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Measurement of RNA-induced PKR Activation in vitro

Featured protocol,  Author: Kobe C. Yuen
Kobe C. YuenAffiliation: Stowers Institute for Medical Research, Kansas City, MO, USA
Present address: 1 DNA Way, South San Francisco, USA
For correspondence: yuenc4@gene.com
Bio-protocol author page: a4260
date: 3/20/2017, 141 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2178.

Brief version appeared in Cell Rep, Jan 2016
Protein kinase R (PKR) is one of the key RNA-activated sensors for innate immunity. PKR is activated by pathogenic or aberrant RNAs such as short double-stranded RNAs or those with imperfect secondary structures, as well as a reduction in the amount and number of RNA modifications. Activation of PKR may be an underlying mechanism for the pathogenesis of human diseases. In this protocol, I describe a method for studying levels of RNA-induced PKR activation in vitro.

Extraction and Analysis of Carotenoids from Escherichia coli in Color Complementation Assays

Featured protocol,  Authors: Andreas Blatt
Andreas BlattAffiliation: Institut für Molekulare Physiologie - Pflanzenbiochemie, Johannes Gutenberg-Universität, Mainz, Germany
Bio-protocol author page: a4223
 and Martin Lohr
Martin LohrAffiliation: Institut für Molekulare Physiologie - Pflanzenbiochemie, Johannes Gutenberg-Universität, Mainz, Germany
For correspondence: lohr@uni-mainz.de
Bio-protocol author page: a4224
date: 3/20/2017, 109 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2179.

Brief version appeared in Plant J, May 2015
A common method to investigate the function of genes putatively involved in carotenoid biosynthesis is the so called color complementation assay in Escherichia coli (see, e.g., Cunningham and Gantt, 2007). In this assay, the gene under investigation is expressed in E. coli strains genetically engineered to synthesize potential carotenoid substrates, followed by analysis of the pigment changes in the carotenogenic bacteria via high-performance liquid chromatography (HPLC). Two crucial steps in this method are (i) the quantitative extraction of the carotenoids out of E. coli and (ii) the reproducible and complete separation of the pigments by HPLC.

Determination of the Predatory Capability of Bdellovibrio bacteriovorus HD100

Featured protocol,  Authors: Cristina Herencias
Cristina HerenciasAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Bio-protocol author page: a4258
M. Auxiliadora Prieto
M. Auxiliadora PrietoAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Bio-protocol author page: a4259
 and Virginia Martínez
Virginia MartínezAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Present address: Evolva Biotech A/S, Copenhagen, Denmark
For correspondence: virginiaml83@gmail.com
Bio-protocol author page: a4257
date: 3/20/2017, 116 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2177.

Brief version appeared in Sci Rep, Apr 2016
Bdellovibrio bacteriovorus HD100 is an obligate predator that preys upon a wide variety of Gram negative bacteria. The biphasic growth cycle of Bdellovibrio includes a free-swimming attack phase and an intraperiplasmic growth phase, where the predator replicates its DNA and grows using the prey as a source of nutrients, finally dividing into individual cells (Sockett, 2009). Due to its obligatory predatory lifestyle, manipulation of Bdellovibrio requires two-member culturing techniques using selected prey microorganisms (Lambert et al., 2003). In this protocol, we describe a detailed workflow to grow and quantify B. bacteriovorus HD100 and its predatory ability, to easily carry out these laborious and time-consuming techniques.

Extraction, Purification and Quantification of Diffusible Signal Factor Family Quorum-sensing Signal Molecules in Xanthomonas oryzae pv. oryzae

Featured protocol,  Authors: Lian Zhou
Lian ZhouAffiliation 1: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Affiliation 2: Zhiyuan Innovative Research Center, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4225
Xing-Yu Wang
Xing-Yu WangAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4226
Wei Zhang
Wei ZhangAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4227
Shuang Sun
Shuang SunAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4228
 and Ya-Wen He
Ya-Wen HeAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
For correspondence: yawenhe@sjtu.edu.cn
Bio-protocol author page: a4229
date: 3/20/2017, 108 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2190.

Brief version appeared in Mol Plant Microbe Interact, Mar 2016
Bacteria use quorum-sensing (QS) systems to monitor and regulate their population density. Bacterial QS involves small molecules that act as signals for bacterial communication. Many Gram-negative bacterial pathogens use a class of widely conserved molecules, called diffusible signal factor (DSF) family QS signals. The measurement of DSF family signal molecules is essential for understanding DSF metabolic pathways, signaling networks, as well as regulatory roles. Here, we describe a method for the extraction of DSF family signal molecules from Xanthomonas oryzae pv. oryzae (Xoo) cell pellets and Xoo culture supernatant. We determined the levels of DSF family signals using ultra-performance liquid chromatographic system (UPLC) coupled with accurate mass time-of-flight mass spectrometer (TOF-MS). With the aid of UPLC/MS system, the detection limit of DSF was as low as 1 µM, which greatly improves the ability to detect DSF DSF family signal molecules in bacterial cultures and reaction mixtures.

Isolation of Ribosomal Particles from the Unicellular Cyanobacterium Synechocystis sp. PCC 6803

Featured protocol,  Authors: Carla V. Galmozzi
Carla V. GalmozziAffiliation 1: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Sevilla, Spain
Affiliation 2: Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany
Bio-protocol author page: a4172
 and M. Isabel Muro-Pastor
M. Isabel Muro-Pastor Affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Sevilla, Spain
For correspondence: imuro@ibvf.csic.es
Bio-protocol author page: a4173
date: 3/20/2017, 107 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2176.

Brief version appeared in PLoS One, Jul 2016
Isolation of ribosomal particles is an essential step in the study of ribosomal components as well as in the analysis of trans-acting factors that interact with the ribosome to regulate protein synthesis and modulate the expression profile of the cell in response to different environmental conditions. In this protocol, we describe a procedure for the isolation of 70S ribosomes from the unicellular cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We have successfully used this protocol in our study of the cyanobacterial ribosomal-associated protein LrtA, which is a homologue of bacterial HPF (hibernation promoting factor) (Galmozzi et al., 2016).

Assays for the Detection of Rubber Oxygenase Activities

Featured protocol,  Authors: Wolf Röther
Wolf RötherAffiliation: Institute of Microbiology, University Stuttgart, Stuttgart, Germany
Bio-protocol author page: a4273
Jakob Birke
Jakob BirkeAffiliation: Institute of Microbiology, University Stuttgart, Stuttgart, Germany
Bio-protocol author page: a4274
 and Dieter Jendrossek
Dieter JendrossekAffiliation: Institute of Microbiology, University Stuttgart, Stuttgart, Germany
For correspondence: dieter.jendrossek@imb.uni-stuttgart.de
Bio-protocol author page: a4275
date: 3/20/2017, 117 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2188.

Brief version appeared in BMC Microbiol, May 2016
Microbial biodegradation of rubber relies on extracellular rubber oxygenases that catalyze the oxidative cleavage of the double bond of the polyisoprene backbone into oligo-isoprenoids. This protocol describes the determination of rubber oxygenase activities by an online measurement of molecular oxygen consumption via a non-invasive fluorescence-based assay. The produced oligo-isoprenoid cleavage products with terminal keto- and aldehyde-groups are identified qualitatively and quantitatively by HPLC. Our method allows for the characterization of homologue rubber oxygenases, and can likely be adapted to assay other oxygenases consuming dioxygen. Here we describe the determination of rubber oxygenase activities at the examples of the so far two known types of rubber oxygenases, namely rubber oxygenase A (RoxA) and latex clearing protein (Lcp).

In Gel Kinase Assay

Featured protocol,  Authors: Gaston A. Pizzio
Gaston A. PizzioAffiliation: Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG; consortium CSIC-IRTA-UAB-UB), Barcelona, Spain
For correspondence: gapizzio@gmail.com
Bio-protocol author page: a2769
 and Pedro L. Rodriguez
Pedro L. RodriguezAffiliation: Instituto de Biologia Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientificas-Universidad Politecnica de Valencia, Valencia, Spain
Bio-protocol author page: a4211
date: 3/5/2017, 233 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2170.

Brief version appeared in Plant Cell, Jun 2012
Proper spatiotemporal regulation of protein phosphorylation in cells and tissues is required for normal development and homeostasis. We present the protocol ‘In Gel Kinase Assay’, which is useful for protein kinase activity measurements from crude protein extracts. We have successfully used ‘In Gel Kinase Assay’ protocol to show that the Arabidopsis thaliana sextuple mutant in the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR-ABA receptors; line pyr/pyl112458) is impaired in ABA-mediated activation of SnRK2.2, SnRK2.3 and OST1/SnRK2.6, as much as the triple mutant snrk2.2/2.3/2.6 (Gonzalez-Guzman et al., 2012).

Surface Inoculation and Quantification of Pseudomonas syringae Population in the Arabidopsis Leaf Apoplast

Featured protocol,  Authors: Cristián Jacob*
Cristián JacobAffiliation: Department of Plant Sciences, University of California, Davis, USA
For correspondence: cjjacob@ucdavis.edu
Bio-protocol author page: a4204
Shweta Panchal*
Shweta PanchalAffiliation: Centre for Genome Research, Faculty of Science, the Maharaja Sayajirao University of Baroda, Baroda, India
For correspondence: shwetapanchal84@gmail.com
Bio-protocol author page: a4158
 and Maeli Melotto
Maeli MelottoAffiliation: Department of Plant Sciences, University of California, Davis, USA
For correspondence: melotto@ucdavis.edu
Bio-protocol author page: a4160
 (*contributed equally to this work) date: 3/5/2017, 239 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2167.

Brief version appeared in Front Plant Sci, Jun 2016
Bacterial pathogens must enter the plant tissue in order to cause a successful infection. Foliar bacterial pathogens that are not able to directly penetrate the plant epidermis rely on wounds or natural openings to internalize leaves. This protocol describes a procedure to estimate the population size of Pseudomonas syringae in the leaf apoplast after surface inoculation of Arabidopsis rosettes.

An HPLC-based Method to Quantify Coronatine Production by Bacteria

Featured protocol,  Authors: Shweta Panchal
Shweta PanchalAffiliation: Centre for Genome Research, Department of Microbiology and Biotechnology Centre, the Maharaja Sayajirao University of Baroda, Baroda, India
For correspondence: shwetapanchal84@gmail.com
Bio-protocol author page: a4158
Zachary S. Breitbach
Zachary S. BreitbachAffiliation: Department of Chemistry, University of Texas, Arlington, USA
For correspondence: zachbreitbach@yahoo.com
Bio-protocol author page: a4159
 and Maeli Melotto
Maeli MelottoAffiliation: Department of Plant Sciences, University of California, Davis, USA
For correspondence: melotto@ucdavis.edu
Bio-protocol author page: a4160
date: 3/5/2017, 232 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2147.

Brief version appeared in Front Plant Sci, Jun 2016
Coronatine is a polyketide phytotoxin produced by several pathovars of the plant pathogenic bacterium Pseudomonas syringae. It is one of the most important virulence factors determining the success of bacterial pathogenesis in the plant at both epiphytic and endophytic stages of the disease cycle. This protocol describes an optimized procedure to culture bacterial cells for coronatine production and to quantify the amount of coronatine secreted in the culture medium using an HPLC-based method.

Isolation of Outer Membrane Vesicles from Phytopathogenic Xanthomonas campestris pv. campestris

Featured protocol,  Authors: Gideon Mordukhovich
Gideon MordukhovichAffiliation 1: Department of Plant Pathology and Weed Research, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel
Affiliation 2: Robert H. Smith Faculty of Agriculture, Food, and Environment, Hebrew University of Jerusalem, Rehovot, Israel
For correspondence: gideon.mordukhovic@mail.huji.ac.il
Bio-protocol author page: a4144
 and Ofir Bahar
Ofir BaharAffiliation: Department of Plant Pathology and Weed Research, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel
For correspondence: ofirb@agri.gov.il
Bio-protocol author page: a4145
date: 3/5/2017, 163 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2160.

Brief version appeared in Mol Plant Microbe Interact, May 2016
Gram-negative bacteria naturally release outer membrane vesicles (OMVs) to the surrounding environment. OMVs contribute to multiple processes, such as cell-cell communication, delivery of enzymes and toxins, resistance to environmental stresses and pathogenesis. Little is known about OMVs produced by plant-pathogenic bacteria, and their interactions with host plants. The protocol described below discusses the isolation process of OMVs from Xanthomonas campestris pv. campestris strain 33913, a bacterial pathogen of Crucifiers. Nevertheless, this protocol can be used and/or adapted for isolation of OMVs from other phytopathogenic bacteria to promote the study of OMVs in the context of plant-microbe interactions.

Measurement of RNA-induced PKR Activation in vitro

Author: Kobe C. Yuen
Kobe C. YuenAffiliation: Stowers Institute for Medical Research, Kansas City, MO, USA
Present address: 1 DNA Way, South San Francisco, USA
For correspondence: yuenc4@gene.com
Bio-protocol author page: a4260
date: 3/20/2017, 141 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2178.

[Abstract] Protein kinase R (PKR) is one of the key RNA-activated sensors for innate immunity. PKR is activated by pathogenic or aberrant RNAs such as short double-stranded RNAs or those with imperfect secondary structures, as well as a reduction in the amount and number of RNA modifications. Activation of PKR ...

Extraction and Analysis of Carotenoids from Escherichia coli in Color Complementation Assays

Authors: Andreas Blatt
Andreas BlattAffiliation: Institut für Molekulare Physiologie - Pflanzenbiochemie, Johannes Gutenberg-Universität, Mainz, Germany
Bio-protocol author page: a4223
 and Martin Lohr
Martin LohrAffiliation: Institut für Molekulare Physiologie - Pflanzenbiochemie, Johannes Gutenberg-Universität, Mainz, Germany
For correspondence: lohr@uni-mainz.de
Bio-protocol author page: a4224
date: 3/20/2017, 109 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2179.

[Abstract] A common method to investigate the function of genes putatively involved in carotenoid biosynthesis is the so called color complementation assay in Escherichia coli (see, e.g., Cunningham and Gantt, 2007). In this assay, the gene under investigation is expressed in E. coli strains genetically engineered ...

Determination of the Predatory Capability of Bdellovibrio bacteriovorus HD100

Authors: Cristina Herencias
Cristina HerenciasAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Bio-protocol author page: a4258
M. Auxiliadora Prieto
M. Auxiliadora PrietoAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Bio-protocol author page: a4259
 and Virginia Martínez
Virginia MartínezAffiliation: Environmental Biology Department, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain
Present address: Evolva Biotech A/S, Copenhagen, Denmark
For correspondence: virginiaml83@gmail.com
Bio-protocol author page: a4257
date: 3/20/2017, 116 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2177.

[Abstract] Bdellovibrio bacteriovorus HD100 is an obligate predator that preys upon a wide variety of Gram negative bacteria. The biphasic growth cycle of Bdellovibrio includes a free-swimming attack phase and an intraperiplasmic growth phase, where the predator replicates its DNA and grows using the prey as a ...

Extraction, Purification and Quantification of Diffusible Signal Factor Family Quorum-sensing Signal Molecules in Xanthomonas oryzae pv. oryzae

Authors: Lian Zhou
Lian ZhouAffiliation 1: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Affiliation 2: Zhiyuan Innovative Research Center, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4225
Xing-Yu Wang
Xing-Yu WangAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4226
Wei Zhang
Wei ZhangAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4227
Shuang Sun
Shuang SunAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4228
 and Ya-Wen He
Ya-Wen HeAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
For correspondence: yawenhe@sjtu.edu.cn
Bio-protocol author page: a4229
date: 3/20/2017, 108 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2190.

[Abstract] Bacteria use quorum-sensing (QS) systems to monitor and regulate their population density. Bacterial QS involves small molecules that act as signals for bacterial communication. Many Gram-negative bacterial pathogens use a class of widely conserved molecules, called diffusible signal factor (DSF) family ...

Isolation of Ribosomal Particles from the Unicellular Cyanobacterium Synechocystis sp. PCC 6803

Authors: Carla V. Galmozzi
Carla V. GalmozziAffiliation 1: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Sevilla, Spain
Affiliation 2: Zentrum für Molekulare Biologie der Universität Heidelberg, Heidelberg, Germany
Bio-protocol author page: a4172
 and M. Isabel Muro-Pastor
M. Isabel Muro-Pastor Affiliation: Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Sevilla, Spain
For correspondence: imuro@ibvf.csic.es
Bio-protocol author page: a4173
date: 3/20/2017, 107 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2176.

[Abstract] Isolation of ribosomal particles is an essential step in the study of ribosomal components as well as in the analysis of trans-acting factors that interact with the ribosome to regulate protein synthesis and modulate the expression profile of the cell in response to different environmental conditions. ...

Assays for the Detection of Rubber Oxygenase Activities

Authors: Wolf Röther
Wolf RötherAffiliation: Institute of Microbiology, University Stuttgart, Stuttgart, Germany
Bio-protocol author page: a4273
Jakob Birke
Jakob BirkeAffiliation: Institute of Microbiology, University Stuttgart, Stuttgart, Germany
Bio-protocol author page: a4274
 and Dieter Jendrossek
Dieter JendrossekAffiliation: Institute of Microbiology, University Stuttgart, Stuttgart, Germany
For correspondence: dieter.jendrossek@imb.uni-stuttgart.de
Bio-protocol author page: a4275
date: 3/20/2017, 117 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2188.

[Abstract] Microbial biodegradation of rubber relies on extracellular rubber oxygenases that catalyze the oxidative cleavage of the double bond of the polyisoprene backbone into oligo-isoprenoids. This protocol describes the determination of rubber oxygenase activities by an online measurement of molecular ...

In Gel Kinase Assay

Authors: Gaston A. Pizzio
Gaston A. PizzioAffiliation: Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG; consortium CSIC-IRTA-UAB-UB), Barcelona, Spain
For correspondence: gapizzio@gmail.com
Bio-protocol author page: a2769
 and Pedro L. Rodriguez
Pedro L. RodriguezAffiliation: Instituto de Biologia Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientificas-Universidad Politecnica de Valencia, Valencia, Spain
Bio-protocol author page: a4211
date: 3/5/2017, 233 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2170.

[Abstract] Proper spatiotemporal regulation of protein phosphorylation in cells and tissues is required for normal development and homeostasis. We present the protocol ‘In Gel Kinase Assay’, which is useful for protein kinase activity measurements from crude protein extracts. We have successfully used ‘In Gel ...

Surface Inoculation and Quantification of Pseudomonas syringae Population in the Arabidopsis Leaf Apoplast

Authors: Cristián Jacob*
Cristián JacobAffiliation: Department of Plant Sciences, University of California, Davis, USA
For correspondence: cjjacob@ucdavis.edu
Bio-protocol author page: a4204
Shweta Panchal*
Shweta PanchalAffiliation: Centre for Genome Research, Faculty of Science, the Maharaja Sayajirao University of Baroda, Baroda, India
For correspondence: shwetapanchal84@gmail.com
Bio-protocol author page: a4158
 and Maeli Melotto
Maeli MelottoAffiliation: Department of Plant Sciences, University of California, Davis, USA
For correspondence: melotto@ucdavis.edu
Bio-protocol author page: a4160
 (*contributed equally to this work) date: 3/5/2017, 239 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2167.

[Abstract] Bacterial pathogens must enter the plant tissue in order to cause a successful infection. Foliar bacterial pathogens that are not able to directly penetrate the plant epidermis rely on wounds or natural openings to internalize leaves. This protocol describes a procedure to estimate the population size ...

An HPLC-based Method to Quantify Coronatine Production by Bacteria

Authors: Shweta Panchal
Shweta PanchalAffiliation: Centre for Genome Research, Department of Microbiology and Biotechnology Centre, the Maharaja Sayajirao University of Baroda, Baroda, India
For correspondence: shwetapanchal84@gmail.com
Bio-protocol author page: a4158
Zachary S. Breitbach
Zachary S. BreitbachAffiliation: Department of Chemistry, University of Texas, Arlington, USA
For correspondence: zachbreitbach@yahoo.com
Bio-protocol author page: a4159
 and Maeli Melotto
Maeli MelottoAffiliation: Department of Plant Sciences, University of California, Davis, USA
For correspondence: melotto@ucdavis.edu
Bio-protocol author page: a4160
date: 3/5/2017, 232 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2147.

[Abstract] Coronatine is a polyketide phytotoxin produced by several pathovars of the plant pathogenic bacterium Pseudomonas syringae. It is one of the most important virulence factors determining the success of bacterial pathogenesis in the plant at both epiphytic and endophytic stages of the disease cycle. This ...

Isolation of Outer Membrane Vesicles from Phytopathogenic Xanthomonas campestris pv. campestris

Authors: Gideon Mordukhovich
Gideon MordukhovichAffiliation 1: Department of Plant Pathology and Weed Research, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel
Affiliation 2: Robert H. Smith Faculty of Agriculture, Food, and Environment, Hebrew University of Jerusalem, Rehovot, Israel
For correspondence: gideon.mordukhovic@mail.huji.ac.il
Bio-protocol author page: a4144
 and Ofir Bahar
Ofir BaharAffiliation: Department of Plant Pathology and Weed Research, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel
For correspondence: ofirb@agri.gov.il
Bio-protocol author page: a4145
date: 3/5/2017, 163 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2160.

[Abstract] Gram-negative bacteria naturally release outer membrane vesicles (OMVs) to the surrounding environment. OMVs contribute to multiple processes, such as cell-cell communication, delivery of enzymes and toxins, resistance to environmental stresses and pathogenesis. Little is known about OMVs produced by ...
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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, 85874 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, 78980 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, 40449 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, 16881 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, 13153 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, 11026 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, 10631 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, 10154 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, 8649 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, 8070 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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