Browsing by Subject "Aptamers."
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Item Characterization of the metallo-ß-lactamase from Pseudomonas aeruginosa, IMP-1.(2014-06-11) Solida, Nicole R.; Kim, Sung-Kun, 1968-; Chemistry and Biochemistry.; Baylor University. Dept. of Chemistry and Biochemistry.The rate at which pathogenic bacteria are gaining antibiotic resistance has become increasingly alarming. Major contributors of this antibiotic resistance in microbes are a class of enzymes known as β-lactamases. These enzymes are effective in breaking down the most commonly prescribed antibiotics at present. This work investigates two separate metallo-β-lactamase enzymes, first IMP-1, which provides antibiotic resistance to Pseudomonas aeruginosa, and second Bla2, which grants antibiotic resistance to Bacillus anthracis. The main focus of this work was to investigate and characterize IMP-1 according to its activity and stability in temperature and pH. This was done in an effort to increase the general knowledge for potential inhibitors to be designed. The secondary focus of this work was to examine the ability of novel hydroxamate compounds to inhibit the growth of bacterial cells expressing Bla2. In addition to this work, aptamers were investigated as a potential means of future inhibitor design.Item Development of an aptamer-based detection assay against Bacillus anthracis lethal factor and Escherichia coli.(2013-09-24) Lahousse, Mieke.; Kim, Sung-Kun, 1968-; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.The impressive capacity of bacteria to adapt to their environment has led to the development of mechanisms that can convey resistance to currently FDA approved antibiotics. In an attempt to find a solution to the possibility that soon there will be no antibiotic treatment available for bacterial infections, new approaches are being explored. One such approach relies on the effective detection and/or diagnosis of pathogens or unique bacterial proteins to aid in prevention or better treatment of infectious diseases. Within this work we describe the development of ssDNA aptamers by SELEX that can bind to their target with high affinity and can be used to develop colorimetric assays that can positively detect the presence of the target. Our targets for aptamer development were whole E. coli cells, which are currently used in water quality assessment, and a unique protein toxin (lethal factor) produced by B. anthracis, the etiological agent of anthrax. As a result of the SELEX experiments we recovered one aptamer that binds to LF protein with high affinity and we demonstrated that its use in the development of an aptamer-based colorimetric assay for LF was successful. We also demonstrate that the aptamer found for LF protein can act as an inhibitor of the catalytic activity of LF, suggests its potential use as a therapeutic agent. The SELEX experiment using whole E. coli cells generated a diverse number of aptamers with binding affinities in the low µM range. Additionally, studies of aptamer specificity showed that three aptamers had no binding affinity for other coliform strains, making them suitable aptamers for further characterization. This work also describes the characterization of a glutaredoxin protein expressed by Synechocystis sp. involved in arsenate reduction. Elucidation of the mechanism by which cyanobacteria can survive in highly arsenic contaminated environments could enlighten possible remediation approaches. In conclusion, we successfully developed ssDNA aptamers that can bind with high affinity and specificity to its targets with demonstrated potential as detecting agents and contributed to the enrichment of the knowledge available for glutaredoxin GrxA involved in arsenate oxidoreduction by Synechocystis sp.Item Selection of aptamers against live E.coli cells using Cell SELEX.(2011-01-05T19:45:04Z) Shipley, Sheena.; Kim, Sung-Kun, 1968-; Chemistry and Biochemistry.; Baylor University. Dept. of Chemistry and Biochemistry.Aptamers are oligonucleotides that bind with great specificity and affinity to a target molecule. These oligonucleotides are produced through the course of Systemic Evolution of Ligands by Exponential Enrichment (SELEX). SELEX is a combinatorial chemistry technique used to generate a random DNA or RNA library, which is then incubated with a target molecule. The binding aptamers are divided from the nonbinding random pool DNA/aptamers, and then amplified via polymerase chain reaction (PCR). Double stranded DNA molecules have been used to select against purified target molecules; in this study we have developed a selection technique using live Escherichia coli cells as a target and using Bacillus subtilis and Enterobacter aerogenes as negative controls. Aptamer pools obtained from approximately 8 rounds of Cell SELEX demonstrated an affinity for E .coli cells when tested via fluorescence detection.