Team:East Chapel Hill/Demonstrate

Demonstration That Our Part Works!

Our project pertains to measuring concentrations of fluoride and characterizing fluoride riboswitches with higher affinity to fluoride; these technologies can be used to determine methods to sequester, bioremediate, and detect fluoride.
We developed the "Fluoride Riboswitch Regulated Chloramphenicol Acetyltransferase Operon" (CHOP) regulated by the fluoride riboswitch as a system to characterize the fluoride riboswitch.
Since bacteria does not grow in the presence of chloramphenicol, the protein bacteria needs the chloramphenicol acetyltransferase protein in order to grow on chloramphenicol. We plated serial dilutions of CHOP and ΔcrcB, which is our control E. coli without the resistance to chloramphenicol, onto plates with varying concentrations of fluoride in order to test the best level the fluoride that CHOP can stimulate growth.

Our results demonstrated that with no fluoride there only growth of CHOP containing bacteria on chloramphenicol plates at the highest dilutions TOP and no growth when plated at lower density BOTTOM. However, as the fluoride concentration of the plates increased, the growth of CHOP containing bacteria also increased. In other words, CHOP's ability to allow E. coli to grow on chloramphenicol is fluoride dependent. This result demonstrates that the fluoride riboswitch was able to regulate the expression of chloramphenicol acetyltransferase as intended; the concentration with the most growth of CHOP would represent the best activating concentration of fluoride for the riboswitch. Since the growth of bacteria containing the CHOP vector is correlated with the activation of the fluoride riboswitch, CHOP can be used to find other riboswitches that have a better affinity to fluoride, by screening for fluoride-dependent bacterial growth at lower concentrations of fluoride.