Prokaryotes

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    Protocols in Current Issue
    Measurement of Ascorbic Acid and Glutathione Content in Cyanobacterium Synechocystis sp. PCC 6803
    Ascorbic acid (AsA) and gluthathione (GSH) are two key components of the antioxidant machinery of eukaryotic and prokaryotic cells. The cyanobacterium Synechocystis sp. PCC 6803 presents both compounds in different concentrations (AsA, ...
    Attachment of a 32P-phosphate to the 3′ Terminus of a DNA Oligonucleotide
    Authors:  Joshua C. Cofsky and Jennifer A. Doudna, date: 10/20/2020, view: 211, Q&A: 0
    Biochemical investigations into DNA-binding and DNA-cutting proteins often benefit from the specific attachment of a radioactive label to one of the two DNA termini. In many cases, it is essential to perform two versions of the same experiment: one ...
    Understanding Docking Complexes of Macromolecules Using HADDOCK: The Synergy between Experimental Data and Computations
    Authors:  Andrea Saponaro, Vincenzo Maione, Alexandre M. J. J. Bonvin and Francesca Cantini, date: 10/20/2020, view: 264, Q&A: 0
    This protocol illustrates the modelling of a protein-peptide complex using the synergic combination of in silico analysis and experimental results. To this end, we use the integrative modelling software HADDOCK, which possesses the powerful ...
    Estimation of the Minimum Number of Replication Origins Per Chromosome in any Organism
    Author:  Marcelo S. da Silva, date: 10/20/2020, view: 305, Q&A: 0
    Eukaryote nuclear genomes predominantly replicate through multiple replication origins. The number of replication origins activated per chromosome during the S-phase duration may vary according to many factors, but the predominant one is replication ...
    Measurement of Ascorbic Acid and Glutathione Content in Cyanobacterium Synechocystis sp. PCC 6803
    [Abstract] Ascorbic acid (AsA) and gluthathione (GSH) are two key components of the antioxidant machinery of eukaryotic and prokaryotic cells. The cyanobacterium Synechocystis sp. PCC 6803 presents both compounds in different concentrations (AsA, 20-100 μM and GSH, 2-5 mM). Therefore, it is important to have precise and sensitive methods to ...
    Attachment of a 32P-phosphate to the 3′ Terminus of a DNA Oligonucleotide
    Authors:  Joshua C. Cofsky and Jennifer A. Doudna, date: 10/20/2020, view: 211, Q&A: 0
    [Abstract] Biochemical investigations into DNA-binding and DNA-cutting proteins often benefit from the specific attachment of a radioactive label to one of the two DNA termini. In many cases, it is essential to perform two versions of the same experiment: one with the 5′ DNA end labeled and one with the 3′ DNA end labeled. While homogeneous 5′-radiolabeling ...
    Understanding Docking Complexes of Macromolecules Using HADDOCK: The Synergy between Experimental Data and Computations
    Authors:  Andrea Saponaro, Vincenzo Maione, Alexandre M. J. J. Bonvin and Francesca Cantini, date: 10/20/2020, view: 264, Q&A: 0
    [Abstract] This protocol illustrates the modelling of a protein-peptide complex using the synergic combination of in silico analysis and experimental results. To this end, we use the integrative modelling software HADDOCK, which possesses the powerful ability to incorporate experimental data, such as NMR Chemical Shift Perturbations and biochemical ...
    Estimation of the Minimum Number of Replication Origins Per Chromosome in any Organism
    Author:  Marcelo S. da Silva, date: 10/20/2020, view: 305, Q&A: 0
    [Abstract] Eukaryote nuclear genomes predominantly replicate through multiple replication origins. The number of replication origins activated per chromosome during the S-phase duration may vary according to many factors, but the predominant one is replication stress. Several studies have applied different approaches to estimate the number and map the ...
    TetR Regulated in vivo Repression Technology to Identify Conditional Gene Silencing in Genetically Engineerable Bacteria Using Vibrio cholerae Murine Infections as Model System
    Authors:  Franz G. Zingl, Fabian Mitterer, Himadri B. Thapa and Stefan Schild, date: 10/05/2020, view: 219, Q&A: 0
    [Abstract] Investigation of bacterial gene regulation upon environmental changes is still a challenging task. For example, Vibrio cholerae, a pathogen of the human gastrointestinal tract, faces diverse transient conditions in different compartments upon oral ingestion. Genetic reporter systems have been demonstrated to be extremely powerful tools to ...
    Affinity Purification of GO-Matryoshka Biosensors from E. coli for Quantitative Ratiometric Fluorescence Analyses
    [Abstract] Genetically encoded biosensors are powerful tools for quantitative visualization of ions and metabolites in vivo. Design and optimization of such biosensors typically require analyses of large numbers of variants. Sensor properties determined in vitro such as substrate specificity, affinity, response range, dynamic range, and ...
    Analysis of Gram-negative Bacteria Peptidoglycan by Ultra-performance Liquid Chromatography
    Authors:  Laura Alvarez, Baptiste Cordier, Sven van Teeffelen and Felipe Cava, date: 10/05/2020, view: 298, Q&A: 0
    [Abstract] Bacteria are surrounded by a protective peptidoglycan cell wall. Provided that this structure and the enzymes involved are the preferred target for our most successful antibiotics, determining its structural and chemical complexity is of the highest interest. Traditionally, high-performance liquid chromatography (HPLC) analyses have been ...
    Assembly and Imaging Set up of PIE-Scope
    [Abstract] Cryo-Electron Tomography (cryo-ET) is a method that enables resolving the structure of macromolecular complexes directly in the cellular environment. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy ...
    Simple Time-lapse Imaging for Quantifying the Hydrostatic Production of Oxygenic Photogranules
    Authors:  Esmee D. Joosten, Jérôme Hamelin and Kim Milferstedt, date: 10/05/2020, view: 306, Q&A: 0
    [Abstract] Oxygenic photogranules (OPGs) are dense, three-dimensional aggregates containing a syntrophic, light-driven microbial community. Their temporal and spatial development interests microbial ecologists working at the bioprocess engineering interface, as this knowledge can be used to optimize biotechnological applications, such as wastewater treatment ...
    Evaluation of Viable Cells in Pseudomonas aeruginosa Biofilms by Colony Count and Live/Dead Staining
    Authors:  Magdalena Pezzoni, Ramón Augusto Pizarro and Cristina Susana Costa, date: 09/20/2020, view: 363, Q&A: 0
    [Abstract] Pseudomonas aeruginosa is a human pathogen capable to form robust biofilms. P. aeruginosa biofilms represent a serious problem because of the adverse effects on human health and industry, from sanitary and economic points of view. Typical strategies to break down biofilms have been long used, such as the use of disinfectants or ...



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