Wild type and ∆crcB were grown directly next to each other at varying levels of fluoride. As described in literature, ∆crcB should not grow when exposed to fluoride levels over 500µM. Unexpectedly, ∆crcB did grow on high levels of fluoride when placed on the same plates as wild type E. coli. Without wild type E. coli, no growth of ∆crcB was seen on 1mM and 2mM fluoride.
In order to figure out the cause of the unexpected growth, we reached out to Dr. Randy Stockbridge at University of Michigan for possible explanations.
Dr. Stockbridge informed us of Fluoride’s dependability of pH. A low pH environment is necessary for Fluoride to enter cells. Therefore, we hypothesized that the wild type altered the pH of the agar to a more basic level, facilitating the growth of ∆crcB.
To confirm our hypothesis, we prepared similar set of plates, but with phenol red, a pH indicator, for visual confirmation.
Phenol red appears red at basic pH and turns yellow at acidic pH. In the absence of a buffer, wild type has a visibly basic pH. Therefore, once the entire plate became basic, ∆crcB showed fair amount of growth. On previous days, ∆crcB grew only at red areas (low pH).
To further confirm our results, we prepared the same plates with a MES buffer, which kept the agar at a 6.5 pH, which is slightly acidic.
Upon the addition of the buffer, the alteration of pH was significantly delayed, so F was able to enter the membrane of ∆crcB, allowing the strain to grow.
CHOP (Chloramphenicol Operon) and ∆crcB (E. coli without Fluoride Channel) were added to 50µ chloramphenicol plates and observed over the course of 4 days.
- The plates had increasing concentrations of F with CHOP and ∆crcB to determine the level of fluoride that leads to the greatest growth of CHOP.
- ∆crcB was part of the control and did not grow because it does not have the fluoride channel to pump out the F.
- As observed, there is most growth of CHOP on 100μM of F, suggesting maximum growth of CHOP at 100μM of F.
- Another set of plates with 10-5 and 10-4 dilutions of Chloramphenicol were prepared.
- The plates were divided into quadrants with CHOP on the top left and bottom left quadrants and crcB on the top right and bottom right.
- CHOP had the most growth at 100μM F at 10-4 dilution.
Future Directions
The CHOP System can be used to improve the affinity and characterization of the fluoride riboswitch.
Green Fluorescent Protein (GFP) system may be used for detecting presence of fluoride; at a certain level of fluoride, it can trigger gene expression of the GFP as the fluoride riboswitch releases terminator. This may provide a better visual mechanism for people to determine if their water can be safe to drink or not.
