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Spore Preparation Protocol for Enrichment of Clostridia from Murine Intestine

Featured protocol,  Authors: Eric M. Velazquez
Eric M. VelazquezAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
Bio-protocol author page: a4551
Fabian Rivera-Chávez
Fabian Rivera-ChávezAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
Bio-protocol author page: a4552
 and Andreas J. Bäumler
Andreas J. BäumlerAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
For correspondence: ajbaumler@ucdavis.edu
Bio-protocol author page: a4553
date: 5/20/2017, 168 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2296.

Brief version appeared in Cell Host Microbe, Apr 2016
In recent years, many spore-forming commensal Clostridia found in the gut have been discovered to promote host physiology, immune development, and protection against infections. We provide a detailed protocol for rapid enrichment of spore-forming bacteria from murine intestine. Briefly, contents from the intestinal cecum are collected aerobically, diluted and finally treated with chloroform to enrich for Clostridia spores.

Ultradeep Pyrosequencing of Hepatitis C Virus to Define Evolutionary Phenotypes

Featured protocol,  Authors: Brendan A. Palmer
Brendan A. PalmerAffiliation: Molecular Virology Diagnostic & Research Laboratory, Department of Medicine, University College Cork, Cork, Ireland
Bio-protocol author page: a4519
Zoya Dimitrova
Zoya DimitrovaAffiliation: Division of Viral Hepatitis, Centers of Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a4520
Pavel Skums
Pavel SkumsAffiliation: Division of Viral Hepatitis, Centers of Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a4521
Orla Crosbie
Orla CrosbieAffiliation: Department of Gastroenterology, Cork University Hospital, Cork, Ireland
Bio-protocol author page: a4522
Elizabeth Kenny-Walsh
Elizabeth Kenny-WalshAffiliation: Department of Gastroenterology, Cork University Hospital, Cork, Ireland
Bio-protocol author page: a4523
 and Liam J. Fanning
Liam J. FanningAffiliation: Molecular Virology Diagnostic & Research Laboratory, Department of Medicine, University College Cork, Cork, Ireland
For correspondence: l.fanning@ucc.ie
Bio-protocol author page: a4524
date: 5/20/2017, 91 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2284.

Brief version appeared in J Virol, Dec 2015
Analysis of hypervariable regions (HVR) using pyrosequencing techniques is hampered by the ability of error correction algorithms to account for the heterogeneity of the variants present. Analysis of between-sample fluctuations to virome sub-populations, and detection of low frequency variants, are unreliable through the application of arbitrary frequency cut offs. Cumulatively this leads to an underestimation of genetic diversity. In the following technique we describe the analysis of Hepatitis C virus (HCV) HVR1 which includes the E1/E2 glycoprotein gene junction. This procedure describes the evolution of HCV in a treatment naïve environment, from 10 samples collected over 10 years, using ultradeep pyrosequencing (UDPS) performed on the Roche GS FLX titanium platform (Palmer et al., 2014). Initial clonal analysis of serum samples was used to inform downstream error correction algorithms that allowed for a greater sequence depth to be reached. PCR amplification of this region has been tested for HCV genotypes 1, 2, 3 and 4.

Escherichia coli Infection of Drosophila

Featured protocol,  Authors: Charles Tracy
Charles TracyAffiliation: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, USA
Bio-protocol author page: a4450
 and Helmut Krämer
Helmut KrämerAffiliation 1: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, USA
Affiliation 2: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, USA
For correspondence: helmut.kramer@utsouthwestern.edu
Bio-protocol author page: a4451
date: 5/5/2017, 170 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2256.

Brief version appeared in Immunity, Aug 2016
Following septic insults, healthy insects, just like vertebrates, mount a complex immune response to contain and destroy pathogens. The failure to efficiently clear bacterial infections in immuno-compromised fly mutants leads to higher mortality rates which provide a powerful indicator for genes with important roles in innate immunity. The following protocol is designed to reproducibly inject a known amount of non-pathogenic E. coli into otherwise sterile flies and to measure the survival of flies after infection. The protocol can be easily adapted to different types of bacteria.

Measuring Cyanobacterial Metabolism in Biofilms with NanoSIMS Isotope Imaging and Scanning Electron Microscopy (SEM)

Featured protocol,  Authors: Rhona K. Stuart
Rhona K. StuartAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
For correspondence: stuart25@llnl.gov
Bio-protocol author page: a4466
Xavier Mayali
Xavier MayaliAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4467
Michael P. Thelen
Michael P. ThelenAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4468
Jennifer Pett-Ridge
Jennifer Pett-RidgeAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4469
 and Peter K. Weber
Peter K. WeberAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4470
date: 5/5/2017, 168 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2263.

Brief version appeared in Mbio, Jun 2016
To advance the understanding of microbial interactions, it is becoming increasingly important to resolve the individual metabolic contributions of microorganisms in complex communities. Organisms from biofilms can be especially difficult to separate, image and analyze, and methods to address these limitations are needed. High resolution imaging secondary ion mass spectrometry (NanoSIMS) generates single cell isotopic composition measurements, and can be used to quantify incorporation and exchange of an isotopically labeled substrate among individual organisms. Here, incorporation of cyanobacterial extracellular organic matter (EOM) by members of a cyanobacterial mixed species biofilm is used as a model to illustrate this method. Incorporation of stable isotope labeled (15N and 13C) EOM by two groups, cyanobacteria and associated heterotrophic microbes, are quantified. Methods for generating, preparing, and analyzing samples for quantifying uptake of stable isotope-labeled EOM in the biofilm are described.

Incubation of Cyanobacteria under Dark, Anaerobic Conditions and Quantification of the Excreted Organic Acids by HPLC

Featured protocol,  Authors: Chika Yasuda
Chika YasudaAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
Bio-protocol author page: a4452
Hiroko Iijima
Hiroko IijimaAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
Bio-protocol author page: a4453
Haruna Sukigara
Haruna SukigaraAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
Bio-protocol author page: a4454
 and Takashi Osanai
Takashi OsanaiAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
For correspondence: tosanai@meiji.ac.jp
Bio-protocol author page: a4455
date: 5/5/2017, 146 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2257.

Brief version appeared in Sci Rep, Aug 2016
Succinate and lactate are commodity chemicals used for producing bioplastics. Recently, it was found that such organic acids are excreted from cells of the unicellular cyanobacterium Synechocystis sp. PCC 6803 under dark, anaerobic conditions. To conduct the dark, anaerobic incubation, cells were concentrated within a vial that was then sealed with a butyl rubber cap, following which N2 gas was introduced into the vial. The organic acids produced were quantified by high-performance liquid chromatography via post-labeling with bromothymol blue as a pH indicator. After separation by ion-exclusion chromatography, the organic acids were identified by comparing their retention time with that of standard solutions. These procedures allow researchers to quantify the organic acids produced by microorganisms, contributing to knowledge about the biology and biotechnology of cyanobacteria.

Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting

Featured protocol,  Authors: Martino Bardelli
Martino BardelliAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
Bio-protocol author page: a4283
Francisco Zarate-Perez
Francisco Zarate-PerezAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
Bio-protocol author page: a4284
Leticia Agundez
Leticia AgundezAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Department of Genetics, University College London Institute of Ophthalmology, London, United Kingdom
Bio-protocol author page: a4285
Nelly Jolinon
Nelly JolinonAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4486
R. Michael Linden
R. Michael LindenAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Genetic Medicine Institute, Pfizer Inc., London, United Kingdom
Bio-protocol author page: a4487
Carlos R. Escalante
Carlos R. EscalanteAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA, USA
Bio-protocol author page: a4288
 and Els Henckaerts
Els HenckaertsAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
For correspondence: els.henckaerts@kcl.ac.uk
Bio-protocol author page: a4289
date: 5/5/2017, 163 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2271.

Brief version appeared in J Virol, Aug 2016
Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975).

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.

In vitro Assays for Measuring Endothelial Permeability by Transwells and Electrical Impedance Systems

Featured protocol,  Authors: Hong-Ru Chen
Hong-Ru ChenAffiliation: The Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
Bio-protocol author page: a4488
 and Trai-Ming Yeh
Trai-Ming YehAffiliation 1: The Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
Affiliation 2: Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
For correspondence: today@mail.ncku.edu.tw
Bio-protocol author page: a4489
date: 5/5/2017, 160 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2273.

Brief version appeared in PLoS Negl Trop Dis, Jul 2016
Vascular leakage is an important feature in several diseases, such as septic shock, viral hemorrhagic fever, cancer metastasis and ischemia-reperfusion injuries. Thus establishing assays for measuring endothelial permeability will provide insight into the establishment or progression of such diseases. Here, we provide transwell permeability assay and electrical impedance sensing assay for studying endothelial permeability in vitro. With these methods, the effect of a molecule on endothelial permeability could be defined.

Preparation of Everted Membrane Vesicles from Escherichia coli Cells

Featured protocol,  Author: Marina Verkhovskaya
Marina VerkhovskayaAffiliation: Institute of Biotechnology, PO Box 65 (Viikinkaari 1) FIN-00014 University of Helsinki, Helsinki, Finland
For correspondence: Marina.Verkhovskaya@Helsinki.Fi
Bio-protocol author page: a4446
date: 5/5/2017, 147 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2254.

Brief version appeared in FEBS Lett, Jun 2016
The protocol for obtaining electrically sealed membrane vesicles from E. coli cells is presented. Proton pumps such as Complex I, quinol oxidase, and ATPase are active in the obtained vesicles. Quality of the preparation was tested by monitoring the electric potential generated by these pumps.

Conjugation Assay for Testing CRISPR-Cas Anti-plasmid Immunity in Staphylococci

Featured protocol,  Authors: Forrest C. Walker
Forrest C. WalkerAffiliation: Department of Biological Sciences, University of Alabama, Tuscaloosa, USA
Bio-protocol author page: a4490
 and Asma Hatoum-Aslan
Asma Hatoum-AslanAffiliation: Department of Biological Sciences, University of Alabama, Tuscaloosa, USA
For correspondence: ahatoum@ua.edu
Bio-protocol author page: a4491
date: 5/5/2017, 218 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2293.

Brief version appeared in J Bacteriol, Jan 2014
CRISPR-Cas is a prokaryotic adaptive immune system that prevents uptake of mobile genetic elements such as bacteriophages and plasmids. Plasmid transfer between bacteria is of particular clinical concern due to increasing amounts of antibiotic resistant pathogens found in humans as a result of transfer of resistance plasmids within and between species. Testing the ability of CRISPR-Cas systems to block plasmid transfer in various conditions or with CRISPR-Cas mutants provides key insights into the functionality and mechanisms of CRISPR-Cas as well as how antibiotic resistance spreads within bacterial communities. Here, we describe a method for quantifying the impact of CRISPR-Cas on the efficiency of plasmid transfer by conjugation. While this method is presented in Staphylococcus species, it could be more broadly used for any conjugative prokaryote.

Adhesion and Invasion Assay Procedure Using Caco-2 Cells for Listeria monocytogenes

Featured protocol,  Authors: Swetha Reddy
Swetha ReddyAffiliation: College of Veterinary Science, Mississippi State University, Starkville, USA
For correspondence: mswethareddy@gmail.com
Bio-protocol author page: a4476
 and Frank Austin
Frank AustinAffiliation: College of Veterinary Science, Mississippi State University, Starkville, USA
Bio-protocol author page: a4477
date: 5/5/2017, 174 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2267.

Brief version appeared in Microb Pathog, Mar 2016
Listeria monocytogenes is an important Gram-positive foodborne pathogen that is a particular problem in ready-to-eat food. It has an ability to survive in harsh conditions like refrigeration temperatures and high salt concentrations and is known to cross intestinal, placental and blood-brain barriers. Several cancerous cell lines like cervical, liver, dendritic, intestinal and macrophages have been used to study in vitro propagation and survival of listeria in human cells. Human intestinal epithelial cells have been used to study how listeria crosses the intestinal barrier and cause infection. The protocol in this articles describes the procedures to grow Caco-2 cells, maintain cells and use them for adhesion and invasion assays. During adhesion assay the cells are incubated with listeria for 30 min but in invasion assay the cell growth is arrested at several time points after infection to monitor the growth and survival rate of listeria in cells.

Immunoprecipitation of Cell Surface Proteins from Gram-negative Bacteria

Featured protocol,  Authors: Carlos Eduardo Pouey Cunha*
Carlos Eduardo Pouey CunhaAffiliation 1: School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
Affiliation 2: Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brasil
Bio-protocol author page: a4431
Jane Newcombe*
Jane NewcombeAffiliation: School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
Bio-protocol author page: a4432
Odir Antonio Dellagostin
Odir Antonio DellagostinAffiliation: Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brasil
Bio-protocol author page: a4433
 and Johnjoe McFadden
Johnjoe McFaddenAffiliation: School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
For correspondence: j.mcfadden@surrey.ac.uk
Bio-protocol author page: a4434
 (*contributed equally to this work) date: 5/5/2017, 199 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2250.

Brief version appeared in Microbiology, Feb 2014
The meningococcus (Neisseria meningitidis) remains an important threat to human health worldwide. This Gram-negative bacterium causes elevated disabilities and mortality in infected individuals. Despite several available vaccines, currently there is no universal vaccine against all circulating meningococcal strains (Vogel et al., 2013). Herein, we describe a new protocol that is capable of identifying only cell surface exposed proteins that play a role in immunity, providing this research field with a more straightforward approach to identify novel vaccine targets. Even though N. meningitidis is used as a model in the protocol herein described, this protocol can be used for any Gram-negative bacteria provided modifications and optimizations are carried out to adapt it to different bacterial and disease characteristics (e.g., membrane fragility, growth methods, serum antibody levels, etc.).

Evaluation of Plasmid Stability by Negative Selection in Gram-negative Bacteria

Featured protocol,  Authors: Damián Lobato Márquez
Damián Lobato MárquezAffiliation: Department of Medicine, Imperial College London, London, UK
For correspondence: d.marquez@imperial.ac.uk
Bio-protocol author page: a4462
 and Laura Molina García
Laura Molina GarcíaAffiliation: Department of Cell and Developmental Biology, University College London, London, UK
Bio-protocol author page: a1812
date: 5/5/2017, 139 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2261.

Brief version appeared in Front Mol Biosci, Oct 2016
Plasmid stability can be measured using antibiotic-resistance plasmid derivatives by positive selection. However, highly stable plasmids are below the sensitivity range of these assays. To solve this problem we describe a novel, highly sensitive method to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells. The assay proposed here is based on an aph-parE cassette. When synthesized in the cell, the ParE toxin induces cell death. ParE synthesis is controlled by a rhamnose-inducible promoter. When bacteria carrying the aph-parE module are grown in media containing rhamnose as the only carbon source, ParE is synthesized and plasmid-containing cells are eliminated. Kanamycin resistance (aph) is further used to confirm the absence of the plasmid in rhamnose grown bacteria.

Spore Preparation Protocol for Enrichment of Clostridia from Murine Intestine

Authors: Eric M. Velazquez
Eric M. VelazquezAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
Bio-protocol author page: a4551
Fabian Rivera-Chávez
Fabian Rivera-ChávezAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
Bio-protocol author page: a4552
 and Andreas J. Bäumler
Andreas J. BäumlerAffiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA
For correspondence: ajbaumler@ucdavis.edu
Bio-protocol author page: a4553
date: 5/20/2017, 168 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2296.

[Abstract] In recent years, many spore-forming commensal Clostridia found in the gut have been discovered to promote host physiology, immune development, and protection against infections. We provide a detailed protocol for rapid enrichment of spore-forming bacteria from murine intestine. Briefly, contents from the intestinal cecum are collected aerobically, ...

Ultradeep Pyrosequencing of Hepatitis C Virus to Define Evolutionary Phenotypes

Authors: Brendan A. Palmer
Brendan A. PalmerAffiliation: Molecular Virology Diagnostic & Research Laboratory, Department of Medicine, University College Cork, Cork, Ireland
Bio-protocol author page: a4519
Zoya Dimitrova
Zoya DimitrovaAffiliation: Division of Viral Hepatitis, Centers of Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a4520
Pavel Skums
Pavel SkumsAffiliation: Division of Viral Hepatitis, Centers of Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a4521
Orla Crosbie
Orla CrosbieAffiliation: Department of Gastroenterology, Cork University Hospital, Cork, Ireland
Bio-protocol author page: a4522
Elizabeth Kenny-Walsh
Elizabeth Kenny-WalshAffiliation: Department of Gastroenterology, Cork University Hospital, Cork, Ireland
Bio-protocol author page: a4523
 and Liam J. Fanning
Liam J. FanningAffiliation: Molecular Virology Diagnostic & Research Laboratory, Department of Medicine, University College Cork, Cork, Ireland
For correspondence: l.fanning@ucc.ie
Bio-protocol author page: a4524
date: 5/20/2017, 91 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2284.

[Abstract] Analysis of hypervariable regions (HVR) using pyrosequencing techniques is hampered by the ability of error correction algorithms to account for the heterogeneity of the variants present. Analysis of between-sample fluctuations to virome sub-populations, and detection of low frequency variants, are unreliable through the application of arbitrary frequency ...

Escherichia coli Infection of Drosophila

Authors: Charles Tracy
Charles TracyAffiliation: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, USA
Bio-protocol author page: a4450
 and Helmut Krämer
Helmut KrämerAffiliation 1: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, USA
Affiliation 2: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, USA
For correspondence: helmut.kramer@utsouthwestern.edu
Bio-protocol author page: a4451
date: 5/5/2017, 170 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2256.

[Abstract] Following septic insults, healthy insects, just like vertebrates, mount a complex immune response to contain and destroy pathogens. The failure to efficiently clear bacterial infections in immuno-compromised fly mutants leads to higher mortality rates which provide a powerful indicator for genes with important roles in innate immunity. The following ...

Measuring Cyanobacterial Metabolism in Biofilms with NanoSIMS Isotope Imaging and Scanning Electron Microscopy (SEM)

Authors: Rhona K. Stuart
Rhona K. StuartAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
For correspondence: stuart25@llnl.gov
Bio-protocol author page: a4466
Xavier Mayali
Xavier MayaliAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4467
Michael P. Thelen
Michael P. ThelenAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4468
Jennifer Pett-Ridge
Jennifer Pett-RidgeAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4469
 and Peter K. Weber
Peter K. WeberAffiliation: Physical and Life Sciences Division, Lawrence Livermore National Laboratory, Livermore, USA
Bio-protocol author page: a4470
date: 5/5/2017, 168 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2263.

[Abstract] To advance the understanding of microbial interactions, it is becoming increasingly important to resolve the individual metabolic contributions of microorganisms in complex communities. Organisms from biofilms can be especially difficult to separate, image and analyze, and methods to address these limitations are needed. High resolution imaging secondary ...

Incubation of Cyanobacteria under Dark, Anaerobic Conditions and Quantification of the Excreted Organic Acids by HPLC

Authors: Chika Yasuda
Chika YasudaAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
Bio-protocol author page: a4452
Hiroko Iijima
Hiroko IijimaAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
Bio-protocol author page: a4453
Haruna Sukigara
Haruna SukigaraAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
Bio-protocol author page: a4454
 and Takashi Osanai
Takashi OsanaiAffiliation: School of Agriculture, Meiji University, Kanagawa, Japan
For correspondence: tosanai@meiji.ac.jp
Bio-protocol author page: a4455
date: 5/5/2017, 146 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2257.

[Abstract] Succinate and lactate are commodity chemicals used for producing bioplastics. Recently, it was found that such organic acids are excreted from cells of the unicellular cyanobacterium Synechocystis sp. PCC 6803 under dark, anaerobic conditions. To conduct the dark, anaerobic incubation, cells were concentrated within a vial that was then sealed with ...

Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting

Authors: Martino Bardelli
Martino BardelliAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
Bio-protocol author page: a4283
Francisco Zarate-Perez
Francisco Zarate-PerezAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
Bio-protocol author page: a4284
Leticia Agundez
Leticia AgundezAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Department of Genetics, University College London Institute of Ophthalmology, London, United Kingdom
Bio-protocol author page: a4285
Nelly Jolinon
Nelly JolinonAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4486
R. Michael Linden
R. Michael LindenAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Genetic Medicine Institute, Pfizer Inc., London, United Kingdom
Bio-protocol author page: a4487
Carlos R. Escalante
Carlos R. EscalanteAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA, USA
Bio-protocol author page: a4288
 and Els Henckaerts
Els HenckaertsAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
For correspondence: els.henckaerts@kcl.ac.uk
Bio-protocol author page: a4289
date: 5/5/2017, 163 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2271.

[Abstract] Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes ...

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 involves a stab inoculation procedure of P. expansum ...

In vitro Assays for Measuring Endothelial Permeability by Transwells and Electrical Impedance Systems

Authors: Hong-Ru Chen
Hong-Ru ChenAffiliation: The Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
Bio-protocol author page: a4488
 and Trai-Ming Yeh
Trai-Ming YehAffiliation 1: The Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
Affiliation 2: Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
For correspondence: today@mail.ncku.edu.tw
Bio-protocol author page: a4489
date: 5/5/2017, 160 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2273.

[Abstract] Vascular leakage is an important feature in several diseases, such as septic shock, viral hemorrhagic fever, cancer metastasis and ischemia-reperfusion injuries. Thus establishing assays for measuring endothelial permeability will provide insight into the establishment or progression of such diseases. Here, we provide transwell permeability assay and ...

Preparation of Everted Membrane Vesicles from Escherichia coli Cells

Author: Marina Verkhovskaya
Marina VerkhovskayaAffiliation: Institute of Biotechnology, PO Box 65 (Viikinkaari 1) FIN-00014 University of Helsinki, Helsinki, Finland
For correspondence: Marina.Verkhovskaya@Helsinki.Fi
Bio-protocol author page: a4446
date: 5/5/2017, 147 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2254.

[Abstract] The protocol for obtaining electrically sealed membrane vesicles from E. coli cells is presented. Proton pumps such as Complex I, quinol oxidase, and ATPase are active in the obtained vesicles. Quality of the preparation was tested by monitoring the electric potential generated by these pumps. ...

Conjugation Assay for Testing CRISPR-Cas Anti-plasmid Immunity in Staphylococci

Authors: Forrest C. Walker
Forrest C. WalkerAffiliation: Department of Biological Sciences, University of Alabama, Tuscaloosa, USA
Bio-protocol author page: a4490
 and Asma Hatoum-Aslan
Asma Hatoum-AslanAffiliation: Department of Biological Sciences, University of Alabama, Tuscaloosa, USA
For correspondence: ahatoum@ua.edu
Bio-protocol author page: a4491
date: 5/5/2017, 218 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2293.

[Abstract] CRISPR-Cas is a prokaryotic adaptive immune system that prevents uptake of mobile genetic elements such as bacteriophages and plasmids. Plasmid transfer between bacteria is of particular clinical concern due to increasing amounts of antibiotic resistant pathogens found in humans as a result of transfer of resistance plasmids within and between species. ...
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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, 90358 views, 31 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, 83488 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, 23596 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, 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....

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] 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, 18071 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, 17637 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, 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] 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, 13639 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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