Microbiology

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    Protocols in Current Issue
    Delivery of the Cas9 or TevCas9 System into Phaeodactylum tricornutum via Conjugation of Plasmids from a Bacterial Donor
    Authors:  Helen Wang, Samuel S. Slattery, Bogumil J. Karas and David R. Edgell, date: 08/20/2018, view: 733, Q&A: 0
    [Abstract] Diatoms are an ecologically important group of eukaryotic microalgae with properties that make them attractive for biotechnological applications such as biofuels, foods, cosmetics and pharmaceuticals. Phaeodactylum tricornutum is a model diatom with defined culture conditions, but routine genetic manipulations are hindered by a lack of ...
    Random Insertional Mutagenesis of a Serotype 2 Dengue Virus Clone
    Authors:  Jeffrey W. Perry and Andrew W. Tai, date: 08/20/2018, view: 498, Q&A: 0
    [Abstract] Protein tagging is a powerful method of investigating protein function. However, modifying positive-strand RNA virus proteins in the context of viral infection can be particularly difficult as their compact genomes and multifunctional proteins mean even small changes can inactivate or attenuate the virus. Although targeted approaches to ...
    CRISPR/Cas Gene Editing of a Large DNA Virus: African Swine Fever Virus
    [Abstract] Gene editing of large DNA viruses, such as African swine fever virus (ASFV), has traditionally relied on homologous recombination of a donor plasmid consisting of a reporter cassette with surrounding homologous viral DNA. However, this homologous recombination resulting in the desired modified virus is a rare event. We recently reported the use of ...
    Method for CRISPR/Cas9 Mutagenesis in Candida albicans
    Authors:  Neta Dean and Henry Ng, date: 04/20/2018, view: 2361, Q&A: 0
    [Abstract] Candida albicans is the most prevalent and important human fungal pathogen. The advent of CRISPR as a means of gene editing has greatly facilitated genetic analysis in C. albicans. Here, we describe a detailed step-by-step procedure to construct and analyze C. albicans deletion mutants. This protocol uses plasmids that ...
    Single-step Precision Genome Editing in Yeast Using CRISPR-Cas9
    [Abstract] Genome modification in budding yeast has been extremely successful largely due to its highly efficient homology-directed DNA repair machinery. Several methods for modifying the yeast genome have previously been described, many of them involving at least two-steps: insertion of a selectable marker and substitution of that marker for the intended ...
    Design of Hybrid RNA Polymerase III Promoters for Efficient CRISPR-Cas9 Function
    Authors:  Joshua Misa, Cory Schwartz and Ian Wheeldon, date: 03/20/2018, view: 1662, Q&A: 0
    [Abstract] The discovery of the CRISPR-Cas9 system from Streptococcus pyogenes has allowed the development of genome engineering tools in a variety of organisms. A frequent limitation in CRISPR-Cas9 function is adequate expression levels of sgRNA. This protocol provides a strategy to construct hybrid RNA polymerase III (Pol III) promoters that ...
    Microbial Mutagenicity Assay: Ames Test
    Authors:  Urvashi Vijay, Sonal Gupta, Priyanka Mathur, Prashanth Suravajhala and Pradeep Bhatnagar, date: 03/20/2018, view: 5822, Q&A: 2
    [Abstract] The Microbial mutagenicity Ames test is a bacterial bioassay accomplished in vitro to evaluate the mutagenicity of various environmental carcinogens and toxins. While Ames test is used to identify the revert mutations which are present in strains, it can also be used to detect the mutagenicity of environmental samples such as drugs, dyes, ...
    Conditional Knockdown of Proteins Using Auxin-inducible Degron (AID) Fusions in Toxoplasma gondii
    Authors:  Kevin M. Brown, Shaojun Long and L. David Sibley, date: 02/20/2018, view: 3450, Q&A: 0
    [Abstract] Toxoplasma gondii is a member of the deadly phylum of protozoan parasites called Apicomplexa. As a model apicomplexan, there is a great wealth of information regarding T. gondii’s 8,000+ protein coding genes including sequence variation, expression, and relative contribution to parasite fitness. However, new tools are needed to ...
    Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli
    Authors:  Enrico König, Francesca Zerbini, Ilaria Zanella, Davide Fraccascia and Guido Grandi, date: 01/20/2018, view: 5880, Q&A: 0
    [Abstract] With the recent implementation of the CRISPR/Cas9 technology as a standard tool for genome editing, laboratories all over the world are undergoing one of the biggest advancements in molecular biology since PCR. The key advantage of this method is its simplicity and universal applicability for species of any phylum. Of particular interest is the ...
    Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions
    Authors:  Amanda Hopes, Vladimir Nekrasov, Nigel Belshaw, Irina Grouneva, Sophien Kamoun and Thomas Mock, date: 12/05/2017, view: 3341, Q&A: 1
    [Abstract] Genome editing in diatoms has recently been established for the model species Phaeodactylum tricornutum and Thalassiosira pseudonana. The present protocol, although developed for T. pseudonana, can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include ...



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