Cavitation enhancement effects on yeast chromatin fragmentation and bacteria spore rupture
Creators: name: Wells, Lindsey
Publisher: name: The University of North Carolina at Chapel Hill University Libraries
Year: 2018

description: Chromatin, the packaging of DNA around proteins, provides essential information about gene expression and gene regulation. DNA is wound tightly around proteins at locations where genes are down regulated and it is wound loosely at locations where genes are actively transcribed. Chromatin assays that look at protein-DNA interactions play a crucial role in studying these chromatin signatures. Saccharomyces cerevisiae (yeast) contain chromosomes and similar cellular pathways to human cells, which make them a valuable tool for these assays. A critical step in these assays is the fragmentation of chromatin using ultrasound; however, current sonication methods are unable to optimize a narrow range of fragment sizes and require a bead-beating step to penetrate the yeast cell wall prior to sonication. Similarly, bacteria spores are difficult to rupture with ultrasound due to their tough spore membrane. Bacillus anthracis, the bacteria responsible for anthrax, is a particular spore-forming bacterium of significance due to the high fatality rate associated with anthrax disease. There is substantial interest in developing a detection kit for these spores. This kit would require rapid isolation of DNA from the spores to detect if a biomarker is present that identifies the pathogen.Nanodroplets are a cavitation-enhancing reagent that has already been shown to improve gDNA shearing when activated by ultrasound. In addition to ultrasound activation of nanodroplets, recently there has been interest in the potential of using laser-generated focused ultrasound (LGFU) for this purpose. We hypothesize that activation of nanodroplets with laser-generated focused ultrasound (LGFU) could improve yeast chromatin fragmentation and increase bacteria spore rupture.To test this hypothesis we first determined if cavitation occurred in samples targeted with LGFU. The results of this study show that the LGFU did not produce efficient cavitation. The current setup for the LGFU should be altered to increase the size of the focal point and to fit a thin-walled tube to confirm if this method shows promise to vaporize nanodroplets. Two laboratory sonicators, the Covaris E110 and the qSonica, were used to determine the effect of the nanodroplet reagent on yeast chromatin fragmentation. The tests done with the yeast model only showed significant application of nanodroplets in the Covaris E110 sonicator to improve chromatin fragmentation for use in downstream applications. Fragmentation in the Covaris happened more quickly and more consistently with the addition of nanodroplets. The Covaris E110 and the qSonica were also used to determine the effect of the nanodroplet reagent on spore rupture. Bacillus subtilis spores were used to model B. anthracis spores because of their similar structure and protein composition. The results with the bacteria model show no significant difference in the amount of spores ruptured with and without the cavitation-enhancing reagent in the presence of ultrasound. Although the results show that sonication with nanodroplets does not provide enough energy to lyse bacteria spores, it is believed that a thermal or chemical treatment prior to sonication could be used to weaken the membrane in order to make the cells more susceptible to lysis during sonication.