Performance of cell extract prepared by freeze-thaw and lysozyme
As part of this work, the simple cell lysis methods of freeze-thaw and enzymatic lysis by lysozyme were also assessed for preparing CFPS extract. However, the extracts prepared using these techniques did not produce measurable amounts of protein in CFPS reactions (results not shown). Lysis efficiencies of 99.609% to 99.976% for freeze-thaw and 99.991% for lysozyme were observed. These efficiencies are comparable to the lysis efficiency obtained after 10 min of sonication (99.988%), suggesting insufficient lysis was not the reason for the unviable extract. In contrast to lysis by a high-pressure homogenization or sonication, the freeze-thaw and lysozyme lysis methods do not involve mechanical shearing, which may be important for viable E. coli cell extract (40, 53). However, freeze-thaw lysis has been reported as a viable method to produce insect cell extract for CFPS (8), which suggests that with further engineering such a method could be developed for E. coli cell extract preparation.
In this work, we have sought to simplify the cell extract preparation method using equipment common to biotechnology laboratories, thus eliminating the need for specialized growth and lysis equipment. This was accomplished by performing cell growth in shake flasks and assessing the alternative E. coli disruption techniques of: (i) sonication, (ii) bead vortex mixing, (iii) enzymatic lysis, and (iv) freeze-thaw cycling. Using sonication, we were able to reproducibly produce high-yielding CFPS extract and, in terms of capital equipment cost, sonication is approximately 60% to 90% less expensive than the developed techniques shown in Figure 1. Also, sonication is well suited for laboratories with access to a sonicator but not a bead mill or a high-pressure homogenizer. Another benefit of sonication is that sample volumes as low as 150 µL can be processed, and 96-well plate sonicators are available for high-throughput applications (Supplementary Table S1). Extracts produced by sonication could also be engineered for other cell-free configurations, such as continuous exchange CFPS, which enables extended reaction durations and higher productivities. For researchers without access to a sonicator, bead vortex mixing can also be used for extract preparation, although the CFPS yields obtained through this lysis method are lower than those obtained using sonication or high-pressure homogenization with higher extract-to-extract variability. In conclusion, the combination of shake flask cell culture, sonication or bead vortex mixing cell lysis technique, and streamlined extract preparation protocol described in this work can significantly reduce the time, effort, and capital cost expended in initial proof-of-concept experiments with CFPS. We believe this simple extract preparation technique could become an economically sound milestone for extract preparation and will enable more scientists and engineers to test CFPS for their desired application.
We would like to thank Dr. William G. Pitt (Brigham Young University, UT, USA) for use of his sonicator and Dr. Peter G. Schultz (Scripps Research Institute, CA, USA) for generous gift of pEVOL-pPrF plasmid harbored in the BL21 Star (DE3) strain used in this work. This work was supported by a Brigham Young University Mentoring Environment Grant and the National Science Foundation grant no. 1115229.
The authors declare no competing interests.
Correspondence Address correspondence to Bradley C. Bundy, Department of Chemical Engineering, Brigham Young University, 350S Clyde Building, Provo, UT, USA. Email: [email protected]">[email protected]References
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