Biological Engineering

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    Protocols in Current Issue
    Incorporation of a Chemically Diverse Set of Non-Standard Amino Acids into a Gram-Positive Organism
    Authors:  Devon A. Stork, Michaela A. Jones, Ethan C. Garner and Aditya M. Kunjapur, date: 09/05/2022, view: 317, Q&A: 0
    [Abstract]

    The incorporation of non-standard amino acids (nsAAs) within proteins and peptides through genetic code expansion introduces novel chemical functionalities such as photo-crosslinking and bioconjugation. Given the utility of Bacillus subtilis in fundamental and applied science, we extended existing nsAA incorporation technology from Escherichia

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    Labelling of Active Transcription Sites with Argonaute NRDE-3—Image Active Transcription Sites in vivo in Caenorhabditis elegans
    Authors:  Antoine Barrière and Vincent Bertrand, date: 06/05/2022, view: 868, Q&A: 0
    [Abstract]

    Live labelling of active transcription sites is critical to our understanding of transcriptional dynamics. In the most widely used method, RNA sequence MS2 repeats are added to the transcript of interest, on which fluorescently tagged Major Coat Protein binds, and labels transcription sites and transcripts. Here we describe another strategy, using

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    Patterned Substrate of Mobile and Immobile Ligands to Probe EphA2 Receptor Clustering
    Authors:  Zhongwen Chen, Kabir H. Biswas and Jay T. Groves, date: 06/05/2022, view: 1279, Q&A: 0
    [Abstract]

    A multitude of membrane-localized receptors are utilized by cells to integrate both biochemical and physical signals from their microenvironment. The clustering of membrane receptors is widely presumed to have functional consequences for subsequent signal transduction. However, it is experimentally challenging to selectively manipulate receptor

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    A Fluorescence-based Assay for Measuring Phospholipid Scramblase Activity in Giant Unilamellar Vesicles
    Authors:  Patricia P. M. Mathiassen and Thomas Günther Pomorski, date: 03/20/2022, view: 1007, Q&A: 0
    [Abstract]

    Transbilayer movement of phospholipids in biological membranes is mediated by a diverse set of lipid transporters. Among them are scramblases that facilitate rapid bi-directional movement of lipids without metabolic energy input. In this protocol, we describe the incorporation of phospholipid scramblases into giant unilamellar vesicles (GUVs)

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    Laser Microirradiation and Real-time Recruitment Assays Using an Engineered Biosensor
    Authors:  Carolina dos Santos Passos, Robert E. Cohen and Tingting Yao, date: 03/05/2022, view: 1924, Q&A: 0
    [Abstract]

    Double-strand breaks (DSBs) are lesions in DNA that, if not properly repaired, can cause genomic instability, oncogenesis, and cell death. Multiple chromatin posttranslational modifications (PTMs) play a role in the DNA damage response to DSBs. Among these, RNF168-mediated ubiquitination of lysines 13 or 15 at the N-terminal tail of histone H2A

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    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: 1582, 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

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    Femtoliter Injection of ESCRT-III Proteins into Adhered Giant Unilamellar Vesicles
    [Abstract]

    The endosomal sorting complex required for transport (ESCRT) machinery mediates membrane fission reactions that exhibit a different topology from that observed in clathrin-coated vesicles. In all of the ESCRT-mediated events, the nascent vesicle buds away from the cytosol. However, ESCRT proteins are able to act upon membranes with different

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    Measuring Oligonucleotide Hydrolysis in Cellular Lysates via Viscosity Measurements
    Authors:  Romel Menacho-Melgar and Michael D. Lynch, date: 01/20/2022, view: 1428, Q&A: 0
    [Abstract]

    Cell lysis, a process that releases host oligonucleotides, is required in many biotechnological applications. However, intact oligonucleotides in crude cellular lysates increase the viscosity of lysates, which complicates downstream processes and routine laboratory workflows. To address this, nucleases that hydrolyze the intact oligonucleotides

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    Anaerobic Expression and Purification of Holo-CCIS, an Artificial Iron-sulfur Protein
    Authors:  Bhanu P. Jagilinki, Irina Paluy, Vikas Nanda and Dror Noy, date: 09/20/2021, view: 1807, Q&A: 0
    [Abstract]

    Iron-sulfur proteins are ubiquitous among all living organisms and are indispensable for almost all metabolic pathways ranging from photosynthesis, respiration, nitrogen, and carbon dioxide cycles. The iron-sulfur clusters primarily serve as electron acceptors and donors and transfer electrons to active sites of various enzymes, thus driving the

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