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

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

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

Bacterial Intracellular Sodium Ion Measurement using CoroNa Green

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

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

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

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

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

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

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

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

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

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

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

Antibiotic Disc Assay for Synechocystis sp. PCC6803

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

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

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

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

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

Bacterial Intracellular Sodium Ion Measurement using CoroNa Green

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

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

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

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

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

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

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

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

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

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

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

Antibiotic Disc Assay for Synechocystis sp. PCC6803

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

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

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, 218 views, 0 Q&A
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 ...

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, 227 views, 0 Q&A
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 ...

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, 217 views, 0 Q&A
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 ...

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, 211 views, 0 Q&A
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 ...
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[Bio101] Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method

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

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

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

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

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

[Bio101] 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, 38791 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, 15990 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.49.

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

[Bio101] Purification of 6x His-tagged Protein (from E. coli)

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 12642 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.8.

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

[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, 10489 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, 10255 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, 9582 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, 8036 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, 7420 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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