Microbiology

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    Protocols in Current Issue
    Site-specific DNA Mapping of Protein Binding Orientation Using Azidophenacyl Bromide (APB)
    Authors:  Himasha M. Perera and Michael A. Trakselis, date: 06/20/2020, view: 627, Q&A: 0
    The orientation of a DNA-binding protein bound on DNA is determinative in directing the assembly of other associated proteins in the complex for enzymatic action. As an example, in a replisome, the orientation of the DNA helicase at the replication ...
    Maintenance and Quantitative Phenotyping of the Oomycete-plant Model Pathosystem Hyaloperonospora arabidopsidisArabidopsis
    Authors:  Fang-Yu Hwu and Martin Parniske, date: 06/20/2020, view: 443, Q&A: 0
    The interaction between the host plant Arabidopsis thaliana (Arabidopsis) and the oomycete Hyaloperonospora arabidopsidis (Hpa) is an established model system for the study of an obligate biotrophic downy mildew ...
    Identification of Buffer Conditions for Optimal Thermostability and Solubility of Herpesviral Protein UL37 Using the Thermofluor Assay
    Authors:  Andrea L. Koenigsberg, Jared D. Pitts and Ekaterina E. Heldwein, date: 06/20/2020, view: 313, Q&A: 0
    Structural and biochemical studies of proteins require high amounts of stable, purified proteins. Protein stability often depends on the buffer composition, which includes pH and concentration of salts or other solutes such as glycerol, hence an ...
    Genomic Edition of Ashbya gossypii Using One-vector CRISPR/Cas9
    Authors:  Gloria Muñoz-Fernández, Alberto Jiménez and José Luis Revuelta, date: 06/20/2020, view: 378, Q&A: 0
    The CRISPR/Cas9 system is a novel genetic tool which allows the precise manipulation of virtually any genomic sequence. In this protocol, we use a specific CRISPR/Cas9 system for the manipulation of Ashbya gossypii. The filamentous fungus ...
    Live Cell Measurement of the Intracellular pH of Yeast by Flow Cytometry Using a Genetically-Encoded Fluorescent Reporter
    Authors:  Catherine G. Triandafillou and D. Allan Drummond, date: 06/20/2020, view: 484, Q&A: 0
    The intracellular pH of yeast is a tightly regulated physiological cue that changes in response to growth state and environmental conditions. Fluorescent reporters, which have altered fluorescence in response to local pH changes, can be used to ...
    Herpes Simplex Virus Type 1 Propagation, Titration and Single-step Growth Curves
    Given the endemic seroprevalence of herpes simplex viruses (HSV), its associated human diseases, and the emergence of acyclovir-resistant strains, there is a continuous need for better antiviral therapies. Towards this aim, identifying mechanistic ...
    Site-specific DNA Mapping of Protein Binding Orientation Using Azidophenacyl Bromide (APB)
    Authors:  Himasha M. Perera and Michael A. Trakselis, date: 06/20/2020, view: 627, Q&A: 0
    [Abstract] The orientation of a DNA-binding protein bound on DNA is determinative in directing the assembly of other associated proteins in the complex for enzymatic action. As an example, in a replisome, the orientation of the DNA helicase at the replication fork directs the assembly of the other associated replisome proteins. We have recently determined ...
    Maintenance and Quantitative Phenotyping of the Oomycete-plant Model Pathosystem Hyaloperonospora arabidopsidisArabidopsis
    Authors:  Fang-Yu Hwu and Martin Parniske, date: 06/20/2020, view: 443, Q&A: 0
    [Abstract] The interaction between the host plant Arabidopsis thaliana (Arabidopsis) and the oomycete Hyaloperonospora arabidopsidis (Hpa) is an established model system for the study of an obligate biotrophic downy mildew interaction. The evaluation of the developmental success of Hpa is often based on the quantification ...
    Identification of Buffer Conditions for Optimal Thermostability and Solubility of Herpesviral Protein UL37 Using the Thermofluor Assay
    Authors:  Andrea L. Koenigsberg, Jared D. Pitts and Ekaterina E. Heldwein, date: 06/20/2020, view: 313, Q&A: 0
    [Abstract] Structural and biochemical studies of proteins require high amounts of stable, purified proteins. Protein stability often depends on the buffer composition, which includes pH and concentration of salts or other solutes such as glycerol, hence an efficient method for identifying optimal buffer conditions for stability would minimize time and ...
    Genomic Edition of Ashbya gossypii Using One-vector CRISPR/Cas9
    Authors:  Gloria Muñoz-Fernández, Alberto Jiménez and José Luis Revuelta, date: 06/20/2020, view: 378, Q&A: 0
    [Abstract] The CRISPR/Cas9 system is a novel genetic tool which allows the precise manipulation of virtually any genomic sequence. In this protocol, we use a specific CRISPR/Cas9 system for the manipulation of Ashbya gossypii. The filamentous fungus A. gossypii is currently used for the industrial production of riboflavin (vitamina B2). In ...
    Live Cell Measurement of the Intracellular pH of Yeast by Flow Cytometry Using a Genetically-Encoded Fluorescent Reporter
    Authors:  Catherine G. Triandafillou and D. Allan Drummond, date: 06/20/2020, view: 484, Q&A: 0
    [Abstract] The intracellular pH of yeast is a tightly regulated physiological cue that changes in response to growth state and environmental conditions. Fluorescent reporters, which have altered fluorescence in response to local pH changes, can be used to measure intracellular pH. While microscopy is often used to make such measurements, it is relatively ...
    Differential Fractionation of Erythrocytes Infected by Plasmodium berghei
    Authors:  Bénédicte Gnangnon, Véronique Peucelle and Christine Pierrot, date: 06/05/2020, view: 369, Q&A: 0
    [Abstract] The study of host/pathogen interactions at the cellular level during Plasmodium intra-erythrocytic cycle requires differential extraction techniques aiming to analyze the different compartments of the infected cell. Various protocols have been proposed in the literature to study specific compartments and/or membranes in the infected ...
    RNA Extraction from Ears and Draining Lymph Nodes of Mice Infected with Leishmania amazonensis
    Authors:  Emilie Giraud and Evie Melanitou, date: 06/05/2020, view: 449, Q&A: 0
    [Abstract] Parasites of the genus Leishmania infect the mammalian hosts, including mice and humans and cause cutaneous or visceral leishmaniasis depending upon the parasite species transmitted by the vector sandfly. Leishmania amazonensis is one of the Leishmania species responsible for the cutaneous form of the disease. We have ...
    Safe DNA-extraction Protocol Suitable for Studying Tree-fungus Interactions
    Authors:  Susanna Keriö, Eeva Terhonen and Jared M. LeBoldus, date: 06/05/2020, view: 957, Q&A: 0
    [Abstract] We present a safe and low-cost method suitable for DNA extraction from mycelium and tree tissue samples. After sample preparation, the extraction takes about 60 min. Method performance was tested by extracting DNA from various tree tissue samples and from mycelium grown on solid and liquid media. DNA was extracted from juvenile and mature host ...
    Bacterial Lawn Avoidance and Bacterial Two Choice Preference Assays in Caenorhabditis elegans
    Authors:  Jogender Singh and Alejandro Aballay, date: 05/20/2020, view: 599, Q&A: 0
    [Abstract] Physical avoidance of pathogens is a crucial defense strategy used by the host to reduce pathogen infection. Hosts display the use of multiple strategies to sense and avoid pathogens, ranging from olfaction to sensing of damage caused by pathogen infection. Understanding various mechanisms of pathogen avoidance has the potential to uncover ...
    A Sensitive Coupled Enzyme Assay for Measuring Kinase and ATPase Kinetics Using ADP-Specific Hexokinase
    Authors:  Ciaran R. McFarlane and James W. Murray, date: 05/05/2020, view: 618, Q&A: 0
    [Abstract] Kinases and ATPases perform essential biological functions in metabolism and regulation. Activity of these enzymes is commonly measured by coupling ATP consumption to the synthesis of a detectable product. For most assay systems the ATP concentration during the reaction is unknown, compromising the precision of the assay.

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