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California Polytechnic State University San Luis Obispo

2 ARTICLES PUBLISHED IN JoVE

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. Jewett^4,5,6,7, Katharine R. Watts^2,3, Javin P. Oza^2,3

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.

Bioengineering

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Philip E. J. Smith^1,2, Taylor Slouka^1,2, Mona Dabbas^1,2, Javin P. Oza^1,2

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.