Protocols in Current Issue
    Candida albicans Culture, Cell Harvesting, and Total RNA Extraction
    Authors:  Max V. Cravener and Aaron P. Mitchell, date: 11/05/2020, view: 511, Q&A: 0

    Transcriptional analysis has become a cornerstone of biological research, and with the advent of cheaper and more efficient sequencing technology over the last decade, there exists a need for high-yield and efficient RNA extraction techniques. Fungi such as the human pathogen Candida albicans present a unique obstacle to RNA purification in the

    Estimation of the Minimum Number of Replication Origins Per Chromosome in any Organism
    Author:  Marcelo S. da Silva, date: 10/20/2020, view: 506, 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: 353, 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 ...
    Whole-genome Identification of Transcriptional Start Sites by Differential RNA-seq in Bacteria
    Authors:  Ramón Cervantes-Rivera and Andrea Puhar, date: 09/20/2020, view: 562, Q&A: 0
    [Abstract] Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene ...
    Efficient Agrobacterium-mediated Transformation of the Elite–Indica Rice Variety Komboka
    [Abstract] Genetic transformation is crucial for both investigating gene functions and for engineering of crops to introduce new traits. Rice (Oryza sativa L.) is an important model in plant research, since it is the staple food for more than half of the world’s population. As a result, numerous transformation methods have been developed for both ...
    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: 886, 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 ...
    Conjugation Protocol Optimised for Roseburia inulinivorans and Eubacterium rectale
    Authors:  Paul O. Sheridan, Jennifer C. Martin and Karen P. Scott, date: 04/05/2020, view: 1218, Q&A: 0
    [Abstract] Roseburia and Eubacterium species of the human gut microbiota play an important role in the maintaince of human health, partly by producing butyrate, the main energy source of our colonic epithelial cells. However, our knowledge of the biochemistry and physiology of these bacteria has been limited by a lack of genetic ...
    CRISPR-Cas9 Genome Editing of Plasmodium knowlesi
    Authors:  Franziska Mohring, Melissa N. Hart, Avnish Patel, David A. Baker and Robert W. Moon, date: 02/20/2020, view: 1767, Q&A: 1
    [Abstract] Plasmodium knowlesi is a zoonotic malaria parasite in Southeast Asia that can cause severe and fatal malaria in humans. The main hosts are Macaques, but modern diagnostic tools reveal increasing numbers of human infections. After P. falciparum, P. knowlesi is the only other malaria parasite capable of being maintained in ...
    Measurement of the Promoter Activity in Escherichia coli by Using a Luciferase Reporter
    Authors:  Yuki Yamanaka, Hiroki Watanabe, Erika Yamauchi, Yukari Miyake and Kaneyoshi Yamamoto, date: 01/20/2020, view: 1420, Q&A: 0
    [Abstract] The reporter system is widely used technique for measuring promoter activity in bacterial cells. Until now, a number of reporter system have been developed, but the bioluminescent reporter constructed from the bacterial luciferase genes is one of the useful systems for measuring in vivo dynamics of gene expression. The introduced ...
    Unbiased and Tailored CRISPR/Cas gRNA Libraries by Synthesizing Covalently-closed-circular (3Cs) DNA
    Authors:  Martin Wegner, Koraljka Husnjak and Manuel Kaulich, date: 01/05/2020, view: 2810, Q&A: 0
    [Abstract] Simplicity, efficiency and versatility of the CRISPR/Cas system greatly contributed to its rapid use in a broad range of fields. Applications of unbiased CRISPR/Cas screenings are increasing and thus there is a growing need for unbiased and tailored CRISPR/Cas gRNA libraries. Conventional methods for gRNA library generation apply PCR and cloning ...

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