Researcher(s)
- Alyssa Perrin, Biological Sciences, University of Delaware
Faculty Mentor(s)
- Jeremy Bird, Biological Sciences, University of Delaware
Abstract
ppGpp, or guanosine tetraphosphate and pentaphosphate, is a signaling molecule that is produced when bacteria is under threat or stressed. Bacteria may produce ppGpp when a cell’s medium is changed, when they are starved of amino acids, and when they are treated with antibiotics. I have used a RNA-based biosensor that detects cellular ppGpp to test the effects of chloramphenicol and kanamycin on ppGpp in E.coli. Chloramphenicol and Kanamycin are both commonly used antibiotics in the lab, and it’s important to know if those antibiotics induce cellular stress without us realizing it. My hypothesis is that I will see more ppGpp in the cell when chloramphenicol or kanamycin is present in the media. To test this, I transformed E. coli with two plasmids. One which expresses a RNA aptamer called Broccoli, which fluoresces when bound to ppGpp, and the other contains a chloramphenicol resistance gene. Therefore, if the cells are stressed in the presence of antibiotics, more ppGpp will be produced, which means we should see more fluorescence. To detect the biosensor and see the cells’ fluorescence, an Axio Observer microscope was used. From what has been seen so far, cells with kanamycin and/or chloramphenicol fluoresced, leading us to believe those antibiotics stress the E.coli cells out. In the future, we hope to quantify the levels of ppGpp and would like to adapt the biosensor expression system, allowing it to work in any E.coli strain. To do that I am cloning the broccoli aptamer into a different plasmid that contains a natural E.coli promoter. This new expression plasmid will allow us to study the effect of ppGpp on NAD capping of RNAs in the future.