Bacteria

Bacterial Survival in Dictyostelium

Authors: Regin Rønn*
Regin RønnAffiliation 1: Department of Biology, University of Copenhagen, Copenhagen, Denmark
Affiliation 2: Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, China
Bio-protocol author page: a4771
, Xiuli Hao*
Xiuli HaoAffiliation: Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, China
Bio-protocol author page: a4772
, Freja Lüthje
Freja LüthjeAffiliation: Department of Biology, University of Copenhagen, Copenhagen, Denmark
Bio-protocol author page: a4773
, Nadezhda A. German
Nadezhda A. GermanAffiliation: Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, TX, USA
Bio-protocol author page: a4774
, Xuanji Li
Xuanji LiAffiliation: Department of Biology, University of Copenhagen, Copenhagen, Denmark
Bio-protocol author page: a4775
, Fuyi Huang
Fuyi HuangAffiliation: Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, China
Bio-protocol author page: a4776
, Javan Kisaka
Javan KisakaAffiliation: Department of Chemistry, Western Michigan University, Kalamazoo, MI, USA
Present address: Division of Infectious Diseases, Washington University School of Medicine, St. Louis, MO, USA
Bio-protocol author page: a4777
, David Huffman
David HuffmanAffiliation 1: Department of Chemistry, Western Michigan University, Kalamazoo, MI, USA
Affiliation 2: Department of Plant and Environmental Sciences, University of Copenhagen, MO, USA
Bio-protocol author page: a4778
, Hend A. Alwathnani
Hend A. AlwathnaniAffiliation: Department of Botany and Microbiology, King Saud University, Riyadh, Saudi Arabia
Bio-protocol author page: a4779
, Yong-Guan Zhu
Yong-Guan ZhuAffiliation: Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, China
Bio-protocol author page: a4780
and Christopher Rensing
Christopher RensingAffiliation 1: Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, China
Affiliation 2: J. Craig Venter Institute, La Jolla, CA, USA
Affiliation 3: Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation, College of Resources and Environment, Fujian Agriculture & Forestry University, Fuzhou, China
For correspondence: rensing@iue.ac.cn
Bio-protocol author page: a4781
(*contributed equally to this work), date: 7/5/2017, 107 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2376.

[Abstract] We performed an assay to test the ability of different E. coli strains to survive inside amoebal cells after ingestion. In the assay we incubated bacteria together with cells of Dictyostelium discoideum for six hours. After co-incubation most of the uningested bacteria were removed by centrifugation ...

RNA Capping by Transcription Initiation with Non-canonical Initiating Nucleotides (NCINs): Determination of Relative Efficiencies of Transcription Initiation with NCINs and NTPs

Authors: Jeremy G. Bird
Jeremy G. BirdAffiliation 1: Department of Genetics and Waksman Institute, Rutgers University, Piscataway, USA
Affiliation 2: Department of Chemistry and Waksman Institute, Rutgers University, Piscataway, USA
Bio-protocol author page: a4666
, Bryce E. Nickels
Bryce E. NickelsAffiliation: Department of Genetics and Waksman Institute, Rutgers University, Piscataway, USA
For correspondence: bnickels@waksman.rutgers.edu
Bio-protocol author page: a4667
and Richard H. Ebright
Richard H. EbrightAffiliation: Department of Chemistry and Waksman Institute, Rutgers University, Piscataway, USA
For correspondence: ebright@waksman.rutgers.edu
Bio-protocol author page: a4668
, date: 6/20/2017, 200 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2336.

[Abstract] It recently has been established that adenine-containing cofactors, including nicotinamide adenine dinucleotide (NAD⁺), reduced nicotinamide adenine dinucleotide (NADH), and 3’-desphospho-coenzyme A (dpCoA), can serve as ‘non-canonical initiating nucleotides’ (NCINs) for transcription initiation by ...

Culturing Bacteria from Caenorhabditis elegans Gut to Assess Colonization Proficiency

Authors: Facundo Rodriguez Ayala
Facundo Rodriguez AyalaAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
For correspondence: facundoayala.foncyt@gmail.com
Bio-protocol author page: a4690
, Sebastián Cogliati
Sebastián CogliatiAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
Bio-protocol author page: a4691
, Carlos Bauman
Carlos BaumanAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
Bio-protocol author page: a4692
, Cecilia Leñini
Cecilia LeñiniAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
Bio-protocol author page: a4693
, Marco Bartolini
Marco BartoliniAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
Bio-protocol author page: a4694
, Juan Manuel Villalba
Juan Manuel VillalbaAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
Bio-protocol author page: a4695
, Federico Argañaraz
Federico ArgañarazAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
Bio-protocol author page: a4696
and Roberto Grau
Roberto GrauAffiliation: Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, CONICET, Rosario, Argentina
Bio-protocol author page: a4697
, date: 6/20/2017, 248 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2345.

[Abstract] Determining an accurate count of intestinal bacteria from Caenorhabditis elegans is one critical way to assess colonization proficiency by a given bacteria. This can be accomplished by culturing appropriate dilutions of worm gut bacteria on selective or differential agarized media. Because of the high ...

Producing GST-Cbx7 Fusion Proteins from Escherichia coli

Authors: Thao Ngoc Huynh
Thao Ngoc HuynhAffiliation: Department of Chemistry, University of Colorado Denver, Denver, CO, USA
Bio-protocol author page: a4652
and Xiaojun Ren
Xiaojun Ren Affiliation: Department of Chemistry, University of Colorado Denver, Denver, CO, USA
For correspondence: xiaojun.ren@ucdenver.edu
Bio-protocol author page: a4653
, date: 6/20/2017, 223 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2333.

[Abstract] This protocol describes the production of GST-Cbx7 fusion proteins from E. coli, originally developed in the recent publication (Zhen et al., 2016). The pGEX-6P-1-GST plasmids encoding the Cbx7 variants were transformed into BL21 competent cells. The fusion protein production was induced by isopropyl-beta-D-thiogalactopyranoside ...

Isolation and Infection of Drosophila Primary Hemocytes

Authors: Charles Tracy
Charles TracyAffiliation: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
Bio-protocol author page: a4450
and Helmut Krämer
Helmut KrämerAffiliation 1: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
Affiliation 2: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
Affiliation 3: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
For correspondence: helmut.kramer@utsouthwestern.edu
Bio-protocol author page: a4451
, date: 6/5/2017, 300 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2300.

[Abstract] Phagocytosis of invading pathogens and their subsequent clearance in lysosomes is important for organismal fitness. We have devised the following protocol to extract phagocytic hemocytes from wild-type and mutant Drosophila larvae and infect the isolated hemocytes with GFP-labeled E. coli to measure ...

Escherichia coli Infection of Drosophila

Authors: Charles Tracy
Charles TracyAffiliation: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, USA
Bio-protocol author page: a4450
and Helmut Krämer
Helmut KrämerAffiliation 1: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, USA
Affiliation 2: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, USA
For correspondence: helmut.kramer@utsouthwestern.edu
Bio-protocol author page: a4451
, date: 5/5/2017, 416 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2256.

[Abstract] Following septic insults, healthy insects, just like vertebrates, mount a complex immune response to contain and destroy pathogens. The failure to efficiently clear bacterial infections in immuno-compromised fly mutants leads to higher mortality rates which provide a powerful indicator for genes with ...

Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes

Authors: Yoshihiro Adachi
Yoshihiro AdachiAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4472
, Kie Itoh
Kie ItohAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4473
, Miho Iijima
Miho IijimaAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
Bio-protocol author page: a4474
and Hiromi Sesaki
Hiromi SesakiAffiliation: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
For correspondence: hsesaki@jhmi.edu
Bio-protocol author page: a4475
, date: 5/5/2017, 511 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2266.

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

Preparation of Everted Membrane Vesicles from Escherichia coli Cells

Author: Marina Verkhovskaya
Marina VerkhovskayaAffiliation: Institute of Biotechnology, PO Box 65 (Viikinkaari 1) FIN-00014 University of Helsinki, Helsinki, Finland
For correspondence: Marina.Verkhovskaya@Helsinki.Fi
Bio-protocol author page: a4446
, date: 5/5/2017, 349 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2254.

[Abstract] The protocol for obtaining electrically sealed membrane vesicles from E. coli cells is presented. Proton pumps such as Complex I, quinol oxidase, and ATPase are active in the obtained vesicles. Quality of the preparation was tested by monitoring the electric potential generated by these pumps. ...

Expression and Purification of Mini G Proteins from Escherichia coli

Authors: Byron Carpenter
Byron CarpenterAffiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge, UK
Present address: Warwick Integrative Synthetic Biology Centre, School of Life Sciences, Gibbet Hill Campus, The University of Warwick, Coventry, UK
For correspondence: b.carpenter@warwick.ac.uk
Bio-protocol author page: a4379
and Christopher G. Tate
Christopher G. TateAffiliation: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick Avenue, Cambridge, UK
Bio-protocol author page: a4380
, date: 4/20/2017, 794 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2235.

[Abstract] Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR–G protein complexes is important to fully decipher the mechanism of signal ...

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, 639 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 ...

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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, 93294 views, 32 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, 86300 views, 47 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, 43050 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....

[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, 14082 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, 11835 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, 11286 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, 11121 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, 9559 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, 9114 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 ...

Acetyl-coenzyme A Synthetase (Acs) Assay

Authors: Sara Castaño-Cerezo
Sara Castaño-CerezoAffiliation: Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Chemistry, University of Murcia, Murcia, Spain
Bio-protocol author page: a94
, Vicente Bernal
Vicente BernalAffiliation: Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Chemistry, University of Murcia, Murcia, Spain
For correspondence: vbernal@um.es
Bio-protocol author page: a95
and Manuel Cánovas
Manuel CánovasAffiliation: Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Chemistry, University of Murcia, Murcia, Spain
Bio-protocol author page: a96
, date: 9/5/2012, 7745 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.256.

[Abstract] Acetyl-coenzyme A synthethase (Acs, E.C.6.2.1.1) is an acetate activating enzyme widely represented in nature from bacteria to human. Its function is important for cellular catabolism, especially in order to support microbial growth at low concentrations of acetate (<10 mM) (Castano Cerezo et al., 2011; Castano Cerezo et al., ...

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Author: Fanglian He Fanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USAFor correspondence: fanglian09@gmail.comBio-protocol author page: a9, date: 2/5/2011, 93294 views, 32 Q&ADOI: https://doi.org/10.21769/BioProtoc.30.

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

Author: Fanglian HeFanglian HeAffiliation: Department of Biology, University of Pennsylvania, Philadelphia, USABio-protocol author page: a9, date: 7/20/2011, 86300 views, 47 Q&ADOI: https://doi.org/10.21769/BioProtoc.97.

Author: Lili Jing Lili JingAffiliation: Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USAFor correspondence: lilijingcn@gmail.comBio-protocol author page: a38, date: 1/20/2012, 43050 views, 4 Q&ADOI: https://doi.org/10.21769/BioProtoc.177.

Author: Xiyan Li Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USAFor correspondence: lixiyan@stanford.eduBio-protocol author page: a13, date: 6/5/2011, 11835 views, 2 Q&ADOI: https://doi.org/10.21769/BioProtoc.76.

Author: Lin Fang Lin FangAffiliation: Department of Pediatrics, School of Medicine, Stanford University, Stanford, USAFor correspondence: cheerfulfang@hotmail.comBio-protocol author page: a20, date: 9/20/2011, 11286 views, 1 Q&ADOI: https://doi.org/10.21769/BioProtoc.132.

Author: Hogune Im, date: 10/20/2011, 11121 views, 1 Q&ADOI: https://doi.org/10.21769/BioProtoc.143.