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Measurements of Free-swimming Speed of Motile Salmonella Cells in Liquid Media

Featured protocol,  Authors: Yusuke V. Morimoto
Yusuke V. MorimotoAffiliation 1: Quantitative Biology Center, RIKEN, Suita, Osaka, Japan
Affiliation 2: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
Bio-protocol author page: a3969
Keiichi Namba
Keiichi NambaAffiliation 1: Quantitative Biology Center, RIKEN, Suita, Osaka, Japan
Affiliation 2: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
Bio-protocol author page: a3970
 and Tohru Minamino
Tohru MinaminoAffiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
For correspondence: tohru@fbs.osaka-u.ac.jp
Bio-protocol author page: a3971
date: 1/5/2017, 82 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2093.

Brief version appeared in PLoS Pathog, Mar 2016
Bacteria such as Escherichia coli and Salmonella enterica swim in liquid media using the bacterial flagella. The flagellum consists of the basal body (rotary motor), the hook (universal joint) and the filament (helical screw). Since mutants with a defect in flagellar assembly and function cannot swim smoothly, motility assay is an easy way to characterize flagellar mutants. Here, we describe how to measure free-swimming speeds of Salmonella motile cells in liquid media. Free-swimming behavior under a microscope shows a significant variation among bacterial cells.

Bacterial Intracellular Sodium Ion Measurement using CoroNa Green

Featured protocol,  Authors: Yusuke V. Morimoto
Yusuke V. MorimotoAffiliation 1: Quantitative Biology Center, RIKEN, Suita, Osaka, Japan
Affiliation 2: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
Bio-protocol author page: a3969
Keiichi Namba
Keiichi NambaAffiliation 1: Quantitative Biology Center, RIKEN, Suita, Osaka, Japan
Affiliation 2: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
Bio-protocol author page: a3970
 and Tohru Minamino
Tohru MinaminoAffiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
For correspondence: tohru@fbs.osaka-u.ac.jp
Bio-protocol author page: a3971
date: 1/5/2017, 109 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2092.

Brief version appeared in PLoS Pathog, Mar 2016
The bacterial flagellar type III export apparatus consists of a cytoplasmic ATPase complex and a transmembrane export gate complex, which are powered by ATP and proton motive force (PMF) across the cytoplasmic membrane, respectively, and transports flagellar component proteins from the cytoplasm to the distal end of the growing flagellar structure where their assembly occurs (Minamino, 2014). The export gate complex can utilize sodium motive force in addition to PMF when the cytoplasmic ATPase complex does not work properly. A transmembrane export gate protein FlhA acts as a dual ion channel to conduct both H+ and Na+ (Minamino et al., 2016). Here, we describe how to measure the intracellular Na+ concentrations in living Escherichia coli cells using a sodium-sensitive fluorescent dye, CoroNa Green (Minamino et al., 2016). Fluorescence intensity measurements of CoroNa Green by epi-fluorescence microscopy allows us to measure the intracellular Na+ concentration quantitatively.

Pilot-scale Columns Equipped with Aqueous and Solid-phase Sampling Ports Enable Geochemical and Molecular Microbial Investigations of Anoxic Biological Processes

Featured protocol,  Authors: Dina M. Drennan
Dina M. DrennanAffiliation: Department of Civil and Environmental Engineering Colorado School of Mines, Colorado, United States
Bio-protocol author page: a3938
Robert Almstrand
Robert AlmstrandAffiliation: Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden
Bio-protocol author page: a3939
Ilsu Lee
Ilsu LeeAffiliation: Freeport McMoRan Inc., Oro Valley, Arizona, United States
Bio-protocol author page: a3940
Lee Landkamer
Lee LandkamerAffiliation: Department of Civil and Environmental Engineering Colorado School of Mines, Colorado, United States
Bio-protocol author page: a3941
Linda Figueroa
Linda FigueroaAffiliation: Department of Civil and Environmental Engineering Colorado School of Mines, Colorado, United States
Bio-protocol author page: a3942
 and Jonathan O. Sharp
Jonathan O. SharpAffiliation: Department of Civil and Environmental Engineering Colorado School of Mines, Colorado, United States
For correspondence: jsharp@mines.edu
Bio-protocol author page: a3943
date: 1/5/2017, 94 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2083.

Brief version appeared in Environ Sci Technol, Jan 2016
Column studies can be employed to query systems that mimic environmentally relevant flow-through processes in natural and built environments. Sampling these systems spatially throughout operation, while maintaining the integrity of aqueous and solid-phase samples for geochemical and microbial analyses, can be challenging particularly when redox conditions within the column differ from ambient conditions. Here we present a pilot-scale column design and sampling protocol that is optimized for long-term spatial and temporal sampling. We utilized this experimental set-up over approximately 2 years to study a biologically active system designed to precipitate zinc-sulfides during sulfate reducing conditions; however, it can be adapted for the study of many flow-through systems where geochemical and/or molecular microbial analyses are desired. Importantly, these columns utilize retrievable solid-phase bags in conjunction with anoxic microbial techniques to harvest substrate samples while minimally disrupting column operation.

Fluorescence in situ Localization of Gene Expression Using a lacZ Reporter in the Heterocyst-forming Cyanobacterium Anabaena variabilis

Featured protocol,  Authors: Brenda S. Pratte
Brenda S. PratteAffiliation: Department of Biology, University of Missouri – St. Louis, St. Louis, MO, USA
Bio-protocol author page: a3944
 and Teresa Thiel
Teresa ThielAffiliation: Department of Biology, University of Missouri – St. Louis, St. Louis, MO, USA
For correspondence: thiel@umsl.edu
Bio-protocol author page: a3945
date: 1/5/2017, 71 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2084.

Brief version appeared in Mol Microbiol, Jun 2016
One of the most successful fluorescent proteins, used as a reporter of gene expression in many bacterial, plant and animals, is green fluorescent protein and its modified forms, which also function well in cyanobacteria. However, these fluorescent proteins do not allow rapid and economical quantitation of the reporter gene product, as does the popular reporter gene lacZ, encoding the enzyme β-galactosidase. We provide here a protocol for the in situ localization of β-galactosidase activity in cyanobacterial cells. This allows the same strain to be used for both a simple, quantitative, colorimetric assay with the substrate ortho-nitrophenyl-β-galactoside (ONPG) and for sensitive, fluorescence-based, cell-type localization of gene expression using 5-dodecanolyaminofluorescein di-β-D-galactopyranoside (C12-FDG).

Inoculation of Rice with Different Pathogens: Sheath Blight (Rhizoctonia solani), Damping off Disease (Pythium graminicola) and Barley Powdery Mildew (Blumeria graminis f. sp. hordei)

Featured protocol,  Authors: Rhoda Delventhal
Rhoda DelventhalAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3900
Marco Loehrer
Marco LoehrerAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3899
Denise Weidenbach
Denise WeidenbachAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3901
 and Ulrich Schaffrath
Ulrich SchaffrathAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
For correspondence: schaffrath@bio3.rwth-aachen.de
Bio-protocol author page: a3902
date: 12/20/2016, 147 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2070.

Brief version appeared in Mol Plant, Apr 2016
To prevent yield losses in plant cultivation due to plant pathogens, it is an important task to find new disease resistance mechanisms. Recently, Weidenbach et al. (2016) reported about the capacity of the rice gene OsJAC1 to enhance resistance in rice and barley against a broad spectrum of different pathogens. Here, we describe the respective protocols used by Weidenbach and colleagues for inoculation of rice with the basidiomycete Rhizoctonia solani, the oomycete Pythium graminicola and the ascomycete Blumeria graminis f. sp. hordei (Bgh).

Bacterial Growth Inhibition Assay for Xanthomonoas oryzae pv. oryzae or Escherichia coli K12 Grown together with Plant Leaf Extracts

Featured protocol,  Authors: Marco Loehrer
Marco LoehrerAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3899
Rhoda Delventhal
Rhoda DelventhalAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3900
Denise Weidenbach
Denise WeidenbachAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3901
 and Ulrich Schaffrath
Ulrich SchaffrathAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
For correspondence: schaffrath@bio3.rwth-aachen.de
Bio-protocol author page: a3902
date: 12/20/2016, 171 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2069.

Brief version appeared in Mol Plant, Apr 2016
We performed a growth inhibition assay to test antibacterial compounds in leaf extracts from transgenic rice plants. The assay is based on over-night co-incubation of a defined concentration of colony forming units (cfu) of the respective bacteria together with aqueous extracts of ground leaf tissue. 

Assessment of Wheat Resistance to Fusarium graminearum by Automated Image Analysis of Detached Leaves Assay

Featured protocol,  Authors: Alexandre Perochon
Alexandre PerochonAffiliation: University College Dublin Earth Institute and School of Biology and Environmental Science, College of Science, University College Dublin, Belfield, Dublin, Ireland
For correspondence: alexandre.perochon@ucd.ie
Bio-protocol author page: a3890
 and Fiona M. Doohan
Fiona M. DoohanAffiliation: University College Dublin Earth Institute and School of Biology and Environmental Science, College of Science, University College Dublin, Belfield, Dublin, Ireland
Bio-protocol author page: a2308
date: 12/20/2016, 340 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2065.

Brief version appeared in Plant Physiol, Dec 2015
Fusarium head blight (FHB) caused by Fusarium pathogens is a globally important cereal disease. To study Fusarium pathogenicity and host disease resistance, robust methods for disease assessment and quantification are needed. Here we describe the procedure of a detached leaves assay emphasizing the image analysis. The protocol provides the different steps of a rapid, automatic and quantitative image analysis to evaluate leaf area infected by Fusarium graminearum.

Highly Accurate Real-time Measurement of Rapid Hydrogen-peroxide Dynamics in Fungi

Featured protocol,  Authors: Michael Mentges
Michael MentgesAffiliation: Biocenter Klein Flottbek, Department of Molecular Phytopathology, University of Hamburg, Hamburg, Germany
Bio-protocol author page: a3933
 and Jörg Bormann
Jörg BormannAffiliation: Biocenter Klein Flottbek, Department of Molecular Phytopathology, University of Hamburg, Hamburg, Germany
For correspondence: joerg.bormann@uni-hamburg.de
Bio-protocol author page: a3387
date: 12/20/2016, 179 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2080.

Brief version appeared in Mol Microbiol, Dec 2015
Reactive oxygen species (ROS) are unavoidable by-products of aerobic metabolism. Despite beneficial aspects as a signaling molecule, ROS are principally recognized as harmful agents that act on nucleic acids, proteins and lipids. Reactive oxygen species, and, in particular, hydrogen peroxide (H2O2), are deployed as defense molecules across kingdoms, e.g., by plants in order to defeat invading pathogens like fungi. Necrotrophic plant pathogenic fungi themselves secrete H2O2 to induce host cell death and facilitate infection. Hydrogen peroxide is, to a certain extent, freely diffusible through membranes. To be able to monitor intracellular hydrogen peroxide dynamics in fungi, we recently established the versatile HyPer-imaging technique in the filamentous plant pathogen Fusarium graminearum (Mentges and Bormann, 2015). HyPer consists of a circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain (RD) of the prokaryotic H2O2-sensing protein, OxyR. The OxyR domain renders the sensor highly specific for H2O2. Oxidation of HyPer increases fluorescence of cpYFP excited at 488 nm and decreases fluorescence excited at 405 nm, thereby facilitating ratiometric readouts (Belousov et al., 2006). HyPer turned out to be pH-sensitive. A single amino acid mutation in the H2O2-sensing domain of HyPer renders the sensor insensitive to H2O2. This reporter is called SypHer and serves as a control for pH changes.

Antibiotic Disc Assay for Synechocystis sp. PCC6803

Featured protocol,  Authors: Otilia Cheregi
Otilia CheregiAffiliation: Department of Chemistry, Umeå University, Umeå, Sweden
For correspondence: Otilia.cheregi@gmail.com
Bio-protocol author page: a3903
 and Christiane Funk
Christiane FunkAffiliation: Department of Chemistry, Umeå University, Umeå, Sweden
For correspondence: Christiane.funk@umu.se
Bio-protocol author page: a3904
date: 12/20/2016, 149 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2071.

Brief version appeared in J Photochem Photobiol B, Nov 2015
This protocol describes how to investigate the integrity of the outer cell wall in the cyanobacterium Synechocystis sp. PCC6803 using antibiotics. It is adapted to the agar diffusion test (Bauer et al., 1966), in which filter paper discs impregnated with specified concentrations of antibiotics were placed on agar plates inoculated with bacteria. The antibiotics we tested, interfering with the biosynthesis/function of bacterial cell walls, will diffuse into the agar and produce a zone of cyanobacterial growth inhibition around the disc(s). The size of the inhibition zone reflects the sensitivity of the strain to the action of antibiotics, e.g., a mutation in a protein functioning within the cell wall or its construction would render the mutant strain more sensitive to the respective antibiotic. The method has proven to be useful for phenotyping a mutant of Synechocystis sp. PCC6803 lacking all three genes encoding Deg proteases. Deletion of these ATP-independent serine proteases was shown to have impact on the outer cell layers of Synechocystis cells (Cheregi et al., 2015).

Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol

Featured protocol,  Authors: Simon David Brown
Simon David BrownAffiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
Bio-protocol author page: a3916
 and Alexander Lorenz
Alexander LorenzAffiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
For correspondence: a.lorenz@abdn.ac.uk
Bio-protocol author page: a3917
date: 12/20/2016, 316 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2075.

Brief version appeared in Yeast, Dec 2015
The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells often requires strain construction from scratch. To overcome this problem we and other groups have created transformation cassettes with flanking homologies to ura4+ and kanMX6 which enable an efficient and time-saving way to exchange markers in existing mutated or tagged fission yeast strains.

Measurements of Free-swimming Speed of Motile Salmonella Cells in Liquid Media

Authors: Yusuke V. Morimoto
Yusuke V. MorimotoAffiliation 1: Quantitative Biology Center, RIKEN, Suita, Osaka, Japan
Affiliation 2: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
Bio-protocol author page: a3969
Keiichi Namba
Keiichi NambaAffiliation 1: Quantitative Biology Center, RIKEN, Suita, Osaka, Japan
Affiliation 2: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
Bio-protocol author page: a3970
 and Tohru Minamino
Tohru MinaminoAffiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
For correspondence: tohru@fbs.osaka-u.ac.jp
Bio-protocol author page: a3971
date: 1/5/2017, 82 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2093.

[Abstract] Bacteria such as Escherichia coli and Salmonella enterica swim in liquid media using the bacterial flagella. The flagellum consists of the basal body (rotary motor), the hook (universal joint) and the filament (helical screw). Since mutants with a defect in flagellar assembly and function cannot swim smoothly, motility assay is an easy way to characterize ...

Bacterial Intracellular Sodium Ion Measurement using CoroNa Green

Authors: Yusuke V. Morimoto
Yusuke V. MorimotoAffiliation 1: Quantitative Biology Center, RIKEN, Suita, Osaka, Japan
Affiliation 2: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
Bio-protocol author page: a3969
Keiichi Namba
Keiichi NambaAffiliation 1: Quantitative Biology Center, RIKEN, Suita, Osaka, Japan
Affiliation 2: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
Bio-protocol author page: a3970
 and Tohru Minamino
Tohru MinaminoAffiliation: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan
For correspondence: tohru@fbs.osaka-u.ac.jp
Bio-protocol author page: a3971
date: 1/5/2017, 109 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2092.

[Abstract] The bacterial flagellar type III export apparatus consists of a cytoplasmic ATPase complex and a transmembrane export gate complex, which are powered by ATP and proton motive force (PMF) across the cytoplasmic membrane, respectively, and transports flagellar component proteins from the cytoplasm to the distal end of the growing flagellar structure ...

Pilot-scale Columns Equipped with Aqueous and Solid-phase Sampling Ports Enable Geochemical and Molecular Microbial Investigations of Anoxic Biological Processes

Authors: Dina M. Drennan
Dina M. DrennanAffiliation: Department of Civil and Environmental Engineering Colorado School of Mines, Colorado, United States
Bio-protocol author page: a3938
Robert Almstrand
Robert AlmstrandAffiliation: Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden
Bio-protocol author page: a3939
Ilsu Lee
Ilsu LeeAffiliation: Freeport McMoRan Inc., Oro Valley, Arizona, United States
Bio-protocol author page: a3940
Lee Landkamer
Lee LandkamerAffiliation: Department of Civil and Environmental Engineering Colorado School of Mines, Colorado, United States
Bio-protocol author page: a3941
Linda Figueroa
Linda FigueroaAffiliation: Department of Civil and Environmental Engineering Colorado School of Mines, Colorado, United States
Bio-protocol author page: a3942
 and Jonathan O. Sharp
Jonathan O. SharpAffiliation: Department of Civil and Environmental Engineering Colorado School of Mines, Colorado, United States
For correspondence: jsharp@mines.edu
Bio-protocol author page: a3943
date: 1/5/2017, 94 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2083.

[Abstract] Column studies can be employed to query systems that mimic environmentally relevant flow-through processes in natural and built environments. Sampling these systems spatially throughout operation, while maintaining the integrity of aqueous and solid-phase samples for geochemical and microbial analyses, can be challenging particularly when redox conditions ...

Fluorescence in situ Localization of Gene Expression Using a lacZ Reporter in the Heterocyst-forming Cyanobacterium Anabaena variabilis

Authors: Brenda S. Pratte
Brenda S. PratteAffiliation: Department of Biology, University of Missouri – St. Louis, St. Louis, MO, USA
Bio-protocol author page: a3944
 and Teresa Thiel
Teresa ThielAffiliation: Department of Biology, University of Missouri – St. Louis, St. Louis, MO, USA
For correspondence: thiel@umsl.edu
Bio-protocol author page: a3945
date: 1/5/2017, 71 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2084.

[Abstract] One of the most successful fluorescent proteins, used as a reporter of gene expression in many bacterial, plant and animals, is green fluorescent protein and its modified forms, which also function well in cyanobacteria. However, these fluorescent proteins do not allow rapid and economical quantitation of the reporter gene product, as does the popular ...

Inoculation of Rice with Different Pathogens: Sheath Blight (Rhizoctonia solani), Damping off Disease (Pythium graminicola) and Barley Powdery Mildew (Blumeria graminis f. sp. hordei)

Authors: Rhoda Delventhal
Rhoda DelventhalAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3900
Marco Loehrer
Marco LoehrerAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3899
Denise Weidenbach
Denise WeidenbachAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3901
 and Ulrich Schaffrath
Ulrich SchaffrathAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
For correspondence: schaffrath@bio3.rwth-aachen.de
Bio-protocol author page: a3902
date: 12/20/2016, 147 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2070.

[Abstract] To prevent yield losses in plant cultivation due to plant pathogens, it is an important task to find new disease resistance mechanisms. Recently, Weidenbach et al. (2016) reported about the capacity of the rice gene OsJAC1 to enhance resistance in rice and barley against a broad spectrum of different pathogens. Here, we describe the respective protocols ...

Bacterial Growth Inhibition Assay for Xanthomonoas oryzae pv. oryzae or Escherichia coli K12 Grown together with Plant Leaf Extracts

Authors: Marco Loehrer
Marco LoehrerAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3899
Rhoda Delventhal
Rhoda DelventhalAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3900
Denise Weidenbach
Denise WeidenbachAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3901
 and Ulrich Schaffrath
Ulrich SchaffrathAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
For correspondence: schaffrath@bio3.rwth-aachen.de
Bio-protocol author page: a3902
date: 12/20/2016, 171 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2069.

[Abstract] We performed a growth inhibition assay to test antibacterial compounds in leaf extracts from transgenic rice plants. The assay is based on over-night co-incubation of a defined concentration of colony forming units (cfu) of the respective bacteria together with aqueous extracts of ground leaf tissue. ...

Assessment of Wheat Resistance to Fusarium graminearum by Automated Image Analysis of Detached Leaves Assay

Authors: Alexandre Perochon
Alexandre PerochonAffiliation: University College Dublin Earth Institute and School of Biology and Environmental Science, College of Science, University College Dublin, Belfield, Dublin, Ireland
For correspondence: alexandre.perochon@ucd.ie
Bio-protocol author page: a3890
 and Fiona M. Doohan
Fiona M. DoohanAffiliation: University College Dublin Earth Institute and School of Biology and Environmental Science, College of Science, University College Dublin, Belfield, Dublin, Ireland
Bio-protocol author page: a2308
date: 12/20/2016, 340 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2065.

[Abstract] Fusarium head blight (FHB) caused by Fusarium pathogens is a globally important cereal disease. To study Fusarium pathogenicity and host disease resistance, robust methods for disease assessment and quantification are needed. Here we describe the procedure of a detached leaves assay emphasizing the image analysis. The protocol provides the different ...

Highly Accurate Real-time Measurement of Rapid Hydrogen-peroxide Dynamics in Fungi

Authors: Michael Mentges
Michael MentgesAffiliation: Biocenter Klein Flottbek, Department of Molecular Phytopathology, University of Hamburg, Hamburg, Germany
Bio-protocol author page: a3933
 and Jörg Bormann
Jörg BormannAffiliation: Biocenter Klein Flottbek, Department of Molecular Phytopathology, University of Hamburg, Hamburg, Germany
For correspondence: joerg.bormann@uni-hamburg.de
Bio-protocol author page: a3387
date: 12/20/2016, 179 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2080.

[Abstract] Reactive oxygen species (ROS) are unavoidable by-products of aerobic metabolism. Despite beneficial aspects as a signaling molecule, ROS are principally recognized as harmful agents that act on nucleic acids, proteins and lipids. Reactive oxygen species, and, in particular, hydrogen peroxide (H2O2), are deployed as defense molecules across kingdoms, ...

Antibiotic Disc Assay for Synechocystis sp. PCC6803

Authors: Otilia Cheregi
Otilia CheregiAffiliation: Department of Chemistry, Umeå University, Umeå, Sweden
For correspondence: Otilia.cheregi@gmail.com
Bio-protocol author page: a3903
 and Christiane Funk
Christiane FunkAffiliation: Department of Chemistry, Umeå University, Umeå, Sweden
For correspondence: Christiane.funk@umu.se
Bio-protocol author page: a3904
date: 12/20/2016, 149 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2071.

[Abstract] This protocol describes how to investigate the integrity of the outer cell wall in the cyanobacterium Synechocystis sp. PCC6803 using antibiotics. It is adapted to the agar diffusion test (Bauer et al., 1966), in which filter paper discs impregnated with specified concentrations of antibiotics were placed on agar plates inoculated with bacteria. The ...

Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol

Authors: Simon David Brown
Simon David BrownAffiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
Bio-protocol author page: a3916
 and Alexander Lorenz
Alexander LorenzAffiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
For correspondence: a.lorenz@abdn.ac.uk
Bio-protocol author page: a3917
date: 12/20/2016, 316 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2075.

[Abstract] The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described recently, but introducing these into yeast cells ...
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[Bio101] Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method

Author: Fanglian He
Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: fanglian09@gmail.com
Bio-protocol author page: a9
date: 2/5/2011, 79911 views, 30 Q&A
DOI: https://doi.org/10.21769/BioProtoc.30.

[Abstract] This is a quick and efficient way to extract E. coli plasmid DNA without using commercial kits....

[Bio101] E. coli Genomic DNA Extraction Updates
The author made some updates (highlighted in blue) to the protocol on 09/12/2016.

Author: Fanglian He
Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a9
date: 7/20/2011, 72739 views, 46 Q&A
DOI: https://doi.org/10.21769/BioProtoc.97.

[Abstract] This protocol uses phenol/chloroform method to purify genomic DNA without using commercial kits....

[Bio101] Lentivirus Production

Author: Nabila Aboulaich date: 3/5/2011, 21964 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.39.

[Abstract] Lentivirus is a common tool used to introduce a gene into mammalian or other animal cells.This protocol is to produce lentivirus stocks from hairpin-pLKO.1 plasmid....

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, 20654 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, 19906 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....

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, 17059 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] Purification of Adenovirus by Cesium Chloride Density Gradients

Author: Huan Pang
Huan PangAffiliation: Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, USA
For correspondence: pang_huan@hotmail.com
Bio-protocol author page: a48
date: 4/5/2012, 16840 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.201.

[Abstract] Adenovirus are efficient gene delivery systems. The standard method for purification of adenoviral vectors is based on using a cesium chloride (CsCl) density gradient combined with ultracentrifugation. This method is suitable for small-scale purification and is less expensive than column chromatography ...

Culture and Detection of Mycobacterium tuberculosis (MTB) and Mycobacterium bovis (BCG)

Author: Ran Chen
Ran ChenAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: rcchen@jfkbio.com
Bio-protocol author page: a34
date: 1/20/2012, 15990 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.49.

[Abstract] Mycobacterium tuberculosis (MTB) is the bacterial pathogen responsible for tuberculosis, a human pulmonary infectious disease. Mycobacterium bovis (BCG) is the causative agent of tuberculosis in cattle, and is often used as the vaccine stain in humans. Specific recipes and methods for culture of MTB ...

[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, 13761 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] Purification of 6x His-tagged Protein (from E. coli)

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, 12642 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.8.

[Abstract] A polyhistidine-tag is an amino acid motif that contains at least six histidine (His) residues, usually at the N- or C-terminus of the protein. This tag can also be referred to as a hexa histidine-tag or a 6x His-tag. The protocol described here has been developed to purify His-tagged proteins from ...
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