Karlsruhe Institute of Technology View Institution's Website 20 articles published in JoVE Chemistry In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology Kateryna Horbatok*1,2, Tetyana Makhnii*2, Viktoriia Kosach2, Volodymyr Danko1, Andrey Kovalenko1, Stanislav Fatiushchenkov1, Petro Borysko2, Iryna Pishel2, Oleg Babii3,4, Anne S. Ulrich3, Tim Schober4, Sergii Afonin3, Igor V. Komarov1,2,4 1Taras Shevchenko National University of Kyiv, 2Bienta/Enamine, 3Karlsruhe Institute of Technology, 4Lumobiotics This protocol presents a set of experiments adopted for the evaluation of photoswitchable anticancer peptides, that can be used in the preclinical screening of such compounds. This includes cytotoxicity assessment in 2D and 3D cell cultures, the evaluation of ex vivo (model tissue) photoisomerization efficiency, and in vivo efficacy. Biology Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna Nora Weber1, Michael Hofmeister1, Nadja Wunsch1, Anja Kohler1, Anne-Kristin Kaster2, Jon Vollmers2, Ben Kachel3, Matthias Mack3, Tilman Lamparter1 1Botanical Institute, Karlsruhe Institute of Technology KIT, 2Institute for Biological Interfaces 5 (IBG 5), Karlsruhe Institute of Technology KIT, 3Institut für Technische Mikrobiologie, Hochschule Mannheim Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods. Environment Automated 3D Optical Coherence Tomography to Elucidate Biofilm Morphogenesis Over Large Spatial Scales Anna Depetris1, Antoine Wiedmer2, Michael Wagner3, Sebastian Schäfer4, Tom J. Battin1, Hannes Peter1 1Stream Biofilm and Ecosystem Research Laboratory, School of Architecture, Civil and Environmental Engineering, Ecole Polytechnique Fédérale de Lausanne, 2Plateforme technique, School of Architecture, Civil and Environmental Engineering, Ecole Polytechnique Fédérale de Lausanne, 3Water Chemistry and Water Technology, Engler-Bunte-Institut, Karlsruhe Institute of Technology, 4Thorlabs GmbH Microbial biofilms form complex architectures at interphases and develop into highly scale-dependent spatial patterns. Here, we introduce an experimental system (hard- and software) for the automated acquisition of 3D optical coherence tomography (OCT) datasets. This toolset allows the non-invasive and multi-scale characterization of biofilm morphogenesis in space and time. Chemistry Synthesis of Bimetallic Pt/Sn-based Nanoparticles in Ionic Liquids Christine Dietrich1,2, Georgios Uzunidis1,2, Yannick Träutlein1, Silke Behrens1,2 1Karlsruhe Institute of Technology (KIT), 2Ruprecht-Karls University of Heidelberg A protocol for the synthesis of bimetallic nanoparticles in ionic liquids and the procedure of their catalytic testing in the selective hydrogenation of unsaturated aldehydes are described. Developmental Biology Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes Irene U. Wacker1,2, Lisa Veith3, Waldemar Spomer2,4, Andreas Hofmann2,4, Marlene Thaler5, Stefan Hillmer6, Ulrich Gengenbach2,4, Rasmus R. Schröder1,2,3 1Cryo Electron Microscopy, Centre for Advanced Materials, Universität Heidelberg, 2Heidelberg Karlsruhe Research Partnership (HEiKA), 3Cryo Electron Microscopy, BioQuant, Universitätsklinikum Heidelberg, 4Institute for Automation and Applied Computer Science, Karlsruhe Institute of Technology (KIT), 5Carl Zeiss Microscopy GmbH, 6Electron Microscopy Core Facility, Universität Heidelberg This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM. Engineering Challenges in Rheological Characterization of Highly Concentrated Suspensions — A Case Study for Screen-printing Silver Pastes Ceren Yüce1, Norbert Willenbacher1 1Applied Mechanics Group, Institute for Mechanical Process Engineering and Mechanics, Department of Chemical Engineering, Karlsruhe Institute of Technology A protocol for a robust and application relevant rheological characterization of highly concentrated suspensions is presented. Silver pastes used for screen-printing application in solar cell production are employed as model systems. Genetics Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions Kristin Bösch*1,2, Lamprinos Frantzeskakis*1, Miroslav Vraneš3, Jörg Kämper3, Kerstin Schipper1,2, Vera Göhre1,2,4 1Institute for Microbiology, Heinrich-Heine University Düsseldorf, 2Bioeconomy Science Center (BioSC), 3Department of Genetics, Institute of Applied Biosciences, Karlsruhe Institute of Technology, 4Cluster of Excellence in Plant Sciences (CEPLAS), Heinrich-Heine University Düsseldorf We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag. Bioengineering Fast Pyrolysis of Biomass Residues in a Twin-screw Mixing Reactor Axel Funke1, Daniel Richter1, Andreas Niebel1, Nicolaus Dahmen1, Jörg Sauer1 1Institute of Catalysis Research and Technology (IKFT), Karlsruhe Institute of Technology (KIT) A procedure for thermochemical conversion of biomass residues is presented that aims at maximizing the yield of liquid products (fast pyrolysis). It is based on a technology proven on an industrial scale and especially suitable for treating a straw type of biomass. Engineering Adsorption Device Based on a Langatate Crystal Microbalance for High Temperature High Pressure Gas Adsorption in Zeolite H-ZSM-5 Wenjin Ding1, Giulia Baracchini1, Michael Klumpp2, Wilhelm Schwieger2, Roland Dittmeyer1 1Institute for Micro Process Engineering, Karlsruhe Institute of Technology (KIT), 2Institute of Chemical Reaction Engineering, University Erlangen-Nürnberg (FAU) A protocol for high-temperature and high-pressure gas adsorption measurements on zeolite H-ZSM-5 using an adsorption measurement device based on a langatate crystal microbalance is presented. Prior to the adsorption measurements, the synthesis of zeolite H-ZSM-5 on the langatate crystal microbalance sensor by the steam-assisted crystallization (SAC) method is demonstrated. Biology Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts Johannes F. Buyel1,2, Jürgen Hubbuch3, Rainer Fischer1,2 1Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V., 2Institute for Molecular Biotechnology, RWTH Aachen University, 3Department of Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT) Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 °C. Biology Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes Stefanie Arndt*1, Heidi-Kristin Walter*1, Hans-Achim Wagenknecht1 1Institute of Organic Chemistry, Karlsruhe Institute of Technology Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer. Neuroscience Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons Satoru Yamagishi1, Gandhervin Kesavamoorthy1, Martin Bastmeyer2, Kohji Sato1 1Anatomy and Neuroscience, Hamamatsu University School of Medicine, 2Cell and Neurobiology, Zoological Institute, Karlsruhe Institute of Technology (KIT) Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons. Chemistry Layer-by-layer Synthesis and Transfer of Freestanding Conjugated Microporous Polymer Nanomembranes Peter Lindemann1, Yannick Träutlein1, Christof Wöll1, Manuel Tsotsalas1 1Institute of Functional Interfaces, Karlsruhe Institute of Technology In this paper we describe the interfacial synthesis of conjugated microporous polymers (CMP) on sacrificial substrates, and the dissolution of the substrate for the preparation of freestanding CMP nanomembranes. In addition, we will describe how the fragile nanomembranes can be transferred to other substrates. Neuroscience Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration Rebecca Schmidt1, Tanja Beil1, Uwe Strähle1, Sepand Rastegar1 1Institute of Toxicology and Genetics, Karlsruhe Institute of Technology To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization. Biology A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System Benjamin D. Weger1, Meltem Weger1, Nicole Jung2, Christin Lederer1, Stefan Bräse1,3, Thomas Dickmeis1 1Institute of Toxicology and Genetics, Karlsruhe Institute of Technology - Campus North, 2Institute of Organic Chemistry, Karlsruhe Institute of Technology - Campus North, 3Institute of Organic Chemistry, Karlsruhe Institute of Technology - Campus South We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production. Immunology and Infection Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish Christine Wittmann1, Markus Reischl2, Asmi H. Shah1, Ralf Mikut2, Urban Liebel1,2, Clemens Grabher1 1Institute for Toxicology and Genetics, Karlsruhe Institute of Technology, Karlsruhe, Germany, 2Institute for Applied Informatics, Karlsruhe Institute of Technology, Karlsruhe, Germany Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage. Biology Intracellular Refolding Assay Tamara Vanessa Walther1, Danilo Maddalo1 1Institute of Toxicology and Genetics, Karlsruhe Institute of Technology In this protocol a method to measure intracellular protein refolding after heat shock is described. This method can be used to study foldases like molecular chaperones and their co-factors or compounds able to influence their activity. Firefly luciferase activity is used as reporter to measure chaperone refolding activity. Biology Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors Eric Gottwald1, Brigitte Lahni1, David Thiele1, Stefan Giselbrecht1, Alexander Welle1, Karl-Friedrich Weibezahn1 1Institute for Biological Interfaces, Forschungszentrum Karlsruhe We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop. Biology Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research Karl-Friedrich Weibezahn1 1Institute for Biological Interfaces, Karlsruhe Institute of Technology In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures. Biology Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation Stefan Giselbrecht1, Eric Gottwald1, Roman Truckenmueller2, Christina Trautmann3, Alexander Welle1, Andreas Guber4, Volker Saile4, Thomas Gietzelt5, Karl-Friedrich Weibezahn1 1Institute for Biological Interfaces, Karlsruhe Research Centre, 2Institute for BioMedical Technology, University of Twente, 3Department of Materials Research, Institute for Heavy Ion Research, 4Institute of Microstructure Technology, Karlsruhe Research Centre, 5Institute for Micro Process Engineering, Karlsruhe Research Centre We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.