California Polytechnic State University View Institution's Website 6 articles published in JoVE Bioengineering From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow Philip E. J. Smith1,2, Taylor Slouka1,2, Mona Dabbas1,2, Javin P. Oza1,2 1Department of Chemistry and Biochemistry, California Polytechnic State University San Luis Obispo, 2Center for Application in Biotechnology, California Polytechnic State University San Luis Obispo This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained. Immunology and Infection Determination of Immune Cell Identity and Purity Using Epigenetic-Based Quantitative PCR Suman K. Pradhan1, Jerry Guzman1, Carl Dargitz1, Stephanie Switalski2, Mark Landon1, Sven Olek3, Bjoern Samans3, Ulrich Hoffmueler3, Uma Lakshmipathy1 1Cell Biology, Life Sciences Solutions, Thermo Fisher Scientific, 2California Polytechnic State University (Cal Poly), 3Epiontis GmbH, Precision for Medicine Here we describe a robust method of determining immune cell identity and purity through epigenetic signatures detected using quantitative PCR (qPCR). DNA demethylation at a specific locus serves as a unique identifier for a particular cell type and allows for identification of CD8+, regulatory, or Th17 T cells. Chemistry Microwave-Assisted Preparation of 1-Aryl-1H-pyrazole-5-amines Jarvis Law*1, Aashrita Manjunath*1, Ryan Schioldager*1, Scott Eagon1 1Department of Chemistry and Biochemistry, California Polytechnic State University 1-Aryl-1H-pyrazole-5-amines are prepared from aryl hydrazines combined with either 3-aminocrotononitrile or an α-cyanoketone in a 1 M HCl solution using a microwave reactor. Most reactions are done in 10-15 minutes and pure product can be obtained via vacuum filtration with typical isolated yields of 70-90%. Chemistry Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology Max Z. Levine*1,2, Nicole E. Gregorio*2,3, Michael C. Jewett4,5,6,7, Katharine R. Watts2,3, Javin P. Oza2,3 1Department of Biological Sciences, California Polytechnic State University, San Luis Obispo, 2Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo, 3Department of Chemistry and Biochemistry, California Polytechnic State University, 4Department of Chemical and Biological Engineering, Northwestern University, 5Chemistry of Life Processes Institute, Northwestern University, 6Center for Synthetic Biology, Northwestern University, 7Interdisciplinary Biological Sciences Program, Northwestern University This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized. Immunology and Infection Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species Marie Yeung1, Trevor Thorsen1 1Biological Sciences Department, California Polytechnic State University Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium. Medicine Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling Alexander Michael Guendel*1, Kyle S. Martin*1, Joshua Cutts2, Patricia L. Foley3, Alexander M. Bailey1, Feilim Mac Gabhann4, Trevor R. Cardinal2, Shayn M. Peirce1 1Department of Biomedical Engineering, University of Virginia, 2Department of Biomedical Engineering, California Polytechnic State University, 3Office of Animal Welfare, University of Virginia, 4Department of Biomedical Engineering & Institute for Computational Medicine, Johns Hopkins University We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.