Simple Extract Preparation Methods for E. coli-Based Cell-Free Expression

Alissa C Mullin; Taylor Slouka; Javin P Oza

doi:10.1007/978-1-0716-1998-8_2

Simple Extract Preparation Methods for E. coli-Based Cell-Free Expression

Methods Mol Biol. 2022:2433:51-64. doi: 10.1007/978-1-0716-1998-8_2.

Authors

Alissa C Mullin¹, Taylor Slouka¹, Javin P Oza²

Affiliations

¹ Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, CA, USA.
² Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, CA, USA. joza@calpoly.edu.

PMID: 34985736
DOI: 10.1007/978-1-0716-1998-8_2

Abstract

Cell-free protein synthesis (CFPS) is a powerful platform for synthetic biology, allowing for the controlled expression of proteins without reliance on living cells. However, the process of producing the cell extract, a key component of cell-free reactions, can be a bottleneck for new users to adopt CFPS as it requires technical knowledge and significant researcher oversight. Here, we provide a detailed method for implementing a simplified cell extract preparation workflow using CFAI media. We also provide a detailed protocol for the alternative, 2x YPTG media-based preparation process, as it represents a useful benchmark within the cell-free community.

Keywords: 2xYTPG; Cell-free autoinduction (CFAI); Cell-free expression (CFE); Cell-free protein synthesis (CFPS); In vitro transcription/translation (TX-TL); Synthetic biology.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Cell-Free System / metabolism
Escherichia coli* / genetics
Escherichia coli* / metabolism
Plant Extracts / metabolism
Plant Extracts / pharmacology
Protein Biosynthesis*
Synthetic Biology / methods

Substances

Plant Extracts