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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.

Exopolysaccharide Quantification for the Plant Pathogen Ralstonia solanacearum

Featured protocol,  Authors: Rémi Peyraud
Rémi PeyraudAffiliation: LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France
Bio-protocol author page: a4530
Timothy P. Denny
Timothy P. DennyAffiliation: Department of Plant Pathology, University of Georgia, Athens, Georgia
Bio-protocol author page: a4531
 and Stéphane Genin
Stéphane GeninAffiliation: LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France
For correspondence: Stephane.Genin@inra.fr
Bio-protocol author page: a4532
date: 5/20/2017, 98 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2289.

Brief version appeared in PLoS Pathog, Oct 2016
Soluble exopolysaccharide is a major virulence factor produced by the plant pathogen Ralstonia solanacearum. Its massive production during plant infection is associated with the arrest of water flow in xylem vessels leading eventually to plant death. The composition of this heavy macromolecule includes mainly N-acetylgalactosamine. Here we describe a colorimetric method for quantitative determination of the soluble exopolysaccharide present in culture supernatant of R. solanacearum.

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.

Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes

Featured protocol,  Authors: Yoshihiro Adachi
Yoshihiro AdachiAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4472
Kie Itoh
Kie ItohAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4473
Miho Iijima
Miho IijimaAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4474
 and Hiromi Sesaki
Hiromi SesakiAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
For correspondence: hsesaki@jhmi.edu
Bio-protocol author page: a4475
date: 5/5/2017, 245 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2266.

Brief version appeared in Mol Cell, Sep 2016
A mitochondrion is a dynamic intracellular organelle that actively divides and fuses to control its size, number and shape in cells. A regulated balance between mitochondrial division and fusion is fundamental to the function, distribution and turnover of mitochondria (Roy et al., 2015). Mitochondrial division is mediated by dynamin-related protein 1 (Drp1), a mechano-chemical GTPase that constricts mitochondrial membranes (Tamura et al., 2011). Mitochondrial membrane lipids such as phosphatidic acid and cardiolipin bind Drp1, and Drp1-phospholipid interactions provide key regulatory mechanisms for mitochondrial division (Montessuit et al., 2010; Bustillo-Zabalbeitia et al., 2014; Macdonald et al., 2014; Stepanyants et al., 2015; Adachi et al., 2016). Here, we describe biochemical experiments that quantitatively measure interactions of Drp1 with lipids using purified recombinant Drp1 and synthetic liposomes with a defined set of phospholipids. This assay makes it possible to define the specificity of protein-lipid interaction and the role of the head group and acyl chains.

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.

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 ...

Exopolysaccharide Quantification for the Plant Pathogen Ralstonia solanacearum

Authors: Rémi Peyraud
Rémi PeyraudAffiliation: LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France
Bio-protocol author page: a4530
Timothy P. Denny
Timothy P. DennyAffiliation: Department of Plant Pathology, University of Georgia, Athens, Georgia
Bio-protocol author page: a4531
 and Stéphane Genin
Stéphane GeninAffiliation: LIPM, Université de Toulouse, INRA, CNRS, Castanet-Tolosan, France
For correspondence: Stephane.Genin@inra.fr
Bio-protocol author page: a4532
date: 5/20/2017, 98 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2289.

[Abstract] Soluble exopolysaccharide is a major virulence factor produced by the plant pathogen Ralstonia solanacearum. Its massive production during plant infection is associated with the arrest of water flow in xylem vessels leading eventually to plant death. The composition of this heavy macromolecule includes ...

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 ...

Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes

Authors: Yoshihiro Adachi
Yoshihiro AdachiAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4472
Kie Itoh
Kie ItohAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4473
Miho Iijima
Miho IijimaAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4474
 and Hiromi Sesaki
Hiromi SesakiAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
For correspondence: hsesaki@jhmi.edu
Bio-protocol author page: a4475
date: 5/5/2017, 245 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2266.

[Abstract] A mitochondrion is a dynamic intracellular organelle that actively divides and fuses to control its size, number and shape in cells. A regulated balance between mitochondrial division and fusion is fundamental to the function, distribution and turnover of mitochondria (Roy et al., 2015). Mitochondrial ...

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 ...

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 ...

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 ...

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

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.

[Abstract] 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. ...

Immunoprecipitation of Cell Surface Proteins from Gram-negative Bacteria

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.

[Abstract] 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 ...
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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] GST-Pull Down Protocol

Author: Lili Jing
Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA
For correspondence: lilijingcn@gmail.com
Bio-protocol author page: a38
date: 1/20/2012, 41875 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.177.

[Abstract] GST-Pull down assay is an effective way to examine the direct binding of two proteins in vitro. This protocol is based on GST pull down system from GE healthcare, and uses the binding of unplugged/MuSK receptor and Wnt ligand as an example to illustrate the detailed procedure....

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] 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 ...

[Bio101] RbCl Super Competent Cells

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 6/5/2011, 11449 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.76.

[Abstract] This method is used to inexpensively prepare home-made competent cells of E. coli. The transformation efficiency is at the high end of chemical-efficient competent cells, and close to library-efficient competent cells....

[Bio101] Expression and Purification of GST-tagged Proteins from E. coli

Author: Lin Fang
Lin FangAffiliation: Department of Pediatrics, School of Medicine, Stanford University, Stanford, USA
For correspondence: cheerfulfang@hotmail.com
Bio-protocol author page: a20
date: 9/20/2011, 10965 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.132.

[Abstract] This protocol describes a method for the small and large-scale expression and purification of GST proteins. Due to the diverse nature of proteins, a small-scale expression and purification test is always recommended....

[Bio101] The Inoue Method for Preparation and Transformation of Competent E. coli: "Ultra Competent" Cells

Author: Hogune Im date: 10/20/2011, 10701 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.143.

[Abstract] This protocol differs from other procedures in that the bacterial culture is grown at 18 °C rather than the conventional 37 °C. Otherwise, the protocol is unremarkable and follows a fairly standard course. Why growing the cells at low temperature should affect the efficiency of transformation is unknown. ...

KMnO4 Footprinting

Authors: Ümit Pul
Ümit PulAffiliation: Molecular Biology of Bacteria, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
Bio-protocol author page: a137
Reinhild Wurm
Reinhild WurmAffiliation: Molecular Biology of Bacteria, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
Bio-protocol author page: a138
 and Rolf Wagner
Rolf WagnerAffiliation: Molecular Biology of Bacteria, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany
For correspondence: r.wagner@rz.uni-duesseldorf.de
Bio-protocol author page: a139
date: 11/5/2012, 9143 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.280.

[Abstract] The KMnO4 footprinting method offers a rapid and easy way to detect and localize single-stranded regions within a duplex DNA molecule, such as it occurs for instance within an actively transcribing RNA polymerase-DNA complex or during R-loop formation in DNA-RNA hybrid structures. The method is based ...

Colony Immunoblotting Assay for Detection of Bacterial Cell-surface or Extracellular Proteins

Authors: Timo A. Lehti
Timo A. LehtiAffiliation: Department of Biosciences, University of Helsinki, Helsinki, Finland
For correspondence: timo.lehti@helsinki.fi
Bio-protocol author page: a809
 and Benita Westerlund-Wikström
Benita Westerlund-WikströmAffiliation: Department of Biosciences, University of Helsinki, Helsinki, Finland
Bio-protocol author page: a810
date: 9/5/2013, 8656 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.888.

[Abstract] This simple protocol describes how to detect antigens from agar-grown bacterial colonies transferred to nitrocellulose using specific antibodies. The protocol is well suitable for detection of bacterial proteins exposed on the cell surface or secreted to the extracellular space and it can be modified ...
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