Molecular Biology


    Protocols in Current Issue
    A New Tool for the Flexible Genetic Manipulation of Geobacillus kaustophilus
    Authors:  Ryotaro Amatsu, Kotaro Mori, Shu Ishikawa, Wilfried J. J. Meijer and Ken-ichi Yoshida, date: 09/05/2022, view: 328, Q&A: 0

    Geobacillus kaustophilus, a thermophilic Gram-positive bacterium, is an attractive host for the development of high-temperature bioprocesses. However, its reluctance against genetic manipulation by standard methodologies hampers its exploitation. Here, we describe a simple methodology in which an artificial DNA segment on the chromosome of

    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: 2839, 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 ...
    Preparation of a Bacteriophage T4-based Prokaryotic-eukaryotic Hybrid Viral Vector for Delivery of Large Cargos of Genes and Proteins into Human Cells
    Authors:  Jingen Zhu, Pan Tao, Marthandan Mahalingam and Venigalla B. Rao, date: 04/05/2020, view: 3114, Q&A: 0
    [Abstract] A viral vector that can safely and efficiently deliver large and diverse molecular cargos into cells is the holy grail of curing many human diseases. Adeno-associated virus (AAV) has been extensively used but has a very small capacity. The prokaryotic virus T4 has a large capacity but lacks natural mechanisms to enter mammalian cells. Here, we ...
    Measuring Homologous Recombination Rates between Chromosomal Locations in Salmonella
    Authors:  Gerrit Brandis, Sha Cao and Diarmaid Hughes, date: 02/05/2019, view: 6293, Q&A: 0
    [Abstract] Homologous recombination between two similar DNA molecules, plays an important role in the repair of double-stranded DNA breaks. Recombination can occur between two sister chromosomes, or between two locations of similar sequence identity within the same chromosome. The assay described here is designed to measure the rate of homologous ...
    Analysis of DNA Exchange Using Thymidine Analogs (ADExTA) in Trypanosoma cruzi
    [Abstract] Trypanosoma cruzi is a protozoan parasite belonging to the Trypanosomatidae family. Although the trypanosomatids multiply predominantly by clonal generation, the presence of DNA exchange in some of them has been puzzling researchers over the years, mainly because it may represent a novel form that these organisms use to gain variability. ...
    Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery
    Authors:  Giel Vandemoortele, Delphine De Sutter and Sven Eyckerman, date: 04/05/2017, view: 20595, Q&A: 0
    [Abstract] The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair ...
    Efficient AAV-mediated Gene Targeting Using 2A-based Promoter-trap System
    [Abstract] Adeno-associated virus (AAV)-based targeting vectors have 1-4-log higher gene targeting efficiencies compared with plasmid-based targeting vectors. The efficiency of AAV-mediated gene targeting is further increased by introducing a promoter-trap system into targeting vectors. In addition, we found that the use of ribosome-skipping 2A peptide ...
    Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol
    Authors:  Simon David Brown and Alexander Lorenz, date: 12/20/2016, view: 8083, Q&A: 0
    [Abstract] The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described ...
    FLP/FRT Induction of Mitotic Recombination in Drosophila Germline
    Authors:  Pedro Prudêncio and Leonardo G. Guilgur, date: 05/05/2015, view: 27596, Q&A: 0
    [Abstract] The FLP/FRT system is a site-directed recombination technology based on the targeting of a recombination enzyme (flipase - FLP) to specific DNA regions designated as flipase recognition target (FRT) sites. Initially identified in Saccharomyces cerevisiae, the yeast FLP-enzyme and its FRT recombination targets were successfully transferred ...
    Measuring Homologous Recombination Frequency in Arabidopsis Seedlings
    Authors:  Marisa Rosa and Ortrun Mittelsten Scheid, date: 04/05/2014, view: 11768, Q&A: 0
    [Abstract] Somatic homologous recombination (SHR) is a major pathway of DNA double-strand break (DSB) repair, in which intact homologous regions are used as a template for the removal of lesions. Its frequency in plants is generally low, as most DSB are removed by non-homologous mechanisms in higher eukaryotes. Nevertheless, SHR frequency has been shown to ...

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