Technion - Israel Institute of Technology 19 articles published in JoVE Biology Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling Johannes Venezian1, Hila Zilberman1, Ayala Shiber1 1Faculty of Biology, Technion - Israel Institute of Technology Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae. Bioengineering Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy Einel A. Chaimovitz*1, Evgeniy Reznik*1, Mouna Habib1, Netanel Korin1, Ramez Daniel1 1Department of Biomedical Engineering, Technion - Israel Institute of Technology This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits. Bioengineering In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow Maria Khoury1, Mark Epshtein1, Netanel Korin1 1Department of Biomedical Engineering, Technion - IIT Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system. Neuroscience Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics Caitlin A. Murphy1, Aeyal Raz1,2, Sean M Grady1, Matthew I Banks1 1Department of Anesthesiology, University of Wisconsin, 2Department of Anesthesiology, Rambam Health Care Campus, the Ruth and Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane. Bioengineering Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer Omer Adir*1,2, Noga Sharf-Pauker*1,2, Gal Chen*1,3, Maya Kaduri1, Nitzan Krinsky1,3, Janna Shainsky-Roitman1, Jeny Shklover1, Avi Schroeder1 1Laboratory for Targeted Drug Delivery and Personalized Medicine Technologies, Department of Chemical Engineering, Technion - Israel Institute of Technology, 2The Norman Seiden Multidisciplinary Program for Nanoscience and Nanotechnology, Technion - Israel Institute of Technology, 3The Interdisciplinary Program for Biotechnology, Technion - Israel Institute of Technology This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method. Genetics An Assay for Quantifying Protein-RNA Binding in Bacteria Noa Katz*1, Roni Cohen*1, Orna Atar1, Sarah Goldberg1, Roee Amit1,2 1Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, 2Russell Berrie Nanotechnology Institute, Technion-Israel Institute of Technology In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene. Bioengineering Designing Porous Silicon Films as Carriers of Nerve Growth Factor Michal Rosenberg1, Neta Zilony2,3, Orit Shefi2,3, Ester Segal1,4 1Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, 2Faculty of Engineering, Bar-Ilan University, 3Bar-Ilan Institute of Nanotechnologies and Advanced Materials, 4Russell Berrie Nanotechnology Institute, Technion - Israel Institute of Technology Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers. Engineering Design and Fabrication of an Optical Fiber Made of Water Mark L. Douvidzon1, Shai Maayani2, Leopoldo L. Martin3, Tal Carmon4 1Department of Nanoscience and Nanotechnology, Russell Berrie Nanotechnology Institute (RBNI), Technion - Israel Institute of Technology, 2Department of Material Sciences and Engineering, MIT, 3Centro de Tecnologia Nanofotónica, Universitat Politècnica de València, 4Department of Mechanical Engineering, Technion - Israel Institute of Technology This protocol describes the design and manufacture of a water bridge and its activation as a water fiber. The experiment demonstrates that capillary resonances of the water fiber modulate its optical transmission. Immunology and Infection Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro Lena Lavie1, Larissa Dyugovskaya1, Andrey Polyakov1, Oksana Rogovoy1, Eva Leder1 1The Lloyd Rigler Sleep Apnea Research Laboratory, Unit of Anatomy and Cell Biology, The Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Insitute of Technology We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation. Genetics Novel RNA-Binding Proteins Isolation by the RaPID Methodology Nitzan Samra1,2, Yoav Arava1 1Faculty of Biology, Technion - Israel Institute of Technology, 2Department of Biomolecular Sciences, Weizmann Institute of Science RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell. Biology High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications Yulia Fridman1, Neta Holland1, Rivka Elbaum2, Sigal Savaldi-Goldstein1 1Faculty of Biology, Technion-Israel Institute of Technology, 2Smith Institute of Plant Sciences and Genetics in Agriculture, Hebrew University of Jerusalem Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution. Bioengineering A Microfluidic Model of Biomimetically Breathing Pulmonary Acinar Airways Rami Fishler1, Josué Sznitman1 1Department of Biomedical Engineering, Technion - Israel Institute of Technology Soft-lithography was utilized to produce a representative true-scale model of pulmonary alveolated airways that expand and contract periodically, mimicking physiological breathing motion. This platform recreates respiratory acinar flows on a chip, and is anticipated to facilitate experimental investigation of inhaled aerosol dynamics and deposition in the pulmonary acinus. Developmental Biology Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin Despina Soteriou1, Lana Kostic1, Egor Sedov1, Yahav Yosefzon1, Hermann Steller2, Yaron Fuchs1 1Department of Biology and Lokey Center, Laboratory of Stem Cell Biology and Regenerative Medicine, Technion Israel Institute of Technology, 2Strang Laboratory of Apoptosis and Cancer Biology, Howard Hughes Medical Institute, The Rockefeller University An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis. Chemistry Photochemical Oxidative Growth of Iridium Oxide Nanoparticles on CdSe@CdS Nanorods Philip Kalisman1, Yifat Nakibli1, Lilac Amirav1 1Schulich Faculty of Chemistry, The Russell Berrie Nanotechnology Institute, The Nancy and Stephen Grand Technion Energy Program, Technion – Israel Institute of Technology A protocol for the photochemical oxidative growth of small crystalline iridium oxide nanoparticles on the surface of CdSe@CdS seeded rod nanoparticles is presented. Bioengineering Engineered Vascularized Muscle Flap Dana Egozi*1, Yulia Shandalov*2, Alina Freiman2,3, Dekel Rosenfeld2, David Ben-Shimol4, Shulamit Levenberg2 1Department of Plastic Surgery, Kaplan Medical Center, 2Biomedical Engineering, Technion Israel Institute of Technology, 3Interdepartmental Program in Biotechnology, Technion Israel Institute of Technology, 4Medicine Department, Technion Israel Institute of Technology To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps. Developmental Biology A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice Aya Amitai-Lange*1, Eran Berkowitz*2, Anna Altshuler1, Noora Dbayat2, Waseem Nasser1, Edith Suss-Toby3, Beatrice Tiosano2, Ruby Shalom-Feuerstein1 1Department of Genetics and Developmental Biology, The Ruth and Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, 2Department of Ophthalmology, Hillel Yaffe Medical Center, 3Bioimging Center, Biomedical Core Facility, The Ruth and Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest. Biology Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR) Nurit Livnat Levanon1, Elena Vigonsky1, Oded Lewinson1 1Department of Biochemistry, The Bruce and Ruth Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique. Biology Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation Chen Lesnik1, Yoav Arava1 1Department of Biology, Technion - Israel Institute of Technology Many mRNAs encoding mitochondrial proteins are associated with the mitochondria outer membrane. We describe a subcellular fractionation procedure aimed at isolation of yeast mitochondria with its associated mRNAs and ribosomes. This protocol can be applied to cells grown under diverse conditions in order to reveal mechanisms of mRNA localization and localized translation near the mitochondria. Bioengineering Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors Naama Massad-Ivanir*1, Giorgi Shtenberg*2, Ester Segal1,3 1Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, 2The Inter-Departmental Program of Biotechnology, Technion - Israel Institute of Technology, 3The Russell Berrie Nanotechnology Institute, Technion - Israel Institute of Technology A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).
