Keywords

sonication, vortex mixing, bead mill, Escherichia coli, extracts preparation, cell-free, cell-free protein synthesis, CFPS, in-vitro protein synthesis

Abstract

Escherichia coli-based cell extract is a vital component of inexpensive and high-yielding cell-free protein synthesis reactions. However, effective preparation of E. coli cell extract is limited to high-pressure (French press-style or impinge-style) or bead mill homogenizers, which all require a significant capital investment. Here we report the viability of E. coli cell extract prepared using equipment that is both common to biotechnology laboratories and able to process small volume samples. Specifically, we assessed the low-capital-cost lysis techniques of: (i) sonication, (ii) bead vortex mixing, (iii) freeze-thaw cycling, and (iv) lysozyme incubation to prepare E. coli cell extract for cell-free protein synthesis (CFPS). We also used simple shake flask fermentations with a commercially available E. coli strain. In addition, RNA polymerase was overexpressed in the E. coli cells prior to lysis, thus eliminating the need to add independently purified RNA polymerase to the CFPS reaction. As a result, high-yielding E. coli-based extract was prepared using equipment requiring a reduced capital investment and common to biotechnology laboratories. To our knowledge, this is the first successful prokaryote-based CFPS reaction to be carried out with extract prepared by sonication or bead vortex mixing.

Original Publication Citation

Shrestha, P., Holland, T. M., & Bundy, B. C. (2012). Streamlined Extract Preparation for Escherichia Coli-Based Cell-Free Protein Synthesis by Sonication or Bead Vortex Mixing. BioTechniques, 53(3), 163–174. https://doi.org/10.2144/0000113924

Document Type

Peer-Reviewed Article

Publication Date

2018-04-03

Publisher

BioTechniques

Language

English

College

Ira A. Fulton College of Engineering

Department

Chemical Engineering

University Standing at Time of Publication

Full Professor

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