Browsing by Subject "Vibrio cholerae"
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Item A comparison of Lyse-It to other cellular sample preparation, bacterial lysing, and DNA fragmentation technologies(PLOS, 2019-07-23) Santaus, Tonya M.; Li, Shan; Saha, Lahari; Chen, Wilbur H.; Bhagat, Siya; Stine, O. Colin; Geddes, Chris D.The ability for safe and rapid pathogenic sample transportation and subsequent detection is an increasing challenge throughout the world. Herein, we describe and use bead-beating, vortex, sonication, 903 protein saver cards, and Lyse-It methods, aiming to inactivate Grampositive and -negative bacteria with subsequent genome DNA (quantitative Polymerase Chain Reaction) qPCR detection. The basic concepts behind the four chosen technologies is their versatility, cost, and ease of use in developed and underdeveloped countries. The four methods target the testing of bacterial resilience, cellular extraction from general and complex media and subsequent DNA extraction for qPCR detection and amplification. These results demonstrate that conventional high temperature heating, 903 protein saver cards, and Lyse-It are all viable options for inactivating bacterial growth for safe shipping. Additionally, Lyse-It was found to be particularly useful as this technology can inactivate bacteria, extract cells from 903 protein saver cards, lyse bacterial cells, and additionally keep genomic DNA viable for qPCR detection.Item Effects of Lyse-It on endonuclease fragmentation, function and activity(PLOS, 2019-09-30) Santaus, Tonya M.; Zhang, Fan; Li, Shan; Stine, O. Colin; Geddes, Chris D.Nucleases are enzymes that can degrade genomic DNA and RNA that decrease the accuracy of quantitative measures of those nucleic acids. Here, we study conventional heating, standard microwave irradiation, and Lyse-It, a microwave-based lysing technology, for the potential to fragment and inactivate DNA and RNA endonucleases. Lyse-It employs the use of highly focused microwave irradiation to the sample ultimately fragmenting and inactivating RNase A, RNase B, and DNase I. Nuclease size and fragmentation were determined visually and quantitatively by SDS polyacrylamide gel electrophoresis and the mini-gel Agilent 2100 Bioanalyzer system, with a weighted size calculated to depict the wide range of nuclease fragmentation. Enzyme activity assays were conducted, and the rates were calculated to determine the effect of various lysing conditions on each of the nucleases. The results shown in this paper clearly demonstrate that Lyse-It is a rapid and highly efficient way to degrade and inactivate nucleases so that nucleic acids can be retained for down-stream detection.Item Focused Microwaves for Cellular Lysing, DNA/RNA and Protein/Enzyme Fragmentation and Inactivation(2019-01-01) Santaus, Tonya; Geddes, Christopher D; Chemistry & Biochemistry; ChemistryRapid sample preparation is one of the main hindrances to infectious bacterial identification. To aid in rapid and efficient sample preparation, gold microwave lysing triangles (MLTs) was developed. Gold MLTs serve as a platform to focus microwaves directly to the center of a sample subsequently increasing the electromagnetic energy and temperature. This increase in energy and temperature allows for rapid cellular lysis followed by DNA/RNA fragmentation as well as protein/enzyme degradation and substantially lower activity. The first part of this research explores alterations to gold MLTs to ultimately lower the cost of the technology. Various alterations were investigated including but not limited to � the metal that was used, the microwave focusing geometry, and the gap size (distance) between the geometries. Additionally, standard microwave conditions were determined for Gram-positive and �negative bacteria, in particular L. monocytogenes and V. cholerae. The second part of this research was devoted to the investigation of gold MLTs as compared to other methods like vortex, sonication, and bead-beating as a platform for inactivating infectious pathogens. It was found that by utilizing gold MLTs, pathogenic bacteria was rendered inactive and thus safe for domestic and international shipping. The third and most compelling part of this work was dedicated to the elucidation of the non-thermal mechanism behind the gold MLTs. It was first noticed that DNA/RNA and proteins were being extracted at low microwave settings and subsequently being degraded at higher settings. Further investigation led to the discovery that as microwave conditions were held constant and thus temperature was constant, that genomic DNA and proteins were being degraded as oxygen content increased. This led to investigating reactive oxygen species (ROS). Specific fluorescent ROS probes were utilized to detect singlet oxygen, hydroxyl radicals, and superoxide anion radicals. It was observed that as oxygen concentration increased or microwave irradiation settings increased, there were more ROS generated with the use of gold MLTs; therefore, ROS serves as one part of the non-thermal mechanism. Finally, microwave effects on DNA/RNA nucleases structure and activity was investigated. It was found that with the use of gold MLTs, nucleases can be rendered inactive while still allowing for intracellular component detection.Item Regulation of Ribosomal Protein Synthesis in Vibrio cholerae(American Society for Microbiology, 2004-09) Allen, Todd D.; Watkins, Tonya; Lindahl, Lasse; Zengel, Janice M.We have investigated the regulation of the S10 and spc ribosomal protein (r-protein) operons in Vibrio cholerae. Both operons are under autogenous control; they are mediated by r-proteins L4 and S8, respectively. Our results suggest that Escherichia coli-like strategies for regulating r-protein synthesis extend beyond the enteric members of the gamma subdivision of proteobacteria.