Welcome guest, Sign in

Home

Microbiology

Preparation of Purified Gram-positive Bacterial Cell Wall and Detection in Placenta and Fetal Tissues

Featured protocol,  Authors: Beth Mann
Beth MannAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3796
Lip Nam Loh
Lip Nam LohAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3797
Geli Gao
Geli GaoAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3798
 and Elaine Tuomanen
Elaine TuomanenAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
For correspondence: Elaine.tuomanen@stjude.org
Bio-protocol author page: a3799
date: 12/5/2016, 34 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2037.

Brief version appeared in Cell Host Microbe, Mar 2016
Cell wall is a complex biopolymer on the surface of all Gram-positive bacteria. During infection, cell wall is recognized by the innate immune receptor Toll-like receptor 2 causing intense inflammation and tissue damage. In animal models, cell wall traffics from the blood stream to many organs in the body, including brain, heart, placenta and fetus. This protocol describes how to prepare purified cell wall from Streptococcus pneumoniae, detect its distribution in animal tissues, and study the tissue response using the placenta and fetal brain as examples.

Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection

Featured protocol,  Authors: Jean Kaoru Millet
Jean Kaoru MilletAffiliation: Department of Microbiology and Immunology, Cornell University, Ithaca NY, United States
For correspondence: jkm248@cornell.edu
Bio-protocol author page: a3793
 and Gary R. Whittaker
Gary R. WhittakerAffiliation: Department of Microbiology and Immunology, Cornell University, Ithaca NY, United States
For correspondence: gary.whittaker@cornell.edu
Bio-protocol author page: a942
date: 12/5/2016, 32 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2035.

Brief version appeared in PNAS, Oct 2014
Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors. The basis for their generation lies in the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins of other viruses into a pseudotyped virus particle. These can be engineered to contain reporter genes such as luciferase, enabling quantification of virus entry events upon pseudotyped particle infection with susceptible cells. Here, we detail a protocol enabling generation of MLV-based pseudotyped particles, using the Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) as an example of a heterologous envelope glycoprotein to be incorporated. We also describe how these particles are used to infect susceptible cells and to perform a quantitative infectivity readout by a luciferase assay.

Mouse Model of Dengue Virus Infection with Serotypes 1 and 2 Clinical Isolates

Featured protocol,  Authors: Satoru Watanabe
Satoru Watanabe Affiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
For correspondence: satoru.watanabe@duke-nus.edu.sg
Bio-protocol author page: a3270
Kitti Wing Ki Chan
Kitti Wing Ki ChanAffiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
Bio-protocol author page: a3810
 and Subhash G. Vasudevan
Subhash G. VasudevanAffiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
For correspondence: subhash.vasudevan@duke-nus.edu.sg
Bio-protocol author page: a3811
date: 12/5/2016, 33 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2040.

Brief version appeared in Antiviral Res, Mar 2016
Dengue is a global public health threat caused by infection with any of the 4 related dengue virus serotypes (DENV1-4). Clinical manifestations range from self-limiting febrile illness, known as dengue fever (DF), to life-threatening severe diseases, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Most cases of DHF/DSS are associated with secondary heterotypic infections through a phenomenon that is described as antibody-dependent enhancement of infection (ADE). There are an estimated 400 million human infections and several hundred thousand cases of severe dengue occurring yearly. At present, however, there are no approved antiviral drugs against DENV infection. The lack of a suitable animal model has hampered the evaluation of novel antiviral candidates for DENV infection. Since DENV poorly establishes infection in immunocompetent mice, AG129 mice (lacking type I and II IFN [interferon] receptors) and mouse-adapted DENV2 strains have been applied to dengue animal models that enable to reproduce several of the major pathologies of human infection. Recently, we developed new mouse models with clinical isolates DENV1 and DENV2 that would be useful for drug testing and dengue pathogenesis studies (Watanabe et al., 2016). Here we describe the details to establish dengue mouse models of clinical isolates; from in vitro preparation of the materials to in vivo virus infection. Of note, since infectivity of DENV in mice differs among virus strains, not all clinical isolates can induce severe dengue.

In vitro Autophosphorylation and Phosphotransfer Assay of Cyanobacterial Histidine Kinase 2

Featured protocol,  Author: Iskander M. Ibrahim
Iskander M. IbrahimAffiliation: Faculty of Engineering and Science, University of Greenwich, Chatham Maritime, Kent, ME4 4TB, UK
For correspondence: I.M.Ibrahim@greenwich.ac.uk
Bio-protocol author page: a3795
date: 12/5/2016, 29 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2036.

Brief version appeared in Front Plant Sci, Feb 2016
This is a detailed protocol of an autophosphorylation and phosphotransfer activities of Synechocystis sp. PCC 6803 full-length Histidine Kinase 2 (Hik2) protein described by Ibrahim et al., 2016. In this protocol, radioactively labelled ATP was used to study an autophosphorylation and phosphotransfer activity of the full-length Hik2 protein.

Single Cell Flow Cytometry Assay for Peptide Uptake by Bacteria

Featured protocol,  Authors: Monica Benincasa*
Monica BenincasaAffiliation: Department of Life Sciences, University of Trieste, Trieste, Italy
Bio-protocol author page: a3800
Quentin Barrière*
Quentin BarrièreAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
Bio-protocol author page: a3801
Giulia Runti
Giulia RuntiAffiliation: Department of Life Sciences, University of Trieste, Trieste, Italy
Bio-protocol author page: a3802
Olivier Pierre
Olivier PierreAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
Bio-protocol author page: a3803
Mick Bourge
Mick BourgeAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
Bio-protocol author page: a3804
Marco Scocchi
Marco ScocchiAffiliation: Department of Life Sciences, University of Trieste, Trieste, Italy
For correspondence: mscocchi@units.it
Bio-protocol author page: a3805
 and Peter Mergaert
Peter MergaertAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
For correspondence: peter.mergaert@i2bc.paris-saclay.fr
Bio-protocol author page: a3806
 (*contributed equally to this work) date: 12/5/2016, 33 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2038.

Brief version appeared in MPMI, Nov 2015
Antimicrobial peptides (AMPs) can target the bacterial envelope or alternatively have intracellular targets. The latter requires uptake of the peptide by the bacterial cells. The bacterial internalization of an AMP can be evaluated by a fluorescence-based method that couples the use of the fluorescently labelled AMP to the fluorescence quencher trypan blue. Trypan blue is excluded from the interior of intact cells and the fluorescence of the extracellular peptide or of the peptide bound on the bacterial surface can be quenched by it, while the fluorescence of the internalized peptide is not affected. The uptake of the peptide by the bacteria is determined by measuring the fluorescence in individual cells by flow cytometry.

Pyocyanin Extraction and Quantitative Analysis in Swarming Pseudomonas aeruginosa

Featured protocol,  Authors: Michelle M. King
Michelle M. KingAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
Bio-protocol author page: a3815
Manita Guragain
Manita GuragainAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
Bio-protocol author page: a3812
Svetlana A. Sarkisova
Svetlana A. SarkisovaAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
Bio-protocol author page: a3816
 and Marianna A. Patrauchan
Marianna A. PatrauchanAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
For correspondence: m.patrauchan@okstate.edu
Bio-protocol author page: a3814
date: 12/5/2016, 27 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2042.

Brief version appeared in J Bacteriol, Jan 2016
This protocol describes the quantification of pyocyanin extracted from swarming colonies of Pseudomonas aeruginosa. Pyocyanin is a secondary metabolite and a major virulence factor, whose production is inducible and varies highly under different growth conditions. The protocol is based on the earlier developed chloroform/HCl extraction of pyocyanin from liquid cultures (Frank and Demoss, 1959). Swarming colonies together with the agar they occupy are split into two halves. Pyocyanin is extracted from one of them. Cells are collected from the other half and used to quantify total protein yield and normalize the estimated corresponding pyocyanin quantities.

Measurement of Intracellular Calcium Concentration in Pseudomonas aeruginosa

Featured protocol,  Authors: Manita Guragain
Manita GuragainAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
Bio-protocol author page: a3812
Anthony K. Campbell
Anthony K. CampbellAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
Bio-protocol author page: a3813
 and Marianna A. Patrauchan
Marianna A. PatrauchanAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
For correspondence: m.patrauchan@okstate.edu
Bio-protocol author page: a3814
date: 12/5/2016, 31 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2041.

Brief version appeared in J Bacteriol, Jan 2016
Characterization of the molecular mechanisms of calcium (Ca2+) regulation of bacterial physiology and virulence requires tools enabling measuring and monitoring the intracellular levels of free calcium (Ca2+in). Here, we describe a protocol optimized to use a recombinantly expressed Ca2+-binding protein, aequorin, for detecting Ca2+in in Pseudomonas aeruginosa. Upon binding to free Ca2+, aequorin undergoes chromophore oxidation and emits light, the log of which intensity linearly correlates with the amount of bound Ca2+, and therefore, can be used to measure the concentration of free Ca2+ available for binding. This protocol involves the introduction of the aequorin gene into P. aeruginosa, induction of apoaequorin production, reconstitution of the holoenzyme with its chromophore, and monitoring its luminescence. This protocol allows continuous measuring of Ca2+in concentration in vivo in response to various stimuli.

Heterologous Expression and Purification of the Magnesium Transporter A (MgtA) in Escherichia coli

Featured protocol,  Authors: Saranya Subramani
Saranya SubramaniAffiliation: Norwegian Centre for Molecular Medicine, Nordic EMBL Partnership University of Oslo, Oslo, Norway
Bio-protocol author page: a3695
 and Jens Preben Morth
Jens Preben MorthAffiliation 1: Norwegian Centre for Molecular Medicine, Nordic EMBL Partnership University of Oslo, Oslo, Norway
Affiliation 2: Institute for Experimental Medical Research, Oslo University Hospital, Oslo, Norway
For correspondence: j.p.morth@ncmm.uio.no
Bio-protocol author page: a3696
date: 11/20/2016, 148 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2001.

Brief version appeared in Elife, Jan 2016
The magnesium transporter A (MgtA) is a magnesium transporting P-type ATPase present in prokaryotes and plants (Subramani et al., 2016). In Salmonella typhimurium and Escherichia coli (E. coli), MgtA is expressed only in magnesium limiting conditions and plays an important role in Mg2+ homeostasis (Groisman et al., 2013). The transcription of mgtA is regulated by the two-component system PhoP/PhoQ (Soncini et al., 1996; Kato et al., 1999). The membrane bound histidine kinase, PhoQ, senses low Mg2+ concentration in the periplasmic space and phosphorylates its cognate response regulator, PhoP, which initiates mgtA transcription (Groisman et al., 2013). MgtA is targeted to the plasma membrane and facilitate the bacterial survival under low Mg2+ condition, by importing Mg2+ into the cytoplasm. The MgtA homolog in petunia (PH1) is found in the vacuolar membrane and involved with the coloration of the flower petals (Faraco et al., 2014). As a first step towards understanding the molecular details of MgtA Mg2+ transport, we describe a detailed protocol for the purification of E. coli MgtA that can be used for biochemical and biophysical studies. Recombinant E. coli MgtA with hexa histidine tag at the N-terminus was cloned from E. coli DH5α and over expressed in the E. coli C43(DE3) by fermentation to an OD > 6. Cell lysis was performed in a high pressure homogenizer and the membranes were isolated by ultracentrifugation. Membrane proteins were solubilized with the detergent dodecyl-β-D maltoside. MgtA was purified by affinity and size exclusion chromatography. Final yields of purified MgtA reach ~1 mg MgtA per 3 g of wet cell pellet.

Transfer of Large Contiguous DNA Fragments onto a Low Copy Plasmid or into the Bacterial Chromosome

Featured protocol,  Authors: Analise Z Reeves
Analise Z ReevesAffiliation 1: Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Cambridge, USA
Affiliation 2: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, USA
Bio-protocol author page: a3697
 and Cammie F Lesser
Cammie F LesserAffiliation 1: Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Cambridge, USA
Affiliation 2: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, USA
For correspondence: clesser@mgh.harvard.edu
Bio-protocol author page: a3698
date: 11/20/2016, 110 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2002.

Brief version appeared in ACS Synth Biol, May 2015
Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the bacterial chromosome that incorporates endogenous yeast and λ Red homologous recombination systems. This methodology has been successfully used to isolate and integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of E. coli and its close relatives.

Biochemical Analysis of Caspase-8-dependent Proteolysis of IRF3 in Virus-infected Cells

Featured protocol,  Authors: Gayatri Subramanian
Gayatri SubramanianAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3735
Karen Pan
Karen PanAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3736
Ritu Chakravarti
Ritu ChakravartiAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3737
 and Saurabh Chattopadhyay
Saurabh ChattopadhyayAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
For correspondence: Saurabh.Chattopadhyay@UToledo.edu
Bio-protocol author page: a3738
date: 11/20/2016, 145 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2018.

Brief version appeared in J Biol Chem, Sep 2011
Interferon regulatory factor 3 (IRF3) is a transcription factor, which is critical for the antiviral response against a wide range of viruses (Hiscott, 2007; Ikushima et al., 2013). It gets activated in virus-infected cells via Toll like receptors (TLRs), RIG-I (retinoic acid inducible gene 1) like receptors (RLRs), cyclic GMP-AMP synthase (cGAS) – stimulator of interferon genes (STING), which are sensors of viral components in the cells (Chattopadhyay and Sen, 2014a; 2014b; Hiscott, 2007). IRF3 is a cytoplasmic protein, upon activation by virally activated sensors it gets phosphorylated, translocated to the nucleus and binds to the interferon-sensitive response element (ISRE) of the gene promoters to induce their transcription (Hiscott, 2007). IRF3 has other functions, including direct stimulation of apoptosis in virus-infected cells. In this pathway, the transcriptional activity of IRF3 is not required (Chattopadhyay et al., 2013b; Chattopadhyay et al., 2016; Chattopadhyay et al., 2010; Chattopadhyay and Sen, 2010; Chattopadhyay et al., 2011). These pathways are negatively regulated by host factors as well as by viruses. Our studies indicate that IRF3 can be proteolytically processed by caspase-8-dependent cleavage (Sears et al., 2011). A specific site in IRF3 is targeted by caspase-8, activated in RNA or DNA virus-infected and dsRNA-stimulated cells (Sears et al., 2011). The direct involvement of caspase-8 was confirmed by in vitro cleavage assay using recombinant proteins and in vivo by virus activated caspase-8. The proteolytic cleavage of IRF3 can be inhibited by chemical inhibition or genetic ablation of caspase-8. The cleavage of IRF3 removes the activated pool of IRF3 and thus can be used as a pro-viral mechanism (Figure 1). Using a C-terminally epitope-tagged human IRF3, we analyzed the cleavage of IRF3 in virus-infected cells. Moreover, we used recombinant proteins in vitro to conclude that IRF3 is a substrate of caspase-8 (Sears et al., 2011). In the current protocol, we have outlined a simple and detailed procedure to biochemically analyze the proteolysis of IRF3 in virus-infected cells and the specific role of caspase-8 in this process.

Uptake Assay for Radiolabeled Peptides in Yeast

Featured protocol,  Authors: Melinda Hauser
Melinda HauserAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
Bio-protocol author page: a3756
Houjian Cai
Houjian CaiAffiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, USA
Bio-protocol author page: a3755
Fred Naider
Fred NaiderAffiliation 1: Department of Chemistry and Macromolecular Assembly Institute, College of Staten Island of the City University of New York, Staten Island, New York, USA
Affiliation 2: Ph.D. Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, USA
Bio-protocol author page: a3757
 and Jeffrey M. Becker
Jeffrey M. BeckerAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
For correspondence: jbecker@utk.edu
Bio-protocol author page: a3758
date: 11/20/2016, 117 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2026.

Brief version appeared in Eukaryot Cell, Oct 2007
We describe an assay for measuring the uptake of radioactive peptides into the yeast Saccharomyces cerevisiae. The methods presented here can be adapted to measure a variety of substrates transported into any bacterial or fungal cell via specific carrier-mediated systems.

Halo Assay for Toxic Peptides and Other Compounds in Microorganisms

Featured protocol,  Authors: Houjian Cai
Houjian CaiAffiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, USA
Bio-protocol author page: a3755
Melinda Hauser
Melinda HauserAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
Bio-protocol author page: a3756
Fred Naider
Fred NaiderAffiliation 1: Department of Chemistry and Macromolecular Assembly Institute, College of Staten Island of the City University of New York, Staten Island, USA
Affiliation 2: Ph.D. Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, USA
Bio-protocol author page: a3757
 and Jeffrey M. Becker
Jeffrey M. BeckerAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
For correspondence: jbecker@utk.edu
Bio-protocol author page: a3758
date: 11/20/2016, 122 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2025.

Brief version appeared in Eukaryot Cell, Oct 2007
We describe an assay for determination of toxicity in S. cerevisiae involving spotting of a toxic peptide on a lawn of yeast cells. This assay may be generalized to determine toxicity of a variety of compounds by substituting a putative toxic compound in place of the peptide. The general protocol may also be used to determine toxicity of any small compound toward another microorganism by replacing S. cerevisiae with the target microbe and modifying growth conditions accordingly.

Transformation of Thermus Species by Natural Competence

Featured protocol,  Authors: Alba Blesa
Alba BlesaAffiliation: Department of Molecular Biology, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid - Consejo Superior de Investigaciones Científicas, Madrid, Spain
Bio-protocol author page: a3710
 and José Berenguer
José BerenguerAffiliation: Department of Molecular Biology, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid - Consejo Superior de Investigaciones Científicas, Madrid, Spain
For correspondence: jberenguer@cbm.csic.es
Bio-protocol author page: a3711
date: 11/20/2016, 121 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2007.

Brief version appeared in J Bacteriol, Jan 2015
Many Thermus species harbour genomes scourged with horizontally transferred signatures. Thermus thermophilus (Tth) has been characterized as naturally competent. The transformation protocol described here is based on the maximum DNA uptake rates registered at mid-exponential phase (OD600 0.3-0.4). Here we describe the stepwise protocol followed for transformation of both plasmids and linearized genomic DNA, of which the latter can be employed as an alternative method to electroporation to introduce mutations or to generate gene deletions in Thermus isolates, for instance.

Cell-to-cell DNA Transfer among Thermus Species

Featured protocol,  Authors: Alba Blesa
Alba BlesaAffiliation: Department of Molecular Biology, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid - Consejo Superior de Investigaciones Científicas, Madrid, Spain
Bio-protocol author page: a3710
 and José Berenguer
José BerenguerAffiliation: Department of Molecular Biology, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid - Consejo Superior de Investigaciones Científicas, Madrid, Spain
For correspondence: jberenguer@cbm.csic.es
Bio-protocol author page: a3711
date: 11/20/2016, 109 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2006.

Brief version appeared in J Bacteriol, Jan 2015
The ability to transfer DNA via direct cell-to-cell contact-dependent process similar to conjugation has been described in Thermus thermophilus (Tth). Here, we detail the mating experiment protocol involving the lateral transfer of thermostable antibiotic resistance markers (i.e., kanamycin: KmR; hygromycin: HygR) between Thermus cells, enabling the selection and quantification of the transfer frequencies. Briefly, liquid cultures of both mates are mixed and laid onto a nitrocellulose filter on a TB plate. After incubation at 60 °C, filters are resuspended upon selective plating. The contribution of DNA uptake by transformation is abolished by the addition of DNase I to the mix. This protocol can be used for the transfer of large DNA fragments (> 10 kb) to Thermus species.

Preparation of Purified Gram-positive Bacterial Cell Wall and Detection in Placenta and Fetal Tissues

Authors: Beth Mann
Beth MannAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3796
Lip Nam Loh
Lip Nam LohAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3797
Geli Gao
Geli GaoAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
Bio-protocol author page: a3798
 and Elaine Tuomanen
Elaine TuomanenAffiliation: Department of Infectious Diseases, St Jude Children’s Research Hospital, Memphis TN, USA
For correspondence: Elaine.tuomanen@stjude.org
Bio-protocol author page: a3799
date: 12/5/2016, 34 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2037.

[Abstract] Cell wall is a complex biopolymer on the surface of all Gram-positive bacteria. During infection, cell wall is recognized by the innate immune receptor Toll-like receptor 2 causing intense inflammation and tissue damage. In animal models, cell wall traffics from the blood stream to many organs in the body, including brain, heart, placenta and fetus. ...

Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection

Authors: Jean Kaoru Millet
Jean Kaoru MilletAffiliation: Department of Microbiology and Immunology, Cornell University, Ithaca NY, United States
For correspondence: jkm248@cornell.edu
Bio-protocol author page: a3793
 and Gary R. Whittaker
Gary R. WhittakerAffiliation: Department of Microbiology and Immunology, Cornell University, Ithaca NY, United States
For correspondence: gary.whittaker@cornell.edu
Bio-protocol author page: a942
date: 12/5/2016, 32 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2035.

[Abstract] Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics ...

Mouse Model of Dengue Virus Infection with Serotypes 1 and 2 Clinical Isolates

Authors: Satoru Watanabe
Satoru Watanabe Affiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
For correspondence: satoru.watanabe@duke-nus.edu.sg
Bio-protocol author page: a3270
Kitti Wing Ki Chan
Kitti Wing Ki ChanAffiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
Bio-protocol author page: a3810
 and Subhash G. Vasudevan
Subhash G. VasudevanAffiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
For correspondence: subhash.vasudevan@duke-nus.edu.sg
Bio-protocol author page: a3811
date: 12/5/2016, 33 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2040.

[Abstract] Dengue is a global public health threat caused by infection with any of the 4 related dengue virus serotypes (DENV1-4). Clinical manifestations range from self-limiting febrile illness, known as dengue fever (DF), to life-threatening severe diseases, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Most cases of DHF/DSS are associated ...

In vitro Autophosphorylation and Phosphotransfer Assay of Cyanobacterial Histidine Kinase 2

Author: Iskander M. Ibrahim
Iskander M. IbrahimAffiliation: Faculty of Engineering and Science, University of Greenwich, Chatham Maritime, Kent, ME4 4TB, UK
For correspondence: I.M.Ibrahim@greenwich.ac.uk
Bio-protocol author page: a3795
date: 12/5/2016, 29 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2036.

[Abstract] This is a detailed protocol of an autophosphorylation and phosphotransfer activities of Synechocystis sp. PCC 6803 full-length Histidine Kinase 2 (Hik2) protein described by Ibrahim et al., 2016. In this protocol, radioactively labelled ATP was used to study an autophosphorylation and phosphotransfer activity of the full-length Hik2 protein.
Keywords: ...

Single Cell Flow Cytometry Assay for Peptide Uptake by Bacteria

Authors: Monica Benincasa*
Monica BenincasaAffiliation: Department of Life Sciences, University of Trieste, Trieste, Italy
Bio-protocol author page: a3800
Quentin Barrière*
Quentin BarrièreAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
Bio-protocol author page: a3801
Giulia Runti
Giulia RuntiAffiliation: Department of Life Sciences, University of Trieste, Trieste, Italy
Bio-protocol author page: a3802
Olivier Pierre
Olivier PierreAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
Bio-protocol author page: a3803
Mick Bourge
Mick BourgeAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
Bio-protocol author page: a3804
Marco Scocchi
Marco ScocchiAffiliation: Department of Life Sciences, University of Trieste, Trieste, Italy
For correspondence: mscocchi@units.it
Bio-protocol author page: a3805
 and Peter Mergaert
Peter MergaertAffiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris‐Sud, Université Paris‐Saclay, Gif‐sur‐Yvette cedex, France
For correspondence: peter.mergaert@i2bc.paris-saclay.fr
Bio-protocol author page: a3806
 (*contributed equally to this work) date: 12/5/2016, 33 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2038.

[Abstract] Antimicrobial peptides (AMPs) can target the bacterial envelope or alternatively have intracellular targets. The latter requires uptake of the peptide by the bacterial cells. The bacterial internalization of an AMP can be evaluated by a fluorescence-based method that couples the use of the fluorescently labelled AMP to the fluorescence quencher trypan ...

Pyocyanin Extraction and Quantitative Analysis in Swarming Pseudomonas aeruginosa

Authors: Michelle M. King
Michelle M. KingAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
Bio-protocol author page: a3815
Manita Guragain
Manita GuragainAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
Bio-protocol author page: a3812
Svetlana A. Sarkisova
Svetlana A. SarkisovaAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
Bio-protocol author page: a3816
 and Marianna A. Patrauchan
Marianna A. PatrauchanAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
For correspondence: m.patrauchan@okstate.edu
Bio-protocol author page: a3814
date: 12/5/2016, 27 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2042.

[Abstract] This protocol describes the quantification of pyocyanin extracted from swarming colonies of Pseudomonas aeruginosa. Pyocyanin is a secondary metabolite and a major virulence factor, whose production is inducible and varies highly under different growth conditions. The protocol is based on the earlier developed chloroform/HCl extraction of pyocyanin ...

Measurement of Intracellular Calcium Concentration in Pseudomonas aeruginosa

Authors: Manita Guragain
Manita GuragainAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
Bio-protocol author page: a3812
Anthony K. Campbell
Anthony K. CampbellAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
Bio-protocol author page: a3813
 and Marianna A. Patrauchan
Marianna A. PatrauchanAffiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, US
For correspondence: m.patrauchan@okstate.edu
Bio-protocol author page: a3814
date: 12/5/2016, 31 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2041.

[Abstract] Characterization of the molecular mechanisms of calcium (Ca2+) regulation of bacterial physiology and virulence requires tools enabling measuring and monitoring the intracellular levels of free calcium (Ca2+in). Here, we describe a protocol optimized to use a recombinantly expressed Ca2+-binding protein, aequorin, for detecting Ca2+in in Pseudomonas ...

Heterologous Expression and Purification of the Magnesium Transporter A (MgtA) in Escherichia coli

Authors: Saranya Subramani
Saranya SubramaniAffiliation: Norwegian Centre for Molecular Medicine, Nordic EMBL Partnership University of Oslo, Oslo, Norway
Bio-protocol author page: a3695
 and Jens Preben Morth
Jens Preben MorthAffiliation 1: Norwegian Centre for Molecular Medicine, Nordic EMBL Partnership University of Oslo, Oslo, Norway
Affiliation 2: Institute for Experimental Medical Research, Oslo University Hospital, Oslo, Norway
For correspondence: j.p.morth@ncmm.uio.no
Bio-protocol author page: a3696
date: 11/20/2016, 148 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2001.

[Abstract] The magnesium transporter A (MgtA) is a magnesium transporting P-type ATPase present in prokaryotes and plants (Subramani et al., 2016). In Salmonella typhimurium and Escherichia coli (E. coli), MgtA is expressed only in magnesium limiting conditions and plays an important role in Mg2+ homeostasis (Groisman et al., 2013). The transcription of mgtA ...

Transfer of Large Contiguous DNA Fragments onto a Low Copy Plasmid or into the Bacterial Chromosome

Authors: Analise Z Reeves
Analise Z ReevesAffiliation 1: Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Cambridge, USA
Affiliation 2: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, USA
Bio-protocol author page: a3697
 and Cammie F Lesser
Cammie F LesserAffiliation 1: Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Cambridge, USA
Affiliation 2: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, USA
For correspondence: clesser@mgh.harvard.edu
Bio-protocol author page: a3698
date: 11/20/2016, 110 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2002.

[Abstract] Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the bacterial ...

Biochemical Analysis of Caspase-8-dependent Proteolysis of IRF3 in Virus-infected Cells

Authors: Gayatri Subramanian
Gayatri SubramanianAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3735
Karen Pan
Karen PanAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3736
Ritu Chakravarti
Ritu ChakravartiAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3737
 and Saurabh Chattopadhyay
Saurabh ChattopadhyayAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
For correspondence: Saurabh.Chattopadhyay@UToledo.edu
Bio-protocol author page: a3738
date: 11/20/2016, 145 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2018.

[Abstract] Interferon regulatory factor 3 (IRF3) is a transcription factor, which is critical for the antiviral response against a wide range of viruses (Hiscott, 2007; Ikushima et al., 2013). It gets activated in virus-infected cells via Toll like receptors (TLRs), RIG-I (retinoic acid inducible gene 1) like receptors (RLRs), cyclic GMP-AMP synthase (cGAS) – ...
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 

[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, 77723 views, 30 Q&A, How to cite
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, 70628 views, 46 Q&A, How to cite
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, 21605 views, 6 Q&A, How to cite
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, 20290 views, 0 Q&A, How to cite
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, 19512 views, 2 Q&A, How to cite
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, 16693 views, 3 Q&A, How to cite
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, 16532 views, 1 Q&A, How to cite
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, 15597 views, 4 Q&A, How to cite
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, 13532 views, 1 Q&A, How to cite
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, 12426 views, 1 Q&A, How to cite
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 ...
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40