, Max F. Perutz Laboratories, Department of Medical Biochemistry, A-1030 Vienna, Austria
Bio-protocol author page: a4220
 and Karl Kuchler
Karl KuchlerAffiliation: Medical University Vienna, Max F. Perutz Laboratories, Department of Medical Biochemistry, A-1030 Vienna, Austria
For correspondence: karl.kuchler@meduniwien.ac.at
Bio-protocol author page: a496
date: 4/5/2017, 215 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2228.

[Abstract] Here, we describe a method for a large-scale liquid screening approach in C. glabrata. This liquid media method offers several distinct advantages over solid media approaches. This includes growth measurement on a plate reader instead of comparing growth by eye-sight. Furthermore, the liquid method ...

Wheat Root-dip Inoculation with Fusarium graminearum and Assessment of Root Rot Disease Severity

Authors: Qing Wang
Qing Wang Affiliation: Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and Nutrition, Justus Liebig University, Giessen, Germany
Bio-protocol author page: a4241
 and Sven Gottwald
Sven GottwaldAffiliation: Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and Nutrition, Justus Liebig University, Giessen, Germany
For correspondence: sv.gottwald@t-online.de
Bio-protocol author page: a4240
date: 3/20/2017, 478 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2189.

[Abstract] Fusarium graminearum is one of the most common and potent fungal pathogens of wheat (Triticum aestivum) and other cereals, known for causing devastating yield losses and mycotoxin contaminations of food and feed. The pathogen is mainly considered as a paradigm for the floral disease Fusarium head blight, ...

Xylem Sap Extraction Method from Hop Plants

Authors: Marko Flajšman*
Marko FlajšmanAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Bio-protocol author page: a4167
Stanislav Mandelc*
Stanislav MandelcAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Bio-protocol author page: a4168
Sebastjan Radišek*
Sebastjan RadišekAffiliation: Slovenian Institute of Hop Research and Brewing, Zalec, Slovenia
Bio-protocol author page: a4169
 and Branka Javornik
Branka JavornikAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
For correspondence: branka.javornik@bf.uni-lj.si
Bio-protocol author page: a4170
 (*contributed equally to this work) date: 3/20/2017, 470 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2172.

[Abstract] Verticillium wilt is one of the most important diseases on hop that significantly influence continuation of production on affected areas. It is caused by the soil borne vascular pathogen Verticillium nonalfalfae, which infects plants through the roots and then advances through the vascular (xylem) system. ...

Pathogenicity Assay of Verticillium nonalfalfae on Hop Plants

Authors: Marko Flajšman*
Marko FlajšmanAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Bio-protocol author page: a4167
Sebastjan Radišek*
Sebastjan RadišekAffiliation: Slovenian Institute of Hop Research and Brewing, Zalec, Slovenia
Bio-protocol author page: a4169
 and Branka Javornik
Branka JavornikAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
For correspondence: branka.javornik@bf.uni-lj.si
Bio-protocol author page: a4170
 (*contributed equally to this work) date: 3/20/2017, 408 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2171.

[Abstract] Verticillium nonalfalfae is a soil-borne plant pathogen that infects its hosts through roots. It spreads in the plant’s xylem and causes wilt disease symptoms by secreting different virulence factors. Hop (Humulus lupulus) is a primary host of V. nonalfalfae, so it is used as a model plant for studying ...

Polyethylene Glycol-mediated Transformation of Drechmeria coniospora

Authors: Le D. He
Le D. HeAffiliation: Aix Marseille Univ, CNRS, INSERM, CIML, Centre d’Immunologie de Marseille-Luminy, Marseille, France
Bio-protocol author page: a4195
 and Jonathan J. Ewbank
Jonathan J. EwbankAffiliation: Aix Marseille Univ, CNRS, INSERM, CIML, Centre d’Immunologie de Marseille-Luminy, Marseille, France
For correspondence: ewbank@ciml.univ-mrs.fr
Bio-protocol author page: a4196
date: 3/5/2017, 518 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2157.

[Abstract] Drechmeria coniospora is a nematophagous fungus and potential biocontrol agent. It belongs to the Ascomycota. It is related to Hirsutella minnesotensis, another nematophagous fungus but, phylogenetically, it is currently closest to the truffle parasite Tolypocladium ophioglossoides. Together with its ...

Determination of Elemental Concentrations in Lichens Using ICP-AES/MS

Authors: Liang-Cheng Zhao
Liang-Cheng ZhaoAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4076
Li Wang
Li WangAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4077
Yun-Jun Jiang
Yun-Jun JiangAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4078
Yan-Qiao Hu
Yan-Qiao HuAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4122
Chong-Ying Xu
Chong-Ying XuAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4080
Lei Wang
Lei WangAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4081
Xing Li
Xing Li Affiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4082
Li Wei
Li WeiAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4083
Xiu-Ping Guo
Xiu-Ping GuoAffiliation: Hebei Geological Laboratory, Baoding, China
Bio-protocol author page: a4084
Ai-Qin Liu
Ai-Qin LiuAffiliation: Hebei Geological Laboratory, Baoding, China
For correspondence: laq217510@sina.com
Bio-protocol author page: a4086
 and Hua-Jie Liu
Hua-Jie LiuAffiliation: College of Life Sciences, Hebei University, Baoding, China
For correspondence: liuhuajie@foxmail.com
Bio-protocol author page: a4087
date: 3/5/2017, 436 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2165.

[Abstract] Lichens are good biomonitors for air pollution because of their high enrichment capability of atmospheric chemical elements. To monitor atmospheric element deposition using lichens, it is important to obtain information on the multi-element concentrations in lichen thalli. Because of serious air pollution, ...

Chitin Extraction and Content Measurement in Magnaporthe oryzae

Authors: Xinyu Liu
Xinyu LiuAffiliation: Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
Bio-protocol author page: a4123
 and Zhengguang Zhang
Zhengguang ZhangAffiliation: Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, and Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China
For correspondence: zhgzhang@njau.edu.cn
Bio-protocol author page: a4208
date: 3/5/2017, 521 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2164.

[Abstract] Chitin is a linear polysaccharide composed of β (1→4)-linked N-acetylglucosamine (GlcNAc) residues. In fungi, chitin is an important component of the cell wall. Here, we provide a protocol to measure the chitin content of fungal cells using Magnaporthe oryzae as an example. ...
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In vitro Protein Kinase Assay

Author: Yuehua Wei
Yuehua WeiAffiliation: Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ Robert Wood Johnson Medical School, Piscataway, USA
For correspondence: weiyh.sjtu.edu@gmail.com
Bio-protocol author page: a49
date: 6/5/2012, 22079 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.212.

[Abstract] This protocol will describe experimental procedures for an in vitro kinase assay of the yeast protein kinase Sch9. This protocol can be tailored to detect kinase activity of other yeast protein kinase....

[Bio101] Making Yeast Competent Cells and Yeast Cell Transformation

Author: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Science, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
date: 7/20/2011, 21565 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.96.

[Abstract] This is a quite simple but reliable protocol to make very high transformation efficiency yeast competent cells. By express your gene of interest, protein function can be studied in yeast cells....

[Bio101] Protocol for Whole Cell Lysis of Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 19124 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.14.

[Abstract] This protocol describes how to perform lysis on whole yeast cell samples using NaOH. The lysed cells can then be used for downstream applications such as the extraction of total proteins. ...

Spot Assay for Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2012, 18785 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.16.

[Abstract] This protocol can be used to compare the cell growth rate of yeast under different growth conditions. It involves the serial dilution and spotting of yeast colonies....

[Bio101] Yeast Vacuole Staining with FM4-64

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 14839 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.18.

[Abstract] The lipophilic probe, FM 4-64 does not fluoresce much in water but fluoresces strongly after binding to the outer plasma membrane, providing clear and distinguishable plasma membrane staining. The binding is rapid and reversible. In this protocol vacuoles in yeast cells are stained with the FM4-64 dye, ...

[Bio101] How to Use an Avestin Emulsiflex C3 Homogenizer to Disrupt Cells

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 11837 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.11.

[Abstract] The EmulsiFlex-C3 homogenizer is powered by an electric motor. The pump does not require a compressor for it to run. This equipment can be used to disrupt cells at a large scale. The EmulsiFlex-C3 has a fixed flow-through capacity of 3 L/h. It has the ability to process samples as small as 10 ml. The ...

[Bio101] Large Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicin, Baltimore , USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 11246 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.12.

[Abstract] This protocol can be used to purify membrane proteins from yeast samples under native conditions at a large scale. This protocol has been developed primarily for FLAG-tagged proteins. This protocol can however be slightly modified and applied to other tags, such as GST or HA....

[Bio101] Small Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 10742 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.15.

[Abstract] In this protocol, we show how to purify membrane proteins from yeast using affinity purification under native conditions at a small scale. ...

Illumina Sequencing Library Construction from ChIP DNA

Author: Wei Zheng
Wei ZhengAffiliation: Keck Biotech Services, Yale University, New Haven, USA
For correspondence: wei.zheng.madison@gmail.com
Bio-protocol author page: a10
date: 2/20/2012, 10300 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.91.

[Abstract] The Illumina sequencing platform is very popular among next-generation sequencing platforms. However, the DNA sequencing library construction kit provided by Illumina is considerably expensive. The protocol described here can be used to construct high-quality sequencing libraries from chromatin immunoprecipitated ...

Co-immunoprecipitation in Yeast

Author: Olesya O. Panasenko
Olesya O. PanasenkoAffiliation: Department of Microbiology and Molecular Medicine, University of Geneva, Faculty of Medicine, CMU, Geneva, Switzerland
For correspondence: olesya.panasenko@unige.ch
Bio-protocol author page: a88
date: 8/20/2012, 9677 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.250.

[Abstract] This protocol describes investigation of protein-protein interactions in baker yeast by co-immunoprecipitation (CoIP). CoIP is a technique to identify physiologically relevant protein-protein interactions in the cell. The interesting protein can be isolated out of solution using antibody that specifically ...
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