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Browsing by Subject "cell-free transcription-translation"

Now showing 1 - 3 of 3
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    Data for Membraneless Compartmentalization of Cell-Free Transcription-Translation by Polymer-Assisted Liquid-Liquid Phase Separation
    (2024-11-20) Izri, Ziane; Noireaux, Vincent; noireaux@umn.edu; Noireaux, Vincent; Noireaux Lab
    The data shared in this repository consists mainly of the collection of the original fluorescence microscopy pictures of the PSTXTL system presented in the manuscript together with the product of their processing and analysis. Those pictures were acquired through three different channels: Bright Field, FITC and Texas Red. The bright field images inform on the presence of phase-separated droplets, while the FITC and Texas Red channels inform on the content of the phase-separated droplets, depending on the fluorophores used. The results of the fluorometry analysis are also present in the data collection.
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    Data supporting "PHEIGES, all-cell-free phage synthesis and selection from engineered genomes"
    (2024-01-29) Levrier, Antoine; Karpathakis, Ioannis; Nash, Bruce; Bowden, Steven; Lindner, Ariel; Noireaux, Vincent; noireaux@umn.edu; Noireaux, Vincent; Noireaux lab
    Bacteriophages constitute an invaluable biological reservoir for biotechnology and medicine. The ability to exploit such vast resources is hampered by the lack of methods to rapidly engineer, assemble, package genomes, and select phages. Cell-free transcription-translation (TXTL) offers experimental settings to address such a limitation. Here, we describe PHage Engineering by In vitro Gene Expression and Selection (PHEIGES) using T7 phage genome and Escherichia coli TXTL. Phage genomes are assembled in vitro from PCR-amplified fragments and directly expressed in batch TXTL reactions to produce up to 1011 PFU/ml engineered phages within one day. We further demonstrate a significant genotype-phenotype linkage of phage assembly in bulk TXTL. This enables rapid selection of phages with altered rough lipopolysaccharides specificity from phage genomes incorporating tail fiber mutant libraries. We establish the scalability of PHEIGES by one pot assembly of such mutants with fluorescent gene integration and 10% length-reduced genome.
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    Data supporting Cell-free expression with a quartz crystal microbalance enables rapid, dynamic, and label-free characterization of membrane-interacting proteins
    (2024-07-15) Khakimzhan, Aset; Izri, Ziane; Thompson, Seth; Dmytrenko, Oleg; Fischer, Patrick; Beisel, Chase; Noireaux, Vincent; noireaux@umn.edu; Noireaux, Vincent; Noireaux Lab
    Integral and interacting membrane proteins (IIMPs) constitute a vast family of biomolecules that perform essential functions in all forms of life. However, characterizing their interactions with lipid bilayers remains limited due to challenges in purifying and reconstituting IIMPs in vitro or labeling IIMPs without disrupting their function in vivo. Here, we report cell-free transcription-translation in a quartz crystal microbalance with dissipation (TXTL-QCMD) to dynamically characterize interactions between diverse IIMPs and membranes without protein purification or labeling. As part of TXTL-QCMD, IIMPs are synthesized using cell-free transcription-translation (TXTL), and their interactions with supported lipid bilayers are measured using a quartz crystal microbalance with dissipation (QCMD). TXTL-QCMD reconstitutes known IIMP-membrane dependencies, including specific association with prokaryotic or eukaryotic membranes, and the multiple-IIMP dynamical pattern-forming association of the E. coli division-coordinating proteins MinCDE. Applying TXTL-QCMD to the recently discovered Zorya anti-phage system that is unamenable to labeling, we discovered that ZorA and ZorB integrate within the lipids found at the poles of bacteria while ZorE diffuses freely on the non-pole membrane. These efforts establish the potential of TXTL-QCMD to broadly characterize the large diversity of IIMPs.