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University of Tennessee, Knoxville

8 ARTICLES PUBLISHED IN JoVE

Bioengineering

Bacterial Immobilization for Imaging by Atomic Force Microscopy

David P. Allison^1,2, Claretta J. Sullivan³, Ninell Pollas Mortensen^1,2, Scott T. Retterer^1,4, Mitchel Doktycz^1,4

¹Biological and Nanoscale Systems Group, Biosciences Division, Oak Ridge National Laboratory, ²Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee , ³Department of Surgery, Eastern Virginia Medical School, ⁴Center for Nanophase Materials Sciences Division, Oak Ridge National Laboratory

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

Engineering

Fabrication of Spatially Confined Complex Oxides

Hangwen Guo^1,2, Thomas Z. Ward¹

¹Materials Science and Technology Division, Oak Ridge National Laboratory, ²Department of Physics & Astronomy, University of Tennessee, Knoxville

We describe the use of pulsed laser deposition (PLD), photolithography and wire-bonding techniques to create micrometer scale complex oxides devices. The PLD is utilized to grow epitaxial thin films. Photolithography and wire-bonding techniques are introduced to create practical devices for measurement purposes.

Biology

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Krzysztof Bobik¹, John R. Dunlap², Tessa M. Burch-Smith¹

¹Department of Biochemical, Cellular and Molecular Biology, University of Tennessee, Knoxville, ²Advanced Microscopy and Imaging Facility, University of Tennessee, Knoxville

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

Bioengineering

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Sarah Elizabeth Norred^1,2, Patrick M. Caveney^1,2, Scott T. Retterer^1,2, Jonathan B. Boreyko^1,2, Jason D. Fowlkes^2,3, Charles Patrick Collier², Michael L. Simpson^1,2,3

¹Bredesen Center, University of Tennessee, Knoxville, ²Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, ³Department of Materials Science and Engineering, University of Tennessee, Knoxville

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

Genetics

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Elizabeth M. Dlugosz¹, Scott C. Lenaghan^2,3, C. Neal Stewart, Jr.¹

¹Department of Plant Sciences, University of Tennessee, Knoxville, ²Center for Renewable Carbon, University of Tennessee, Knoxville, ³Department of Mechanical, Aerospace, and Biomedical Engineering, University of Tennessee, Knoxville

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

JoVE Journal

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

Andrea C. Timm¹, Michelle C. Halsted², Jared L. Wilmoth¹, Scott T. Retterer^1,3

¹Biosciences Division, Oak Ridge National Laboratory, ²Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, ³Center for Nanophase Materials Sciences, Oak Ridge National Laboratory

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

Bioengineering

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Jayde A. Aufrecht^1,2, Jennifer M. Ryan³, Sahar Hasim⁴, David P. Allison^2,3, Andreas Nebenführ³, Mitchel J. Doktycz^1,2, Scott T. Retterer^1,2

¹Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, ²Bioscience Division and Center for Nanophase Materials Sciences, Oak Ridge national Laboratory, ³Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, ⁴Department of Microbiology, University of Tennessee

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

Chemistry

Monitoring the Effects of Illumination on the Structure of Conjugated Polymer Gels Using Neutron Scattering

Brian Morgan¹, Samantha J. Rinehart¹, Mark D. Dadmun^1,2

¹Department of Chemistry, University of Tennessee, Knoxville, ²Chemical Sciences Division, Oak Ridge National Laboratory

A protocol for the analysis of gels formed from the optoelectronic conjugated polymer poly(3-hexylthiophene-2,5-diyl) (P3HT) using small and ultra-small angle neutron scattering in both the presence and absence of illumination is presented.