Streamlined Extract Preparation for Escherichia Coli -Based Cell-Free Protein Synthesis by Sonication or Bead Vortex Mixing

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.

Lyophilized Escherichia Coli -Based Cell-Free Systems for Robust, High-Density, Long-Term Storage

“…Lysis efficiency using a high-pressure homogenizer such as that used for this work has been reported to be 99.9996% (26). Prior to lysis, cell concentrations are approximately 600 billion per mL, thus even at 99.9996%, prepared extracts can have upwards of 2.4 million cells per mL extract.…”

“…E. coli cell extracts (Table 1) were prepared as reported previously in preparation for lyophilization (26). Two lyophilized extracts types were prepared: ( i ) standard extract (xSTD lyo ) and ( ii ) standard extract with sucrose as a potential lyoprotectant (xSUC lyo ).…”

“…Cells were grown and extracts were prepared as previously described (3, 26). All growth stages were performed at 37C in LB media with shaking at 280 rpm.…”

“…Following the reaction, 45 L water was added to each well to dilute the reaction over the bottom surface of the well. Resulting GFP fluorescence was measured using a Synergy Mx microplate reader (BioTek Instruments, Inc., Winooski, VT), and protein yield was determined using a linearly correlated calibration curve, as previously detailed (26). …”

Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression

“…Traditionally, the established preparation procedures require complex protocols, special experience, and expensive equipment for mechanical cell disruption (French press or bead beater) that is not readily available in most laboratories and that limits the throughput in terms of sample size and numbers (reviewed in ref. (14 – 16)). Achieving reproducible results using these methods also requires considerable experience, limiting access to the technology.…”