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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.

Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice

Featured protocol,  Authors: Michael A. Schmid
Michael A. SchmidAffiliation: Rega Institute for Medical Research, Virology and Chemotherapy, Department of Immunology and Microbiology, University of Leuven, Leuven, Belgium
For correspondence: michael.alex.schmid@gmail.com
Bio-protocol author page: a4893
Elizabeth Kauffman
Elizabeth KauffmanAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4894
Anne Payne
Anne PayneAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4895
Eva Harris
Eva HarrisAffiliation: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, USA
Bio-protocol author page: a4896
 and Laura D. Kramer
Laura D. KramerAffiliation 1: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Affiliation 2: School of Public Health, State University of New York at Albany, Albany, New York, USA
For correspondence: laura.kramer@health.ny.gov
Bio-protocol author page: a4897
date: 7/20/2017, 29 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2407.

Brief version appeared in PLoS Pathog, Jun 2016
Mosquito-transmitted pathogens are among the leading causes of severe disease and death in humans. Components within the saliva of mosquito vectors facilitate blood feeding, modulate host responses, and allow efficient transmission of pathogens, such as Dengue, Zika, yellow fever, West Nile, Japanese encephalitis, and chikungunya viruses, as well as Plasmodium parasites, among others. Here, we describe standardized methods to assess the impact of mosquito-derived factors on immune responses and pathogenesis in mouse models of infection. This protocol includes the generation of mosquito salivary gland extracts and intradermal inoculation of mouse ears. Ultimately, the information obtained from using these techniques can help reveal fundamental mechanisms of interaction between pathogens, mosquito vectors, and the mammalian host. In addition, this protocol can help establish improved infection challenge models for pre-clinical testing of vaccines or therapeutics that take into account the natural route of transmission via mosquitoes.

Plasmodium Sporozoite Motility on Flat Substrates

Featured protocol,  Authors: Henriette L Prinz
Henriette L PrinzAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4835
Julia M Sattler
Julia M SattlerAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4836
 and Friedrich Frischknecht
Friedrich FrischknechtAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4837
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2395.

Brief version appeared in PLoS Pathog, Jul 2016
Plasmodium sporozoites are the infectious, highly motile forms of the malaria parasite transmitted by Anopheles mosquitoes. Sporozoite motility can be assessed following the dissection of Anopheles salivary glands and isolation of sporozoites in vitro.

Mapping RNA Sequences that Contact Viral Capsid Proteins in Virions

Featured protocol,  Authors: C. Cheng Kao
C. Cheng KaoAffiliation: Department of Molecular & Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA
For correspondence: ckao@indiana.edu
Bio-protocol author page: a4851
Ella Chuang
Ella ChuangAffiliation: Department of Molecular & Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4852
James Ford
James FordAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4853
Jie Huang
Jie HuangAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4854
Ram Podicheti
Ram PodichetiAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4855
 and Doug B. Rusch
Doug B. RuschAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4856
date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2398.

Brief version appeared in J Virol, Aug 2016
We have adapted the methodology of CLIP-seq (Crosslinking-Immunoprecipitation and DNA Sequencing) to map the segments of encapsidated RNAs that contact the protein shells of virions. Results from the protocol report on the RNA sequences that contact the viral capsid.

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.

Using CRISPR/Cas9 for Large Fragment Deletions in Saccharomyces cerevisiae

Featured protocol,  Authors: Huanhuan Hao
Huanhuan HaoAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a4919
Jing Huang
Jing HuangAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a2481
Tongtong Liu
Tongtong LiuAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a4923
Hui Tang
Hui TangAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a4921
 and Liping Zhang
Liping ZhangAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
For correspondence: zhanglphbu@sohu.com
Bio-protocol author page: a4920
date: 7/20/2017, 7 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2415.

Brief version appeared in Anal Biochem, Sep 2016
CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) systems have emerged as a powerful tool for genome editing in many organisms. The wide use of CRISPR/Cas9 systems may be due to the fact that these systems contain a simple guide RNA (sgRNA) that is relatively easy to design and they are very versatile with the ability to simultaneously target multiple genes within a cell (Varshney et al., 2015). We have developed a CRISPR/Cas9 system to delete large genomic fragments (exceeding 30 kb) in Saccharomyces cerevisiae. One application of this technology is to study the effects of large-scale deletions of non-essential genes which may give insight into the function of gene clusters within chromosomes at the molecular level. In this protocol, we describe the general procedures for large fragment deletion in S. cerevisiae using CRISPR/Cas9 including: how to design CRISPR arrays and how to construct Cas9-crRNA expression plasmids as well as how to detect mutations introduced by the system within S. cerevisiae cells.

Vaginal HSV-2 Infection and Tissue Analysis

Featured protocol,  Authors: Marie Beck Iversen
Marie Beck IversenAffiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
Bio-protocol author page: a4818
Søren Riis Paludan
Søren Riis PaludanAffiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
Bio-protocol author page: a4819
 and Christian Kanstrup Holm
Christian Kanstrup HolmAffiliation: Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
For correspondence: holm@biomed.au.dk
Bio-protocol author page: a4820
date: 7/5/2017, 188 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2383.

Brief version appeared in Nat Immunol, Feb 2016
The vaginal murine HSV-2 infection model is very useful in studying mucosal immunity against HSV-2 (Overall et al., 1975; Renis et al., 1976; Parr and Parr, 2003). Histologically, the vagina of Depo-Provera-treated mice is lined by a single layer of mucus secreting columnar epithelial cells overlying two to three layers of proliferative cells. Even though this is morphologically different from the human vagina, it closely resembles the endocervical epithelium, which is thought to be the primary site of HSV-2 infection in women (Parr et al., 1994; Kaushic et al., 2011). In the protocol presented here, mice are pre-treated with Depo-Provera before intra-vaginal inoculation with HSV-2. The virus replicates in the mucosal epithelium from where it spreads to and replicates in the CNS including the spinal cord, brain stem, cerebrum and cerebellum. Cytokine responses can be detected in vaginal washings using ELISA or in vaginal tissues using qPCR. Further, the recruitment of leukocytes to the vagina can be determined by flow cytometry. The model is suitable for research of both innate and adaptive immunity to HSV-2 infection.

Assaying Glycogen and Trehalose in Yeast

Featured protocol,  Authors: Yuping Chen
Yuping ChenAffiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, USA
Bio-protocol author page: a4763
 and Bruce Futcher
Bruce FutcherAffiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, USA
For correspondence: bfutcher@gmail.com
Bio-protocol author page: a4764
date: 7/5/2017, 167 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2371.

Brief version appeared in Mol Cell, May 2016
Organisms store carbohydrates in several forms. In yeast, carbohydrates are stored in glycogen (a multi-branched polysaccharide) and in trehalose (a disaccharide). As in other organisms, the amount of stored carbohydrate varies dramatically with physiological state, and accordingly, an assay of stored carbohydrate can help reveal physiological state. Here, we describe relatively easy and streamlined assays for glycogen and trehalose in yeast that can be applied either to a few samples, or in a moderately high-throughput fashion (dozens to hundreds of samples).

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.

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, an emerging paradigm in biology is the importance ...

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 of growth parameters of E. coli cell expressing ...

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 campestris pv. campestris (Xcc) produces ...

Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice

Authors: Michael A. Schmid
Michael A. SchmidAffiliation: Rega Institute for Medical Research, Virology and Chemotherapy, Department of Immunology and Microbiology, University of Leuven, Leuven, Belgium
For correspondence: michael.alex.schmid@gmail.com
Bio-protocol author page: a4893
Elizabeth Kauffman
Elizabeth KauffmanAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4894
Anne Payne
Anne PayneAffiliation: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Bio-protocol author page: a4895
Eva Harris
Eva HarrisAffiliation: Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, California, USA
Bio-protocol author page: a4896
 and Laura D. Kramer
Laura D. KramerAffiliation 1: Wadsworth Center, New York State Department of Health, Albany, New York, USA
Affiliation 2: School of Public Health, State University of New York at Albany, Albany, New York, USA
For correspondence: laura.kramer@health.ny.gov
Bio-protocol author page: a4897
date: 7/20/2017, 29 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2407.

[Abstract] Mosquito-transmitted pathogens are among the leading causes of severe disease and death in humans. Components within the saliva of mosquito vectors facilitate blood feeding, modulate host responses, and allow efficient transmission of pathogens, such as Dengue, Zika, yellow fever, West Nile, Japanese encephalitis, and chikungunya viruses, as well as ...

Plasmodium Sporozoite Motility on Flat Substrates

Authors: Henriette L Prinz
Henriette L PrinzAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4835
Julia M Sattler
Julia M SattlerAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4836
 and Friedrich Frischknecht
Friedrich FrischknechtAffiliation: Integrative Parasitology, Center for Infectious Diseases, Heidelberg University Medical School, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
Bio-protocol author page: a4837
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2395.

[Abstract] Plasmodium sporozoites are the infectious, highly motile forms of the malaria parasite transmitted by Anopheles mosquitoes. Sporozoite motility can be assessed following the dissection of Anopheles salivary glands and isolation of sporozoites in vitro. ...

Mapping RNA Sequences that Contact Viral Capsid Proteins in Virions

Authors: C. Cheng Kao
C. Cheng KaoAffiliation: Department of Molecular & Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA
For correspondence: ckao@indiana.edu
Bio-protocol author page: a4851
Ella Chuang
Ella ChuangAffiliation: Department of Molecular & Cellular Biochemistry, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4852
James Ford
James FordAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4853
Jie Huang
Jie HuangAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4854
Ram Podicheti
Ram PodichetiAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4855
 and Doug B. Rusch
Doug B. RuschAffiliation: The Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA
Bio-protocol author page: a4856
date: 7/20/2017, 11 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2398.

[Abstract] We have adapted the methodology of CLIP-seq (Crosslinking-Immunoprecipitation and DNA Sequencing) to map the segments of encapsidated RNAs that contact the protein shells of virions. Results from the protocol report on the RNA sequences that contact the viral capsid....

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 as a proxy for biomass accumulation in sessile ...

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 and in some cyanobacterial genera also PHB are ...

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 in soil survival was unknown. This soil survival ...

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 required. Usually, the SOR assay is performed in ...
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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] Lentivirus Production

Author: Nabila Aboulaich date: 3/5/2011, 24419 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.39.

[Abstract] Lentivirus is a common tool used to introduce a gene into mammalian or other animal cells.This protocol is to produce lentivirus stocks from hairpin-pLKO.1 plasmid....

In vitro Protein Kinase Assay

Author: Yuehua Wei
Yuehua WeiAffiliation: Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ Robert Wood Johnson Medical School, Piscataway, USA
For correspondence: weiyh.sjtu.edu@gmail.com
Bio-protocol author page: a49
date: 6/5/2012, 22750 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.212.

[Abstract] This protocol will describe experimental procedures for an in vitro kinase assay of the yeast protein kinase Sch9. This protocol can be tailored to detect kinase activity of other yeast protein kinase....

[Bio101] Making Yeast Competent Cells and Yeast Cell Transformation

Author: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Science, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
date: 7/20/2011, 22348 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.96.

[Abstract] This is a quite simple but reliable protocol to make very high transformation efficiency yeast competent cells. By express your gene of interest, protein function can be studied in yeast cells....

Spot Assay for Yeast

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: 1/5/2012, 19642 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.16.

[Abstract] This protocol can be used to compare the cell growth rate of yeast under different growth conditions. It involves the serial dilution and spotting of yeast colonies....

[Bio101] Purification of Adenovirus by Cesium Chloride Density Gradients

Author: Huan Pang
Huan PangAffiliation: Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, USA
For correspondence: pang_huan@hotmail.com
Bio-protocol author page: a48
date: 4/5/2012, 18627 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.201.

[Abstract] Adenovirus are efficient gene delivery systems. The standard method for purification of adenoviral vectors is based on using a cesium chloride (CsCl) density gradient combined with ultracentrifugation. This method is suitable for small-scale purification and is less expensive than column chromatography ...

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] Yeast Vacuole Staining with FM4-64

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, 15404 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.18.

[Abstract] The lipophilic probe, FM 4-64 does not fluoresce much in water but fluoresces strongly after binding to the outer plasma membrane, providing clear and distinguishable plasma membrane staining. The binding is rapid and reversible. In this protocol vacuoles in yeast cells are stained with the FM4-64 dye, ...

[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 ...
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