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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.

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.

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 ...

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 ...

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 ...

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 ...

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, ...

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 ...

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 ...

Transformation of Thermus Species by Natural Competence

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.

[Abstract] 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 ...

Cell-to-cell DNA Transfer among Thermus Species

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.

[Abstract] 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: ...

Quantitation of Cytochromes b559, b6, and f, and the Core Component of Photosystem I P700 in Cyanobacterial Cells

Authors: Motohide Aoki
Motohide AokiAffiliation: School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
Bio-protocol author page: a3671
Mikio Tsuzuki
Mikio Tsuzuki Affiliation: School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
Bio-protocol author page: a3672
 and Norihiro Sato
Norihiro SatoAffiliation: School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan
For correspondence: nsato@ls.toyaku.ac.jp
Bio-protocol author page: a3673
date: 11/5/2016, 166 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1991.

[Abstract] Cytochrome (Cyt) b559, an important and essential core component of photosystem II in the photosynthetic electron transport system, is a heme-bridged heterodimer protein composed of an alpha subunit (PsbE) and a beta subunit (PsbE), and its reduced form has an absorption maximum in the α-band at 559 ...
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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, 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] 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, 38134 views, 4 Q&A, How to cite
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, 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] 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 ...

[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, 10291 views, 2 Q&A, How to cite
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, 10087 views, 1 Q&A, How to cite
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, 9368 views, 1 Q&A, How to cite
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, 7794 views, 1 Q&A, How to cite
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, 7180 views, 1 Q&A, How to cite
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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