Microbiology

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    Protocols in Current Issue
    A Molecular Cloning and Sanger Sequencing-based Protocol for Detecting Site-specific DNA Methylation
    Authors:  Wei Guo, Anthony Cannon and Damon Lisch, date: 05/05/2022, view: 644, Q&A: 0
    [Abstract]

    DNA methylation is a conserved chemical modification, by which methyl groups are added to the cytosine of DNA molecules. Methylation can influence gene expression without changing the sequence of a particular gene. This epigenetic effect is an intriguing phenomenon that has puzzled biologists for years. By probing the temporal and spatial patterns

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    Selection of Vaccinia Virus Recombinants Using CRISPR/Cas9
    Authors:  Anjali Gowripalan, Stewart A. Smith and David C. Tscharke, date: 12/20/2021, view: 1175, Q&A: 0
    [Abstract]

    The engineering of poxvirus genomes is fundamental to primary and applied virology research. Indeed, recombinant poxviruses form the basis for many novel vaccines and virotherapies but producing and purifying these viruses can be arduous. In recent years, CRISPR/Cas9 has become the favoured approach for genome manipulation due to its speed and

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    Estimation of the Minimum Number of Replication Origins Per Chromosome in any Organism
    Author:  Marcelo S. da Silva, date: 10/20/2020, view: 2272, 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 ...
    Quantification of HIV-2 DNA in Whole Blood
    Authors:  Zsófia Szojka, Sara Karlson, Marianne Jansson and Patrik Medstrand, date: 10/20/2019, view: 3832, Q&A: 0
    [Abstract] Time to AIDS infection is longer with HIV-2, compared to HIV-1, but without antiretroviral therapy both infections will cause AIDS-related mortality. In HIV-2 infection, monitoring of antiretroviral treatment (ART) efficacy is challenging since a large proportion of HIV-2-infected individuals displays low or undetectable plasma RNA levels. Hence, ...
    Strand-specific Single-stranded DNA Sequencing (4S-seq) of E. coli genomes
    Authors:  Takahiro Masuda, Nobuaki Kono, Masaru Tomita and Kazuharu Arakawa, date: 08/05/2019, view: 5429, Q&A: 0
    [Abstract] Most bacterial genomes have biased nucleotide composition, and the asymmetry is considered to be caused by a single-stranded DNA (ssDNA) deamination arising from the bacterial replication machinery. In order to evaluate the relationship experimentally, the position and frequency of ssDNA formed during replication must be verified clearly. Although ...
    CRISPR/Cas9-mediated ssDNA Recombineering in Corynebacterium glutamicum
    Authors:  Jiao Liu, Yu Wang, Ping Zheng and Jibin Sun, date: 10/05/2018, view: 5901, Q&A: 0
    [Abstract] Corynebacterium glutamicum is a versatile workhorse for industrial bioproduction of many kinds of chemicals and fuels, notably amino acids. Development of advanced genetic engineering tools is urgently demanded for systems metabolic engineering of C. glutamicum. Recently unveiled clustered regularly interspaced short palindromic ...
    ChIP-seq Experiment and Data Analysis in the Cyanobacterium Synechocystis sp. PCC 6803
    Authors:  Joaquín Giner-Lamia, Miguel A. Hernández-Prieto and Matthias E. Futschik, date: 06/20/2018, view: 10666, Q&A: 0
    [Abstract] Nitrogen is an essential nutrient for all living organisms. In cyanobacteria, a group of oxygenic photosynthetic bacteria, nitrogen homeostasis is maintained by an intricate regulatory network around the transcription factor NtcA. Although mechanisms controlling NtcA activity appear to be well understood, the sets of genes under its control (i.e. ...
    Targeted Genome Editing of Virulent Phages Using CRISPR-Cas9
    Authors:  Marie-Laurence Lemay, Ariane C. Renaud, Geneviève M. Rousseau and Sylvain Moineau, date: 01/05/2018, view: 13717, Q&A: 1
    [Abstract] This protocol describes a straightforward method to generate specific mutations in the genome of strictly lytic phages. Briefly, a targeting CRISPR-Cas9 system and a repair template suited for homologous recombination are provided inside a bacterial host, here the Gram-positive model Lactococcus lactis MG1363. The CRISPR-Cas9 system is ...
    Markerless Gene Editing in the Hyperthermophilic Archaeon Thermococcus kodakarensis
    Authors:  Alexandra M. Gehring, Travis J. Sanders and Thomas J. Santangelo, date: 11/20/2017, view: 6677, Q&A: 0
    [Abstract] The advent of single cell genomics and the continued use of metagenomic profiling in diverse environments has exponentially increased the known diversity of life. The recovered and assembled genomes predict physiology, consortium interactions and gene function, but experimental validation of metabolisms and molecular pathways requires more ...
    Method for Multiplexing CRISPR/Cas9 in Saccharomyces cerevisiae Using Artificial Target DNA Sequences
    Authors:  Rachael M. Giersch and Gregory C. Finnigan, date: 09/20/2017, view: 10280, Q&A: 0
    [Abstract] Genome manipulation has become more accessible given the advent of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing technology. The Cas9 endonuclease binds a single stranded (single guide) RNA (sgRNA) fragment that recruits the complex to a corresponding genomic target sequence where it induces a double stranded ...



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