Microbiology

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    Protocols in Current Issue
    CRISPR/Cas9-mediated ssDNA Recombineering in Corynebacterium glutamicum
    Authors:  Jiao Liu, Yu Wang, Ping Zheng and Jibin Sun, date: 10/05/2018, view: 1169, 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: 2661, 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: 5345, 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: 3292, 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: 5348, 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 ...
    Detection of Pathogens and Ampicillin-resistance Genes Using Multiplex Padlock Probes
    Authors:  Rick Conzemius and Ivan Barišić, date: 08/20/2017, view: 3707, Q&A: 0
    [Abstract] Diagnostic assays for pathogen identification and characterization are limited either by the number of simultaneously detectable targets, which rely on multiplexing methods, or by time constraints due to cultivation-based techniques. We recently presented a 100-plex method for human pathogen characterization to identify 75 bacterial and fungal ...
    Selection of Genetically Modified Bacteriophages Using the CRISPR-Cas System
    Authors:  Miriam Manor and Udi Qimron, date: 08/05/2017, view: 4712, Q&A: 0
    [Abstract] We present a CRISPR-Cas based technique for deleting genes from the T7 bacteriophage genome. A DNA fragment encoding homologous arms to the target gene to be deleted is first cloned into a plasmid. The T7 phage is then propagated in Escherichia coli harboring this plasmid. During this propagation, some phage genomes undergo homologous ...
    Using CRISPR/Cas9 for Large Fragment Deletions in Saccharomyces cerevisiae
    Authors:  Huanhuan Hao, Jing Huang, Tongtong Liu, Hui Tang and Liping Zhang, date: 07/20/2017, view: 5512, Q&A: 0
    [Abstract] CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) systems have emerged as a powerful tool for genome editing in many organisms. The wide use of CRISPR/Cas9 systems may be due to the fact that these systems contain a simple guide RNA (sgRNA) that is relatively easy to design and they are very ...
    Single Genome Sequencing of Expressed and Proviral HIV-1 Envelope Glycoprotein 120 (gp120) and nef Genes
    [Abstract] The current study provides detailed protocols utilized to amplify the complete HIV-1 gp120 and nef genes from single copies of expressed or integrated HIV present in fresh-frozen autopsy tissues of patients who died while on combined antiretroviral therapy (cART) with no detectable plasma viral load (pVL) at death (Lamers et al ...
    Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting
    [Abstract] Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol ...



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