Troy Holland and Dr. Brad Bundy, Department of Chemical Engineering

My ORCA project proposed to test and optimize various low capital cost cell lysis methods, while leaving the internal cell machinery intact and viable for cell-free protein synthesis research. Our cells of interest were a strain of the bacterium E. coli, and their cellular machinery consists of the chemicals and enzymes they use in their every-day life processes, including (and of particular interest to us) the synthesis of proteins. These proteins have an almost limitless number of potential applications in medicine, food services, and chemical production, so the various methods of protein synthesis are of significant importance, both financially, and for the improvement of quality of human life. Cell-free extract preparation is a preliminary step in cell- free protein synthesis; our methods of extract preparation and optimization are detailed below.

We ran preliminary tests on three methods: i) bead vortexing, ii) freeze-thaw lysis, and iii) sonication. Bead vortexing consists of placing a quantity of tiny glass beads in a tube with a suspension of E. coli cells, and placing the tube on a rapidly rotating vortex machine in order to send the beads through the solution at velocities sufficient to tear the cells apart on impact. Freeze-thaw lysis utilizes the natural action of ice crystals formed upon successive freezing and thawing of a cell suspension, which rupture the cell walls and membranes. The sonication method uses an ultrasonic probe to create pressure waves in solution, which in turn creates micro-bubbles; when these bubbles collapse in the wake of the pressure wave, they create local shock waves, effectively disrupting the cells.

We found that the first method produced results with an unacceptably high degree of variability, the second method gave negligible protein yields, and the final method showed significant promise. We naturally focused our work on the sonication method, which had the most promising results. First, we compiled initial results for presentation at the National Conference of Undergraduate Research held at Weber State University in March 2012, where I gave an oral presentation on our work. We then used these initial results to design and execute further experiments.

After we demonstrated that we could replicate our initial results, we continued our investigation into various influential factors, including length of exposure to ultrasound, cooling time, and cooling method. Various volumes of cell suspension and exposure lengths all showed notable success, and we refined and extended our experiments to be able to conclusively show the consistent trends necessary to demonstrate optimization.

Using both water and cell lysate as a test medium , we demonstrated relatively little temperature increase with sonication (as compared to homogenization, the current standard of extract preparation), and therefore extended our experiments to include exposing the cell suspension to ultrasound for longer periods of time; we also noted that our former method of ice cooling is less effective than using an ice bath (surrounding the test vessel with ice water instead of ice cold air, giving us a much improved heat transfer value ), so we can now optimize cooling rates as well.

Our initial sonication experiments consisted of short exposure times (ranging from 10-90 seconds) followed by short cooling times of roughly the same duration. When we demonstrated that longer exposure times of 10 minutes or more did not exceed our acceptable temperature range, we began a series of tests in the 10 minute range with two minutes of cooling time between bursts of sonication exposure. This resulted in more consistent protein yields, as opposed to our initial, shorter exposure times, which were successful, but had very erratic yields.

These erratic yields continued to some degree due to a flaw in our technique. We wanted to minimize the volume of cell suspension necessary for efficient lysis as part of our optimization, but we found that with very small volumes our sonication probe began to interact with the vessel containing our suspension, resulting in significant heat production, and erratic yields. This was overcome by a relatively crude, but effective apparatus we made to hold the vessel absolutely still in the ice water solution, allowing for more precise positioning of the probe relative to the vessel walls.

We were ultimately successful in obtaining consistent yields of our test protein, at a level comparable with homogenization. Our method allows for the rapid preparation of cell extract samples with a capital cost a fraction of the cost of a homogenizer. The compiled results were published in the September 2012 issue of BioTechniques.

Further research could include experimentation with the effect of the amplitude or frequency of the ultrasound on protein yields or sonication times, as our entire battery of experiments were run at a fixed amplitude and frequency. We would also want to more thoroughly investigate the effectiveness of other methods mentioned (freeze-thaw and bead mill) to more conclusively demonstrate that they are not viable (or under what conditions they become viable), as they could represent further decreases in time and capital equipment costs.