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In vitro AMPylation Assays Using Purified, Recombinant Proteins

Featured protocol,  Authors: Matthias C. Truttmann
Matthias C. TruttmannAffiliation: Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA
For correspondence: matthias.truttmann@childrens.harvard.edu
Bio-protocol author page: a4924
 and Hidde L. Ploegh
Hidde L. PloeghAffiliation: Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA
For correspondence: hidde.ploegh@childrens.harvard.edu
Bio-protocol author page: a1563
date: 7/20/2017, 8 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2416.

Brief version appeared in Proc Natl Acad Sci U S A, Jan 2017
Post-translational protein modifications (PTMs) orchestrate the activity of individual proteins and ensure their proper function. While modifications such as phosphorylation or glycosylation are well understood, more unusual modifications, including nitrosylation or AMPylation remain comparatively poorly characterized. Research on protein AMPylation–which refers to the covalent addition of an AMP moiety to the side chains of serine, threonine or tyrosine–has undergone a renaissance (Yarbrough et al., 2009; Engel et al., 2012; Ham et al., 2014; Woolery et al., 2014; Preissler et al., 2015; Sanyal et al., 2015; Truttmann et al., 2016; Truttmann et al., 2017). The identification and characterization of filamentation (fic) domain-containing AMPylases sparked new interest in this PTM (Kinch et al., 2009; Yarbrough et al., 2009). Based on recent in vivo and in vitro studies, we now know that secreted bacterial AMPylases covalently attach AMP to members of the Rho family of GTPases, while metazoan AMPylases modify HSP70 family proteins in the cytoplasm and the endoplasmic reticulum (ER) (Itzen et al., 2011; Hedberg and Itzen, 2015; Truttmann and Ploegh, 2017). AMPylation is thought to trap HSP70 in a primed yet transiently disabled state that cannot participate in protein refolding reactions (Preissler et al., 2015). In vitro AMPylation experiments are key to assess the activity, kinetics and specificity of protein AMPylation catalyzed by pro- and eukaryotic enzymes. These simple assays require recombinant AMPylases, target proteins (Rho GTPases, HSP70s), as well as ATP as a nucleotide source. Here, we describe strategies to qualitatively and quantitatively study protein AMPylation in vitro.

Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis

Featured protocol,  Authors: Liku B Tezera
Liku B TezeraAffiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
Bio-protocol author page: a4926
Magdalena K Bielecka
Magdalena K Bielecka Affiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
Bio-protocol author page: a4927
 and Paul T Elkington
Paul T ElkingtonAffiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
For correspondence: p.elkington@soton.ac.uk
Bio-protocol author page: a4925
date: 7/20/2017, 27 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2418.

Brief version appeared in eLife, Jan 2017
Standard cell culture models have been used to investigate disease pathology and to test new therapies for over fifty years. However, these model systems have often failed to mimic the changes occurring within three-dimensional (3-D) space where pathology occurs in vivo. To truthfully represent this, an emerging paradigm in biology is the importance of modelling disease in a physiologically relevant 3-D environment. One of the approaches for 3-D cell culture is bioelectrospray technology. This technique uses an alginate-based 3-D environment as an inert backbone within which mammalian cells and extracellular matrix can be incorporated. These alginate-based matrices produce highly reproducible results and can be mixed with different extracellular matrix components. This protocol describes a 3-D system incorporating mycobacteria, primary human blood mononuclear cells and collagen-alginate matrix to dissect the host-pathogen interaction in tuberculosis.

Gene Dosage Experiments in Enterobacteriaceae Using Arabinose-regulated Promoters

Featured protocol,  Authors: Sanchari Bhattacharyya
Sanchari BhattacharyyaAffiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge MA, USA
Bio-protocol author page: a4838
Shimon Bershtein
Shimon BershteinAffiliation: Department of Life Sciences, Ben-Gurion University of the Negev, POB 653, Beer-Sheva, Israel
Bio-protocol author page: a4839
 and Eugene I Shakhnovich
Eugene I ShakhnovichAffiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge MA, USA
For correspondence: shakhnovich@chemistry.harvard.edu
Bio-protocol author page: a4840
date: 7/20/2017, 14 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2396.

Brief version appeared in Elife, Dec 2016
This protocol is used to assay the effect of protein over-expression on fitness of E. coli. It is based on a plasmid expression of a protein of interest from an arabinose-regulated pBAD promoter followed by the measurement of the intracellular protein abundance by Western blot along with the measurement of growth parameters of E. coli cell expressing this protein.

Xanthoferrin Siderophore Estimation from the Cell-free Culture Supernatants of Different Xanthomonas Strains by HPLC

Featured protocol,  Authors: Sheo Shankar Pandey
Sheo Shankar PandeyAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4903
Prashantee Singh
Prashantee SinghAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4904
Biswajit Samal
Biswajit SamalAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4905
Raj Kumar Verma
Raj Kumar VermaAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4906
 and Subhadeep Chatterjee
Subhadeep ChatterjeeAffiliation: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
For correspondence: subhadeep@cdfd.org.in
Bio-protocol author page: a4907
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2410.

Brief version appeared in PLoS Pathog, Nov 2016
Xanthomonads can scavenge iron from the extracellular environment by secreting the siderophores, which are synthesized by the proteins encoded by xss (Xanthomonas siderophore synthesis) gene cluster. The siderophore production varies among xanthomonads in response to a limited supply of iron where Xanthomonas campestris pv. campestris (Xcc) produces less siderophores than Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc). Siderophore production can be measured by HPLC and with the CAS (Chrome azurol S)-agar plate assay, however HPLC is a more accurate method over CAS-agar plate assay for siderophore quantification in Xanthomonads. Here we describe how to quantify siderophores from xanthomonads using HPLC.

Quantification of Chlorophyll as a Proxy for Biofilm Formation in the Cyanobacterium Synechococcus elongatus

Featured protocol,  Authors: Eleonora Sendersky
Eleonora SenderskyAffiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
Bio-protocol author page: a4889
Ryan Simkovsky
Ryan SimkovskyAffiliation: Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
Bio-protocol author page: a4890
Susan S. Golden
Susan S. GoldenAffiliation: Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
Bio-protocol author page: a4891
 and Rakefet Schwarz
Rakefet SchwarzAffiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
For correspondence: Rakefet.Schwarz@biu.ac.il
Bio-protocol author page: a4892
date: 7/20/2017, 15 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2406.

Brief version appeared in Sci Rep, Aug 2016
A self-suppression mechanism of biofilm development in the cyanobacterium Synechococcus elongatus PCC 7942 was recently reported. These studies required quantification of biofilms formed by mutants impaired in the biofilm-inhibitory process. Here we describe in detail the use of chlorophyll measurements as a proxy for biomass accumulation in sessile and planktonic cells of biofilm-forming strains. These measurements allow quantification of the total biomass as estimated by chlorophyll level and representation of the extent of biofilm formation by depicting the relative fraction of chlorophyll in planktonic cells.

Quantitative Determination of Poly-β-hydroxybutyrate in Synechocystis sp. PCC 6803

Featured protocol,  Authors: Yvonne Zilliges
Yvonne ZilligesAffiliation: Freie Universität Berlin, Institute of Experimental Physics/Biophysics and Photosynthesis, Arnimallee 14, Berlin, Germany
For correspondence: yvonne.zilliges@fu-berlin.de
Bio-protocol author page: a4874
 and Ramon Damrow
Ramon DamrowAffiliation: Humboldt-Universität zu Berlin, Institute of Biology/Biochemistry of Plants, Chausseestr. 117, Berlin, Germany
Bio-protocol author page: a4875
date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2402.

Brief version appeared in Front Microbiol, Jun 2016
Cyanobacteria synthesize a variety of chemically-different, high-value biopolymers such as glycogen (polyglucose), poly-β-hydroxybutyrate (PHB), cyanophycin (polyamide of arginine and aspartic acid) and volutin (polyphosphate) under excess conditions. Especially under unbalanced C to N ratios, glycogen and in some cyanobacterial genera also PHB are massively accumulated in the progression of the general nitrogen stress response. Several different technologies have been established for in situ and in vitro PHB analysis from different microbial sources. In this protocol, a rapid and reliable spectrophotometric method is described for PHB quantification in the cyanobacterium Synechocystis sp. PCC 6803 upon nitrogen deprivation as described in (Damrow et al., 2016).

Determination of Survival of Wildtype and Mutant Escherichia coli in Soil

Featured protocol,  Authors: Yinka Somorin
Yinka SomorinAffiliation: Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland
Bio-protocol author page: a4917
 and Conor O'Byrne
Conor O'ByrneAffiliation: Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland
For correspondence: conor.obyrne@nuigalway.ie
Bio-protocol author page: a4918
date: 7/20/2017, 24 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2414.

Brief version appeared in Appl Environ Microbiol, Jul 2016
E. coli resides in the gastrointestinal tract of humans and other warm-blooded animals but recent studies have shown that E. coli can persist and grow in various external environments including soil. The general stress response regulator, RpoS, helps E. coli overcome various stresses, however its role in soil survival was unknown. This soil survival assay protocol was developed and used to determine the role of the general stress response regulator, RpoS, in the survival of E. coli in soil. Using this soil survival assay, we demonstrated that RpoS was important for the survival of E. coli in soil. This protocol describes the development of the soil survival assay especially the recovery of E. coli inoculated into soil and can be adapted to allow further investigations into the survival of other bacteria in soil.

The Sulfur Oxygenase Reductase Activity Assay: Catalyzing a Reaction with Elemental Sulfur as Substrate at High Temperatures

Featured protocol,  Authors: Patrick Rühl
Patrick RühlAffiliation: Sulfur Biochemistry and Microbial Bioenergetics, Dept. of Biology, Technische Universität Darmstadt, Darmstadt, Germany
Bio-protocol author page: a4876
 and Arnulf Kletzin
Arnulf KletzinAffiliation: Sulfur Biochemistry and Microbial Bioenergetics, Dept. of Biology, Technische Universität Darmstadt, Darmstadt, Germany
For correspondence: kletzin@bio.tu-darmstadt.de
Bio-protocol author page: a4877
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2403.

Brief version appeared in J Bacteriol, Jan 2017
The sulfur oxygenase reductase (SOR) reaction is a dioxygen-dependent disproportionation of elemental sulfur (S0), catalyzed at optimal temperatures between 65 °C and 85 °C. Thiosulfate and sulfite are formed as oxidized products as well hydrogen sulfide as reduced product. External co-factors are not required. Usually, the SOR assay is performed in a milliliter scale in S0-containing Tris-buffer at high temperatures followed by colorimetric product quantification. In order to make the SOR assay more sensitive and better reproducible, several modifications were implemented compared to the original SOR assay (Kletzin, 1989). Here we present the modified SOR assay and the following quantification of the reaction products.

Determination of NO and CSF Levels Produced by Bacillus subtilis

Featured protocol,  Authors: 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
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
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
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
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
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: 7/5/2017, 151 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2379.

Brief version appeared in Nat Commun, Jan 2017
The cell-to-cell communication and division of labour that occurs inside a beneficial biofilm produce significant differences in gene expression compared with the gene expression pattern of cells grew under planktonic conditions. In this sense, the levels of NO (nitric oxide) and CSF (Competence Sporulation Stimulating Factor) produced in Bacillus subtilis cultures have been measured only under planktonic growth conditions. We sought to determine whether NO and/or CSF production is affected in B. subtilis cells that develop as a biofilm. To measure the production levels of the two prolongevity molecules, we grew B. subtilis cells under planktonic and biofilm supporting condition.

Bacterial Survival in Dictyostelium

Featured protocol,  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, 158 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2376.

Brief version appeared in Mol Microbiol, Nov 2016
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 and the remaining uningested bacteria were killed by gentamicin. Gentamicin is used because it does not penetrate into eukaryotic cells allowing the ingested bacteria to survive the antibiotic treatment, whereas bacteria outside the amoebal cells are killed.

In vitro AMPylation Assays Using Purified, Recombinant Proteins

Authors: Matthias C. Truttmann
Matthias C. TruttmannAffiliation: Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA
For correspondence: matthias.truttmann@childrens.harvard.edu
Bio-protocol author page: a4924
 and Hidde L. Ploegh
Hidde L. PloeghAffiliation: Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA
For correspondence: hidde.ploegh@childrens.harvard.edu
Bio-protocol author page: a1563
date: 7/20/2017, 8 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2416.

[Abstract] Post-translational protein modifications (PTMs) orchestrate the activity of individual proteins and ensure their proper function. While modifications such as phosphorylation or glycosylation are well understood, more unusual modifications, including nitrosylation or AMPylation remain comparatively poorly ...

Bioelectrospray Methodology for Dissection of the Host-pathogen Interaction in Human Tuberculosis

Authors: Liku B Tezera
Liku B TezeraAffiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
Bio-protocol author page: a4926
Magdalena K Bielecka
Magdalena K Bielecka Affiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
Bio-protocol author page: a4927
 and Paul T Elkington
Paul T ElkingtonAffiliation: Clinical and Experimental Sciences, University of Southampton, Southampton, United Kingdom
For correspondence: p.elkington@soton.ac.uk
Bio-protocol author page: a4925
date: 7/20/2017, 27 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2418.

[Abstract] Standard cell culture models have been used to investigate disease pathology and to test new therapies for over fifty years. However, these model systems have often failed to mimic the changes occurring within three-dimensional (3-D) space where pathology occurs in vivo. To truthfully represent this, ...

Gene Dosage Experiments in Enterobacteriaceae Using Arabinose-regulated Promoters

Authors: Sanchari Bhattacharyya
Sanchari BhattacharyyaAffiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge MA, USA
Bio-protocol author page: a4838
Shimon Bershtein
Shimon BershteinAffiliation: Department of Life Sciences, Ben-Gurion University of the Negev, POB 653, Beer-Sheva, Israel
Bio-protocol author page: a4839
 and Eugene I Shakhnovich
Eugene I ShakhnovichAffiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge MA, USA
For correspondence: shakhnovich@chemistry.harvard.edu
Bio-protocol author page: a4840
date: 7/20/2017, 14 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2396.

[Abstract] This protocol is used to assay the effect of protein over-expression on fitness of E. coli. It is based on a plasmid expression of a protein of interest from an arabinose-regulated pBAD promoter followed by the measurement of the intracellular protein abundance by Western blot along with the measurement ...

Xanthoferrin Siderophore Estimation from the Cell-free Culture Supernatants of Different Xanthomonas Strains by HPLC

Authors: Sheo Shankar Pandey
Sheo Shankar PandeyAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4903
Prashantee Singh
Prashantee SinghAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4904
Biswajit Samal
Biswajit SamalAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4905
Raj Kumar Verma
Raj Kumar VermaAffiliation 1: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
Affiliation 2: Graduate studies, Manipal University, Manipal, India
Bio-protocol author page: a4906
 and Subhadeep Chatterjee
Subhadeep ChatterjeeAffiliation: Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad-500001, India
For correspondence: subhadeep@cdfd.org.in
Bio-protocol author page: a4907
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2410.

[Abstract] Xanthomonads can scavenge iron from the extracellular environment by secreting the siderophores, which are synthesized by the proteins encoded by xss (Xanthomonas siderophore synthesis) gene cluster. The siderophore production varies among xanthomonads in response to a limited supply of iron where Xanthomonas ...

Quantification of Chlorophyll as a Proxy for Biofilm Formation in the Cyanobacterium Synechococcus elongatus

Authors: Eleonora Sendersky
Eleonora SenderskyAffiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
Bio-protocol author page: a4889
Ryan Simkovsky
Ryan SimkovskyAffiliation: Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
Bio-protocol author page: a4890
Susan S. Golden
Susan S. GoldenAffiliation: Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
Bio-protocol author page: a4891
 and Rakefet Schwarz
Rakefet SchwarzAffiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
For correspondence: Rakefet.Schwarz@biu.ac.il
Bio-protocol author page: a4892
date: 7/20/2017, 15 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2406.

[Abstract] A self-suppression mechanism of biofilm development in the cyanobacterium Synechococcus elongatus PCC 7942 was recently reported. These studies required quantification of biofilms formed by mutants impaired in the biofilm-inhibitory process. Here we describe in detail the use of chlorophyll measurements ...

Quantitative Determination of Poly-β-hydroxybutyrate in Synechocystis sp. PCC 6803

Authors: Yvonne Zilliges
Yvonne ZilligesAffiliation: Freie Universität Berlin, Institute of Experimental Physics/Biophysics and Photosynthesis, Arnimallee 14, Berlin, Germany
For correspondence: yvonne.zilliges@fu-berlin.de
Bio-protocol author page: a4874
 and Ramon Damrow
Ramon DamrowAffiliation: Humboldt-Universität zu Berlin, Institute of Biology/Biochemistry of Plants, Chausseestr. 117, Berlin, Germany
Bio-protocol author page: a4875
date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2402.

[Abstract] Cyanobacteria synthesize a variety of chemically-different, high-value biopolymers such as glycogen (polyglucose), poly-β-hydroxybutyrate (PHB), cyanophycin (polyamide of arginine and aspartic acid) and volutin (polyphosphate) under excess conditions. Especially under unbalanced C to N ratios, glycogen ...

Determination of Survival of Wildtype and Mutant Escherichia coli in Soil

Authors: Yinka Somorin
Yinka SomorinAffiliation: Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland
Bio-protocol author page: a4917
 and Conor O'Byrne
Conor O'ByrneAffiliation: Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland
For correspondence: conor.obyrne@nuigalway.ie
Bio-protocol author page: a4918
date: 7/20/2017, 24 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2414.

[Abstract] E. coli resides in the gastrointestinal tract of humans and other warm-blooded animals but recent studies have shown that E. coli can persist and grow in various external environments including soil. The general stress response regulator, RpoS, helps E. coli overcome various stresses, however its role ...

The Sulfur Oxygenase Reductase Activity Assay: Catalyzing a Reaction with Elemental Sulfur as Substrate at High Temperatures

Authors: Patrick Rühl
Patrick RühlAffiliation: Sulfur Biochemistry and Microbial Bioenergetics, Dept. of Biology, Technische Universität Darmstadt, Darmstadt, Germany
Bio-protocol author page: a4876
 and Arnulf Kletzin
Arnulf KletzinAffiliation: Sulfur Biochemistry and Microbial Bioenergetics, Dept. of Biology, Technische Universität Darmstadt, Darmstadt, Germany
For correspondence: kletzin@bio.tu-darmstadt.de
Bio-protocol author page: a4877
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2403.

[Abstract] The sulfur oxygenase reductase (SOR) reaction is a dioxygen-dependent disproportionation of elemental sulfur (S0), catalyzed at optimal temperatures between 65 °C and 85 °C. Thiosulfate and sulfite are formed as oxidized products as well hydrogen sulfide as reduced product. External co-factors are not ...

Determination of NO and CSF Levels Produced by Bacillus subtilis

Authors: 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
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
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
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
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
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: 7/5/2017, 151 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2379.

[Abstract] The cell-to-cell communication and division of labour that occurs inside a beneficial biofilm produce significant differences in gene expression compared with the gene expression pattern of cells grew under planktonic conditions. In this sense, the levels of NO (nitric oxide) and CSF (Competence Sporulation ...

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, 158 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 ...
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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, 93841 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, 86877 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, 43232 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, 18418 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, 14182 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, 11912 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, 11344 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, 11190 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, 9646 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, 9206 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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