14 articles published in JoVE

• Biochemistry

  
  Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions Markus Götz¹, Philipp Wortmann¹, Sonja Schmid^1,2, Thorsten Hugel^{1 1}Institute of Physical Chemistry, University of Freiburg, ²Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

• Chemistry

  
  Laser-heating and Radiance Spectrometry for the Study of Nuclear Materials in Conditions Simulating a Nuclear Power Plant Accident Dario Manara¹, Luca Soldi^1,2,4, Sara Mastromarino^1,3,5, Kostantinos Boboridis¹, Davide Robba¹, Luka Vlahovic¹, Rudy Konings^{1 1}European Commission, Joint Research Centre, ²Energy Department, Politecnico di Milano, ³Department of Chemical Physics, Sapienza - Università di Roma, ⁴CEA Saclay, ⁵TU Delft We present experiments in which real nuclear fuel, cladding, and containment materials are laser heated to temperatures beyond 3,000 K while their behavior is studied by radiance spectroscopy and thermal analysis. These experiments simulate, on a laboratory scale, the formation of a lava-phase following a nuclear reactor core meltdown.

• Bioengineering

  
  An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage Vahid Arbabi*^1,2, Behdad Pouran*^1,2, Amir. A. Zadpoor¹, Harrie Weinans^{1,2,3 1}Department of Biomechanical Engineering, Faculty of Mechanical, Maritime, and Materials Engineering, Delft University of Technology (TU Delft), ²Department of Orthopedics, UMC Utrecht, ³Department of Rheumatology, UMC Utrecht We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

• Environment

  
  Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge Simon Felz¹, Salah Al-Zuhairy¹, Olav Andreas Aarstad², Mark C.M. van Loosdrecht¹, Yue Mei Lin^{1 1}Department of Biotechnology, Delft University of Technology, ²Department of Biotechnology, Norwegian Biopolymer Laboratory (NOBIPOL), Norwegian University of Science and Technology The protocol provides a methodology to solubilize aerobic granular sludge in order to extract alginate-like extracellular polymers (ALE).

• Biology

  
  Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets Mathijs Vleugel¹, Sophie Roth¹, Celebrity F. Groenendijk¹, Marileen Dogterom^{1 1}Department of Bionanoscience, Delft University of Technology The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

• Chemistry

  
  Light-driven Enzymatic Decarboxylation Katharina Köninger¹, Marius Grote¹, Ioannis Zachos¹, Frank Hollmann², Robert Kourist^{1 1}Faculty of Biology and Biotechnology, Ruhr Universität Bochum, ²Biology and Biotechnology, Delft University of Technology We describe a protocol for the light-catalyzed generation of hydrogen peroxide — a cofactor for oxidative transformations.

• Engineering

  
  Ultrasonic Welding of Thermoplastic Composite Coupons for Mechanical Characterization of Welded Joints through Single Lap Shear Testing Irene F. Villegas¹, Genevieve Palardy^{1 1}Structural Integrity and Composites, Delft University of Technology A straightforward procedure for ultrasonic welding of thermoplastic composite coupons for basic mechanical testing is described. Key characteristics of this ultrasonic welding process are the use of flat energy directors for simplified process preparation and the use of process data for the fast definition of optimum processing conditions.

• Engineering

  
  Fabrication of Low Temperature Carbon Nanotube Vertical Interconnects Compatible with Semiconductor Technology Sten Vollebregt¹, Ryoichi Ishihara^{1 1}Department of Microelectronics, Delft University of Technology A method for the growth of low temperature vertically-aligned carbon nanotubes, and the subsequent fabrication of vertical interconnect electrical test structures using semiconductor fabrication is presented.

• Biology

  
  EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1 Peter-Leon Hagedoorn¹, Laura van der Weel¹, Wilfred R. Hagen^{1 1}Department of Biotechnology, Delft University of Technology The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

• Bioengineering

  
  Magnetic Tweezers for the Measurement of Twist and Torque Jan Lipfert¹, Mina Lee¹, Orkide Ordu¹, Jacob W. J. Kerssemakers¹, Nynke H. Dekker^{1 1}Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology Magnetic tweezers, a powerful single-molecule manipulation technique, can be adapted for the direct measurements of the twist (using a configuration called freely-orbiting magnetic tweezers) and torque (using a configuration termed magnetic torque tweezers) in biological macromolecules. Guidelines for performing such measurements are given, including applications to the study of DNA and associated nucleo-protein filaments.

• Chemistry

  
  Surface Passivation for Single-molecule Protein Studies Stanley D. Chandradoss¹, Anna C. Haagsma¹, Young Kwang Lee², Jae-Ho Hwang², Jwa-Min Nam², Chirlmin Joo^{1 1}Kavli Institute of NanoScience, Department of BioNanoScience, Delft University of Technology, ²Department of Chemistry, Seoul National University We describe a method for passivating a glass surface using polyethylene glycol (PEG). This protocol covers surface cleaning, surface functionalization, and PEG coating. We introduce a new strategy for treating the surface with PEG molecules over two rounds, which yields superior quality of passivation compared to existing methods.

• Environment

  
  Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun Matthew D. Mattozzi^1,2, Mathias J. Voges^1,2,3, Pamela A. Silver^1,2, Jeffrey C. Way^{1,2 1}Synthetic Biology Platform, Wyss Institute for Biologically Inspired Engineering, Harvard University, ²Department of Systems Biology, Harvard Medical School, ³Department of Biotechnology, Delft University of Technology This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

• Biology

  
  Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein Vlad O. Karas¹, Ilja Westerlaken¹, Anne S. Meyer^{1 1}Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.

• Bioengineering

  
  A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments Eva K. Brinkman¹, Kira Schipper¹, Nadine Bongaerts¹, Mathias J. Voges¹, Alessandro Abate², S. Aljoscha Wahl^{1 1}Department of Biotechnology, Delft University of Technology, ²Delft Center for Systems and Control, Delft University of Technology A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via β-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.