California Institute of Technology View Institution's Website 32 articles published in JoVE Biology Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates Shawn R. Yoshida1,2, Shasha Chong1 1Division of Chemistry and Chemical Engineering, California Institute of Technology, 2Division of Biology and Biological Engineering, California Institute of Technology Many intrinsically disordered proteins have been shown to participate in the formation of highly dynamic biomolecular condensates, a behavior important for numerous cellular processes. Here, we present a single-molecule imaging-based method for quantifying the dynamics by which proteins interact with each other in biomolecular condensates in live cells. Biology Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish Babita Sharma*1,2, Yogaspoorthi J. Subramaniam*1,2, Desingu Ayyappa Raja1,2,3, Ayush Aggarwal1,2, Sridhar Sivasubbu1, Vivek T. Natarajan1,2 1CSIR-Institute of Genomics and Integrative Biology, 2Academy of Scientific and Innovative Research, 3Division of Biology and Biological Engineering, California Institute of Technology Regulators of melanocyte functions govern visible differences in the pigmentation outcome. Deciphering the molecular function of the candidate pigmentation gene poses a challenge. Herein, we demonstrate the use of a zebrafish model system to identify candidates and classify them into regulators of melanin content and melanocyte number. Bioengineering Rapid Characterization of Genetic Parts with Cell-Free Systems John B. McManus1,2, Casey B. Bernhards3,4, Caitlin E. Sharpes3,4, David C. Garcia2,4, Stephanie D. Cole4, Richard M. Murray2, Peter A. Emanuel4, Matthew W. Lux4 1US Army Combat Capabilities Development Command Army Research Laboratory, 2Biology and Biological Engineering, California Institute of Technology, 3Excet, Inc., 4US Army Combat Capabilities Development Command Chemical Biological Center Well-characterized genetic parts are necessary for the design of novel genetic circuits. Here we describe a cost-effective, high-throughput method for rapidly characterizing genetic parts. Our method reduces cost and time by combining cell-free lysates, linear DNA to avoid cloning, and acoustic liquid handling to increase throughput and reduce reaction volumes. Environment Simulation of Early Earth Hydrothermal Chimneys in a Thermal Gradient Environment Ninos Hermis1, Gabriel LeBlanc2, Laura M. Barge1 1NASA Jet Propulsion Laboratory, California Institute of Technology, 2The University of Tulsa The goal of this protocol is to form simulated hydrothermal chimneys via chemical garden injection experiments and introduce a thermal gradient across the inorganic precipitate membrane, using a 3D printable condenser that can be reproduced for educational purposes. Neuroscience WheelCon: A Wheel Control-Based Gaming Platform for Studying Human Sensorimotor Control Quanying Liu1,2,3, Yorie Nakahira2, Zhichao Liang1, Ahkeel Mohideen2, Adam Dai2, Sung Hoon Choi2, Angelina Pan2, Dimitar M. Ho2, John C. Doyle2 1Department of Biomedical Engineering, Southern University of Science and Technology, 2Division of Engineering and Applied Science, California Institute of Technology, 3Neuroscience Center, Huntington Medical Research Institutes WheelCon is a novel, free and open-source platform to design video games that noninvasively simulates mountain biking down a steep, twisting, bumpy trail. It contains components presenting in human sensorimotor control (delay, quantization, noise, disturbance, and multiple feedback loops) and allows researchers to study the layered architecture in sensorimotor control. Engineering Surface Mapping of Earth-like Exoplanets using Single Point Light Curves Siteng Fan1, Yuk L. Yung1 1Division of Geological and Planetary Sciences, California Institute of Technology The protocol extracts information from light curves of exoplanets and constructs their surface maps. It uses light curves of Earth, which serves as a proxy exoplanet, to demonstrate the approach. Developmental Biology Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis Eileen F. Ablondi1, Sudip Paudel2, Morgan Sehdev3, John P. Marken4, Andrew D. Halleran4, Atiqur Rahman5, Peter Kemper5, Margaret S. Saha2 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, 2Department of Biology, College of William and Mary, 3Harvard Medical School, Harvard University, 4Department of Bioengineering, California Institute of Technology, 5Department of Computer Science, College of William and Mary We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level. Chemistry Experimental Methods for Efficient Solar Hydrogen Production in Microgravity Environment Katharina Brinkert1,2, Ömer Akay3, Matthias H. Richter1,4, Janine Liedtke2, Katherine T. Fountaine5,6, Hans-Joachim Lewerenz7, Michael Giersig3,8 1Division of Chemistry and Chemical Engineering, California Institute of Technology, 2European Space Agency/ ESTEC, 3Department of Physics, Freie Universitat Berlin, 4Applied Physics and Sensors, Brandenburg University of Technology Cottbus, 5Resnick Sustainability Institute, California Institute of Technology, 6NG Next, Northrop Grumman Corporation, 7Division of Engineering and Applied Science and Joint Center for Artificial Photosynthesis, California Institute of Technology, 8International Academy of Optoelectronics at Zhaoqing, South China Normal University Efficient solar-hydrogen production has recently been realized on functionalized semiconductor-electrocatalyst systems in a photoelectrochemical half-cell in microgravity environment at the Bremen Drop Tower. Here, we report the experimental procedures for manufacturing the semiconductor-electrocatalyst device, details of the experimental set-up in the drop capsule and the experimental sequence during free fall. Behavior Simultaneous Eye Tracking and Single-Neuron Recordings in Human Epilepsy Patients Shuo Wang1, Nand Chandravadia2, Adam N. Mamelak2, Ueli Rutishauser2,3,4 1Department of Chemical and Biomedical Engineering, and Rockefeller Neuroscience Institute, West Virginia University, 2Departments of Neurosurgery and Neurology, Cedars-Sinai Medical Center, 3Center for Neural Science and Medicine, Department of Biomedical Sciences, Cedars-Sinai Medical Center, 4Division of Biology and Biological Engineering, California Institute of Technology We describe a method to conduct single-neuron recordings with simultaneous eye tracking in humans. We demonstrate the utility of this method and illustrate how we used this approach to obtain neurons in the human medial temporal lobe that encode targets of a visual search. Chemistry Efficient Synthesis of All-Carbon Quaternary Centers via the Conjugate Addition of Functionalized Monoorganozinc Bromides Tyler J. Fulton1, Katherine M. Townsend2, Michael R. Krout2 1Warren and Katharine Schlinger Laboratory for Chemistry and Chemical Engineering, California Institute of Technology, 2Department of Chemistry, Bucknell University A simple and practical protocol for the efficient conjugate addition of functionalized monoorganozinc bromides to cyclic α,β-unsaturated carbonyls to furnish all-carbon quaternary centers was developed. Bioengineering Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM) Manuel Bedrossian1, Casey Barr2, Chris A. Lindensmith3, Kenneth Nealson2, Jay L. Nadeau1 1Department of Medical Engineering, California Institute of Technology, 2Department of Earth Sciences, University of Southern California, 3Jet Propulsion Laboratory, California Institute of Technology Digital holographic microscopy (DHM) is a volumetric technique that allows imaging samples 50-100X thicker than brightfield microscopy at comparable resolution, with focusing performed post-processing. Here DHM is used for identifying, counting, and tracking microorganisms at very low densities and compared with optical density measurements, plate count, and direct count. Behavior Automated Analysis of a Nematode Population-based Chemosensory Preference Assay Cynthia M. Chai1,2, Christopher J. Cronin1,2, Paul W. Sternberg1,2 1Division of Biology and Bioengineering, California Institute of Technology, 2Howard Hughes Medical Institute, California Institute of Technology We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage. Developmental Biology Live Confocal Imaging of Developing Arabidopsis Flowers Nathanaël Prunet1 1Department of Biology and Biological Engineering, California Institute of Technology Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers. Neuroscience Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression Beverley M. Henley1, Bruce N. Cohen1, Charlene H. Kim1, Heather D. Gold1, Rahul Srinivasan1, Sheri McKinney1, Purnima Deshpande1, Henry A. Lester1 1Division of Biology and Biological Engineering, California Institute of Technology (Caltech) In Parkinson's Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq. Developmental Biology In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas Jacob R. Tremblay*1,2, Jeanne M. LeBon*1, Angela Luo1, Janine C. Quijano1, Lena Wedeken1, Kevin Jou1, Arthur D. Riggs1, David A. Tirrell3, H. Teresa Ku1 1Diabetes and Metabolism Research Institute, Beckman Research Institute of City of Hope, 2Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, 3Division of Chemistry and Chemical Engineering, California Institute of Technology In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described. Immunology and Infection Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic Poly(I:C) Ke-Huan Chow*1, Zihao Yan*1,2, Wei-Li Wu1 1Division of Biology and Biological Engineering, California Institute of Technology, 2Harvard Medical School Maternal immune activation (MIA) is a model for an environmental risk factor of autism and schizophrenia. The goal of this article is to provide a step-by-step procedure of how to induce MIA in the pregnant mice in order to enhance the reproducibility of this model. Bioengineering Label-free Single Molecule Detection Using Microtoroid Optical Resonators Judith Su1 1Division of Biology and Biological Engineering, California Institute of Technology We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented. Chemistry Chemical Gardens as Flow-through Reactors Simulating Natural Hydrothermal Systems Laura M. Barge1,2,3, Yeghegis Abedian1,2, Ivria J. Doloboff1,2, Jessica E. Nuñez1,3,4, Michael J. Russell1,2, Richard D. Kidd1, Isik Kanik1,2 1NASA Jet Propulsion Laboratory, California Institute of Technology, 2NASA Astrobiology Institute, Icy Worlds, 3Blue Marble Space Institute of Science, 4Citrus College We describe chemical garden formation via injection experiments that allow for laboratory simulations of natural chemical garden systems that form at submarine hydrothermal vents. Bioengineering An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles Grace Y. Tang1, Melanie A. Pribisko1, Ryan K. Henning1, Punnajit Lim2, John Termini2, Harry B. Gray1, Robert H. Grubbs1 1Division of Chemistry and Chemical Engineering, California Institute of Technology, 2Department of Molecular Medicine, Beckman Research Institute of the City of Hope Gallium(III) 5,10,15-(tris)pentafluorophenylcorrole and its freebase analogue exhibit low micromolar cell cytotoxicity. This manuscript describes an RNA transcription reaction, imaging RNA with an ethidium bromide-stained gel, and quantifying RNA with UV-Vis spectroscopy, in order to assess transcription inhibition by corroles and demonstrates a straightforward method of evaluating anticancer candidate properties. Biology Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters R. Scott McIsaac*1,3, Benjamin L. Oakes*1, David Botstein1,2, Marcus B. Noyes1 1The Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Department of Molecular Biology, Princeton University, 3Division of Chemistry and Chemical Engineering, California Institute of Technology This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters and quantification of the ATFs ability to stimulate GFP expression via flow cytometry. Neuroscience Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition Daniel A. Lee1,2, Juan Salvatierra2, Esteban Velarde3, John Wong3, Eric C. Ford4, Seth Blackshaw2,5 1Division of Biology, California Institute of Technology, 2Solomon H. Snyder Department of Neuroscience, Neurology, and Ophthalamology, Johns Hopkins University School of Medicine, 3Department of Radiation Oncology & Molecular Radiation Sciences, Johns Hopkins University School of Medicine, 4Department of Radiation Oncology, University Of Washington Medical Center, 5Institute for Cell Engineering and High-Throughput Biology Center, Johns Hopkins University School of Medicine The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis. Biology Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology Zachary Z. Sun*1, Clarmyra A. Hayes*2, Jonghyeon Shin3, Filippo Caschera4, Richard M. Murray2, Vincent Noireaux4 1Department of Biology, California Institute of Technology, 2Department of Bioengineering, California Institute of Technology, 3Synthetic Biology Center, Department of Bioengineering, Massachusetts Institute of Technology, 4School of Physics and Astronomy, University of Minnesota This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data. Engineering Dry Oxidation and Vacuum Annealing Treatments for Tuning the Wetting Properties of Carbon Nanotube Arrays Adrianus Indrat Aria1, Morteza Gharib1 1Graduate Aeronautical Laboratories, California Institute of Technology This article describes a simple method to fabricate vertically aligned carbon nanotube arrays by CVD and to subsequently tune their wetting properties by exposing them to vacuum annealing or dry oxidation treatment. Engineering Bringing the Visible Universe into Focus with Robo-AO Christoph Baranec1,2, Reed Riddle1, Nicholas M. Law3, A.N. Ramaprakash4, Shriharsh P. Tendulkar2, Khanh Bui1, Mahesh P. Burse4, Pravin Chordia4, Hillol K. Das4, Jack T.C. Davis1, Richard G. Dekany1, Mansi M. Kasliwal5, Shrinivas R. Kulkarni1,2, Timothy D. Morton2, Eran O. Ofek6, Sujit Punnadi4 1Caltech Optical Observatories, California Institute of Technology, 2Department of Astronomy, California Institute of Technology, 3Dunlap Institute for Astronomy and Astrophysics, University of Toronto, 4Inter-University Centre for Astronomy & Astrophysics, 5Observatories of the Carnegie Institution for Science, 6Benoziyo Center for Astrophysics, Weizmann Institute of Science Light from astronomical objects must travel through the earth's turbulent atmosphere before it can be imaged by ground-based telescopes. To enable direct imaging at maximum theoretical angular resolution, advanced techniques such as those employed by the Robo-AO adaptive-optics system must be used. Bioengineering Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus (SCUVA) Kakani Katija1, Sean P. Colin2,3, John H. Costello3,4, John O. Dabiri5 1Applied Ocean Physics and Engineering, Woods Hole Oceanographic Institution, 2Environmental Science and Marine Biology, Roger Williams University, 3Marine Biology Laboratory, Whitman Center, 4Department of Biology, Providence College, 5Departments of Aeronautics and Bioengineering, California Institute of Technology This protocol provides instructions on how to use a self-contained underwater velocimetry apparatus (SCUVA), which is designed for quantification of in situ animal-generated flows. In addition, this protocol addresses challenges posed by field conditions, and includes operator motion, predicting position of animals, and orientation of SCUVA. Biology High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli Mary F. Farrow1, Frances H. Arnold2 1Department Of Chemistry and Chemical Engineering, California Institute of Technology, 2Chemistry and Chemical Engineering, California Institute of Technology We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants. Neuroscience Assessment of Motor Balance and Coordination in Mice using the Balance Beam Tinh N. Luong*1, Holly J. Carlisle*1, Amber Southwell1, Paul H. Patterson1 1Department of Biology, California Institute of Technology Deficits in fine motor coordination can be assessed with the balance beam test. Performance on the beam is quantified by the speed at which the beam is traversed and the number of times the mouse slips on the beam. Biology Electron Cryotomography of Bacterial Cells Songye Chen1, Alasdair McDowall1,2, Megan J. Dobro1, Ariane Briegel1,2, Mark Ladinsky1,2, Jian Shi2, Elitza I. Tocheva1, Morgan Beeby1,2, Martin Pilhofer1,2, H. Jane Ding1, Zhuo Li1,2, Lu Gan1, Dylan M. Morris1, Grant J. Jensen1,2 1Division of Biology, California Institute of Technology - Caltech, 2Howard Hughes Medical Institute, California Institute of Technology - Caltech We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution. Biology Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining Hyung-Kook (Peter) Lee1, Ashley P. Wright1, Kai Zinn1 1Division of Biology, California Institute of Technology We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo. Biology Extracting DNA from the Gut Microbes of the Termite (Zootermopsis Angusticollis) and Visualizing Gut Microbes Eric Matson1, Elizabeth Ottesen1, Jared Leadbetter1 1Department of Environmental Science and Engineering, California Institute of Technology - Caltech This video illustrates the technique for extracting DNA from the species of microbes resident in the termite hindgut. The preparation of a wet mount slide, which is useful for visualizing the gut microbial community is also illustrated, and a tour through the species-rich gut environment is given. Biology Investigating the Microbial Community in the Termite Hindgut - Interview Jared Leadbetter1 1Department of Environmental Science and Engineering, California Institute of Technology - Caltech Jared Leadbetter explains why the termite-gut microbial community is an excellent system for studying the complex interactions between microbes. The symbiotic relationship existing between the host insect and lignocellulose-degrading gut microbes is explained, as well as the industrial uses of these microbes for degrading plant biomass and generating biofuels. Biology Layers of Symbiosis - Visualizing the Termite Hindgut Microbial Community Jared Leadbetter1 1Department of Environmental Science and Engineering, California Institute of Technology - Caltech Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth. Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.
