Protocols in Current Issue
    Defined Mutant Library Sequencing (DML-Seq) for Identification of Conditional Essential Genes
    Authors:  Shuai Shao, Lifan Wei, Feng Xia, Yuanxing Zhang and Qiyao Wang, date: 03/05/2021, view: 442, Q&A: 0

    Transposon insertion sequencing (TIS) is an emerging technique which utilizes a massive transposon mutant library to screen specific phenotype and determine the conditional essential genetic requirements for bacterial fitness under distinct conditions combined with high-throughput parallel sequencing technology. Compared with a massive mutant

    Bacterial Conjugation Protocol for Ruminant Mycoplasmas
    Authors:  Eveline Sagné, Christine Citti and Emilie Dordet-Frisoni, date: 01/20/2021, view: 574, Q&A: 0

    In Mycoplasma agalactiae, two simultaneous processes of DNA transfer have been described that require direct cell-to-cell contact and are similar to conjugation. One involves the self-transmission of an integrative conjugative element (ICE) while the second concerns the horizontal transfer of large and small fragments of chromosomal DNA. Here, we

    Candida albicans Culture, Cell Harvesting, and Total RNA Extraction
    Authors:  Max V. Cravener and Aaron P. Mitchell, date: 11/05/2020, view: 1018, 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: 994, 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: 747, 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: 1378, 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: 1329, 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: 1750, 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: 2531, 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 ...

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