Microbiology

Categories

    Protocols in Current Issue
    A Modified Fluctuation Assay with a CAN1 Reporter in Yeast
    Authors:  Pengyao Jiang, Anja R. Ollodart and Maitreya J. Dunham, date: 06/05/2022, view: 518, Q&A: 0
    [Abstract]

    Understanding the generation of mutations is fundamental to understanding evolution and genetic disease; however, the rarity of such events makes experimentally identifying them difficult. Mutation accumulation (MA) methods have been widely used. MA lines require serial bottlenecks to fix de novo mutations, followed by whole-genome sequencing.

    ...
    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: 680, Q&A: 1
    [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

    ...
    Phytophthora sojae Transformation Based on the CRISPR/Cas9 System
    Authors:  Jingting Cao, Min Qiu, Wenwu Ye and Yuanchao Wang, date: 03/20/2022, view: 752, Q&A: 0
    [Abstract]

    Phytophthora sojae is a model species for the study of plant pathogenic oomycetes. The initial research on gene function using Phytophthora was mainly based on gene silencing technology. Recently, the CRISPR/Cas9-mediated genome editing technology was successfully established in P. sojae and widely used in oomycetes. In this protocol, we describe

    ...
    RNA-mediated in vivo Directed Evolution in Yeast
    Authors:  Emil D. Jensen and Michael K. Jensen, date: 03/05/2022, view: 1511, Q&A: 0
    [Abstract]

    Directed evolution is a powerful approach to obtain genetically-encoded sought-for traits. Compared to the prolonged adaptation regimes to mutations occurring under natural selection, directed evolution unlocks rapid screening and selection of mutants with improved traits from vast mutated sequence spaces. Many systems have been developed to

    ...
    An in vitro Assay of mRNA 3’ end Using the E. coli Cell-free Expression System
    Authors:  Monford Paul Abishek N and Heon M. Lim, date: 02/20/2022, view: 1367, Q&A: 0
    [Abstract]

    At the end of about 80% of the operon in Escherichia coli, translation termination decouples transcription, leading to Rho-dependent transcription termination (RDT). However, no in vitro or in vivo assay system has proven to be good enough to see the 3’ end of the mRNA generated by RDT. Here, we present a cell-free assay system that

    ...
    Selection of Vaccinia Virus Recombinants Using CRISPR/Cas9
    Authors:  Anjali Gowripalan, Stewart A. Smith and David C. Tscharke, date: 12/20/2021, view: 1194, 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

    ...
    A Nucleocapsid-based Transcomplementation Cell Culture System of SARS-CoV-2 to Recapitulate the Complete Viral Life Cycle
    Authors:  Yanying Yu, Xiaohui Ju and Qiang Ding, date: 11/05/2021, view: 1448, Q&A: 0
    [Abstract]

    The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As this virus is classified as a biosafety level-3 (BSL-3) agent, the development of countermeasures and basic research methods is logistically difficult. Recently, using reverse genetics, we developed a BSL-2 cell culture system for production

    ...
    A Rapid Induction Overexpression System for the Fission Yeast Schizosaccharomyces pombe
    Author:  Angad Garg, date: 10/20/2021, view: 845, Q&A: 0
    [Abstract]

    The fission yeast Schizosaccharomyces pombe is an excellent genetically tractable model organism used in the study of conserved eukaryotic cellular biology. One genetic tool in the assessment of gene function is the in vivo overexpression of proteins. Existing overexpression tools have limitations of induction kinetics, dynamic range, and/or

    ...
    Quantification of RuBisCO Expression and Photosynthetic Oxygen Evolution in Cyanobacteria
    Authors:  Mateusz Kędzior and Betul Kacar, date: 10/20/2021, view: 1085, Q&A: 0
    [Abstract]

    Phototrophic microorganisms are frequently engineered to regulate the expression and the activity of targeted enzymes of interest for specific biotechnological and agricultural applications. This protocol describes a method to evaluate the expression of RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) in the model cyanobacterium

    ...
    Cell-free Translation: Preparation and Validation of Translation-competent Extracts from Saccharomyces cerevisiae
    Authors:  Brandon M. Trainor, Anton A. Komar, Dimitri G. Pestov and Natalia Shcherbik, date: 09/20/2021, view: 1790, Q&A: 0
    [Abstract]

    Cell-free translation is a powerful technique for in vitro protein synthesis. While cell-free translation platforms prepared from bacterial, plant, and mammalian cells are commercially available, yeast-based translation systems remain proprietary knowledge of individual labs. Here, we provide a detailed protocol for simple, fast, and

    ...



    We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.