Guanosine tetraphosphate (ppGpp) is a bacterial signaling molecule involved in activating the stringent response, a cellular reaction to environmental stress that downregulates cell division and metabolism processes to conserve nutrients. The stringent response is implicated in some instances of antibiotic resistance, so broadening the current understanding of ppGpp signaling is useful. This experiment seeks to generate a ppGpp biosensor that will bind ppGpp and emit fluorescent light in its presence which will allow for improved research into the pathways and functions of the signaling molecule. To generate a novel ppGpp biosensor, I converted a biosensor previously used to detect cyclic di-GMP (a different signaling molecule) to contain a binding site transformed to now bind specifically with ppGpp. The genetic sequence for the cyclic di-GMP binding site was replaced with the ppGpp hydrolase domain which has a specific affinity for ppGpp; however, hydrolase activity would provide unwanted breakdown of the ppGpp, so it is mutated further to neutralize hydrolase activity. The desired outcome of this experiment results in a biosensor with an active site that has a specific and sufficient binding affinity for the ppGpp molecule. This sensor is designed to have the active site (ppGpp binding site) flanked by two fluorescent proteins that will interact more closely and transfer fluorescent light. When the first fluorescent protein is activated and the second fluorescent protein is in close proximity, emitted light can be transferred to the second. Binding of ppGpp will cause a conformational shift in the biosensor’s structure, causing the two fluorescent proteins to move further apart. This results in them losing the ability to transfer the fluorescent energy between fluorescent proteins. Using a fluorescent microscope or fluorescent plate reader, I will be able to determine the level of transferred fluorescent energy, and in turn measure the amount of ppGpp in the sample. Having generated the biosensor, I must now determine the ppGpp detection level, intensity of change in fluorescent transfer upon ppGpp binding, and the binding affinity for other nucleotides that might give me an incorrect signal. Using this, we can determine how ppGpp levels are regulated in bacteria under conditions of stress.
| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:asrf-1955 |
| Date | 06 April 2022 |
| Creators | Robinson, Andrew, Robinson, Andrew |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Source | Appalachian Student Research Forum & Jay S. Boland Undergraduate Research Symposium |
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