Antibiotic resistance has become a major problem in the United States. Approximately 2 million people are affected by hospital-acquired infections. Each year about 90,000 people are killed from them. Of the infections 70% of them are resistant to at least one drug. In order to trigger antibiotic resistance in bacteria, the antibiotics need to be detected by sensors in the bacteria. Riboswitches may act as toxin sensors in bacteria. Riboswitches are RNA aptamers that regulate gene expression via allosteric structural changes triggered by binding of a small molecule. Most identified riboswitches specifically recognize the metabolic product of the gene to be regulated. When the concentration of the metabolite reaches its threshold it binds to the riboswitch causing a structural change that in most cases turns off transcription or translation of the metabolite-producing gene. The ykkCD riboswitch appears to recognize the antibiotic, tetracycline to up-regulate expression of an efflux pump (also called ykkCD) that exports toxic drugs from the bacterial cell. In this work we present initial characterization of the previously uncharacterized ykkCD riboswitch. With the help of tetracycline derivatives and mutagenesis studies on the riboswitch we will (1) determine the substrate specificity of this riboswitch; (2) assess the importance of aromatic character and/or functional groups in antibiotic recognition. To achieve this goal we have developed a fluorescent binding assays. The binding assays will measure the binding affinity (Kd) of the riboswitch-antibiotic complex. Since substrates of the efflux pump are toxic to the bacterial cell, we posit that the ykkCD riboswitch might work as a toxin sensor and could serve as a target in the fight against bacterial pathogens.
Afterwards we will evaluate how the ykkCD riboswitch sensor works in vivo. In order to do this we will have to quantify the amount of protein produced in the presence of tetracycline derivatives and mutant sensors. However quantifying the level of a particular protein in the cell is difficult so instead we replace the sequence of the efflux pump with the B-gal gene in B subtilis cell and quantify B-gal enzymatic activity using a colorimetric assay. This is a widely used technique in which the fluorescence corresponds to how much protein is produced. / Department of Chemistry
| Identifer | oai:union.ndltd.org:BSU/oai:cardinalscholar.bsu.edu:123456789/194923 |
| Date | 06 August 2011 |
| Creators | James, Delores M. |
| Contributors | Fernandez, Timea |
| Source Sets | Ball State University |
| Detected Language | English |
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