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    Detection of Reactive Oxygen Species (ROS) in Cyanobacteria Using the Oxidant-sensing Probe 2’,7’-Dichlorodihydrofluorescein Diacetate (DCFH-DA)
    [Abstract] Reactive oxygen species (ROS) are cell signaling molecules synthesized inside the cells as a response to routine metabolic processes. In stress conditions such as ultraviolet radiation (UVR), ROS concentration increases several folds in the cells that become toxic for the cell survival. Here we present the method for in vivo detection of ...
    Snapshots of the Signaling Complex DesK:DesR in Different Functional States Using Rational Mutagenesis and X-ray Crystallography
    [Abstract] We have developed protocols to generate site-specific variants of the histidine-kinase DesK and its cognate response regulator DesR, conducive to trapping different signaling states of the proteins. Co-expression of both partners in E. coli, ensuring an excess of the regulator, was essential for soluble production of the DesK:DesR ...
    Superoxide Dismutase (SOD) and Catalase (CAT) Activity Assay Protocols for Caenorhabditis elegans
    Authors:  Jing Zhang, Rui Chen, Zhenyang Yu and Lili Xue, date: 08/20/2017, view: 386, Q&A: 0
    [Abstract] Assays for superoxide dismutase (SOD) and catalase (CAT) activities are widely employed to indicate antioxidant responses underlying the toxic effects of test chemicals. Yet, earlier studies mainly described the procedures as performed according to manufacturer’s instructions without modifications that are specific to any organisms. The present ...
    Liposome Disruption Assay to Examine Lytic Properties of Biomolecules
    Authors:  John R. Jimah, Paul H. Schlesinger and Niraj H. Tolia, date: 08/05/2017, view: 478, Q&A: 0
    [Abstract] Proteins may have three dimensional structural or amino acid features that suggest a role in targeting and disrupting lipids within cell membranes. It is often necessary to experimentally investigate if these proteins and biomolecules are able to disrupt membranes in order to conclusively characterize the function of these biomolecules. Here, we ...
    Analysis of N-acetylmuramic acid-6-phosphate (MurNAc-6P) Accumulation by HPLC-MS
    Authors:  Marina Borisova and Christoph Mayer, date: 08/05/2017, view: 420, Q&A: 0
    [Abstract] We describe here in detail a high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based method to determine N-acetylmuramic acid-6-phosphate (MurNAc-6P) in bacterial cell extracts. The method can be applied to both Gram-negative and Gram-positive bacteria, and as an example we use Escherichia coli cells in this ...
    Advanced Design of Minimalistic Dumbbell-shaped Gene Expression Vectors
    Authors:  Xiaoou Jiang and Volker Patzel, date: 08/05/2017, view: 298, Q&A: 0
    [Abstract] Minimal DNA vectors exclusively comprising therapeutically relevant sequences hold great promise for the development of novel therapeutic regimen. Dumbbell-shaped vectors represent non-viral non-integrating DNA minimal vectors which have entered an advanced stage of clinical development (Hardee et al., 2017). Spliceable introns and DNA ...
    Observation of Pneumococcal Phase Variation in Colony Morphology
    Authors:  Jing Li, Juanjuan Wang, Fangfang Jiao and Jing-Ren Zhang, date: 08/05/2017, view: 482, Q&A: 0
    [Abstract] Streptococcus pneumoniae (pneumococcus) is an important human pathogen that causes pneumonia, meningitis, sepsis, and otitis media. This bacterium normally resides in the nasopharynx as a commensal, but sometimes disseminates to sterile sites of humans and causes local or systemic inflammation. This biphasic behavior of S. pneumoniae ...
    Digestion of Peptidoglycan and Analysis of Soluble Fragments
    Authors:  Ryan E. Schaub and Joseph P. Dillard, date: 08/05/2017, view: 461, Q&A: 0
    [Abstract] Peptidoglycan (murein) is a vital component of the cell wall of nearly all bacteria, composed of sugars linked by short peptides. This protocol describes the purification of macromolecular peptidoglycan from cultured bacteria and the analysis of enzyme-digested peptidoglycan fragments using high performance liquid chromatography (HPLC). Digested ...
    Measurement of Energy-dependent Rhodamine 6G Efflux in Yeast Species
    Authors:  Yvetta Gbelska, Nora Toth Hervay, Vladimira Dzugasova and Alexandra Konecna, date: 08/05/2017, view: 373, Q&A: 0
    [Abstract] Rhodamine 6G is a highly fluorescent dye often used to determine the transport activity of yeast membrane efflux pumps. The ATP-binding cassette transporter KlPdr5p confers resistance to several unrelated drugs in Kluyveromyces lactis. KlPdr5p also extrudes rhodamine 6G (R6G) from intact yeast cells in an ...
    Quantitative Determination of Poly-β-hydroxybutyrate in Synechocystis sp. PCC 6803
    Authors:  Yvonne Zilliges and Ramon Damrow, date: 07/20/2017, view: 334, Q&A: 0
    [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 ...