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Open AccessProtocol

A Crude Extract Preparation and Optimization from a Genomically Engineered Escherichia coli for the Cell-Free Protein Synthesis System: Practical Laboratory Guideline

1
Department of Biological and Agricultural Engineering, Louisiana State University, Baton Rouge, LA 70803, USA
2
Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Methods Protoc. 2019, 2(3), 68; https://doi.org/10.3390/mps2030068
Received: 2 July 2019 / Revised: 7 August 2019 / Accepted: 7 August 2019 / Published: 9 August 2019
(This article belongs to the Special Issue Cell-Free Synthetic Biology)
PDF [1629 KB, uploaded 9 August 2019]

Abstract

With the advancement of synthetic biology, the cell-free protein synthesis (CFPS) system has been receiving the spotlight as a versatile toolkit for engineering natural and unnatural biological systems. The CFPS system reassembles the materials necessary for transcription and translation and recreates the in vitro protein synthesis environment by escaping a physical living boundary. The cell extract plays an essential role in this in vitro format. Here, we propose a practical protocol and method for Escherichia coli-derived cell extract preparation and optimization, which can be easily applied to both commercially available and genomically engineered E. coli strains. The protocol includes: (1) The preparation step for cell growth and harvest, (2) the thorough step-by-step procedures for E. coli cell extract preparation including the cell wash and lysis, centrifugation, runoff reaction, and dialysis, (3) the preparation for the CFPS reaction components and, (4) the quantification of cell extract and cell-free synthesized protein. We anticipate that the protocol in this research will provide a simple preparation and optimization procedure of a highly active E. coli cell extract.
Keywords: cell-free protein synthesis; E. coli crude extract preparation; genomically engineered E. coli; sonication cell-free protein synthesis; E. coli crude extract preparation; genomically engineered E. coli; sonication
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Kim, J.; Copeland, C.E.; Padumane, S.R.; Kwon, Y.-C. A Crude Extract Preparation and Optimization from a Genomically Engineered Escherichia coli for the Cell-Free Protein Synthesis System: Practical Laboratory Guideline. Methods Protoc. 2019, 2, 68.

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