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.
