Difference between revisions of "Team:East Chapel Hill/results"

Testing for the Effect of Fluoride on CHOP on E. coli growth at different concentrations of Chloramphenicol

In order to test for the concentration of Chloramphenicol that yielded the highest dynamic range for the switch CHOP, CHOP (with grown overnight in the presence of fluoride), and ∆crcB were plated on different concentrations of Chloramphenicol. They were then incubated for several days.

For 5ug/mL of chloramphenicol, both CHOP and delta-crcB grew, while for 125 ug/mL, none of them grew for all three days. However, on 35ug/mL, there was distinct growth between CHOP, CHOP with fluoride, and delta-crcB (which did not grow as it does not have the chloramphenicol resistance gene). This allowed us to determine 35ug/mL as a reasonable concentration of chloramphenicol for future experiments. This means that our CHOP system is able to provide qualitative data of the difference in growth between the presence of fluoride and the absence of fluoride.

Characterizing the Growth of ΔcrcB at Various Levels of Fluoride in the Presence and Absence of the Wild Type E. coli

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 that fluoride can only cross the membrane in acidic conditions as hydrofluoric acid. Therefore, we hypothesized that the wild type altered the pH of the agar to a more basic level, facilitating the growth of ∆crcB.

Testing for the Wild Type’s Effect on pH of LB Agar with Buffers

In order to test the hypothesis that the Wild Type E. coli changes the pH of the agar to allow growth of ΔcrcB, we plated WT and ΔcrcB together, and one without WT. All plates had either no buffer, or 20mM/50mM of MES (pH 6.5, slightly acidic), or 50mM of TRIS at pH 8 (slightly basic). All plates had 500uM of fluoride.

In this experiment, the growth of ΔcrcB depended on the presence of Wild Type. Without the Wild Type, ΔcrcB had very little growth. With Wild Type, ΔcrcB had obvious visual growth by Day 2. With our knowledge that fluoride can only cross the membrane in acidic conditions as hydrofluoric acid, we can infer that the Wild Type is causing the pH to be more basic, preventing fluoride to cross ΔcrcB’s membranes. TRIS buffered plates also showed that at basic pH, ΔcrcB can thrive as fluoride is not entering its membranes. MES buffered plates also showed that at acidic pH, ΔcrcB had slowed growth.

Reconfirming Wild Type’s Effect on PH of LB Agar Using Phenol Red

To confirm our hypothesis from the previous experiment, we prepared similar set of plates, but with the pH indicator phenol red, 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 addition of the buffer, the change in pH was delayed, so fluoride could enter the cells and inhibit the growth of the ∆crcB strain.

Testing for Optimal Fluoride Levels and Dilutions for CHOP Growth

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 because it did not have a Chloramphenicol resistance gene.
• 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.
• High-throughput screening of potential fluoride riboswitches
• Using CHOP, different riboswitches can be screened by having the Chloramphenicol Operon inserted into their genes downstream, where they can be observed over the course of ~4 days on varying concentrations of fluoride to see what concentration they can grow most effectively in - which would correspond to the effectiveness and affinity of the riboswitch.
• CHOP can be used for other transcriptional riboswitches that bind to different ligands or molecules to determine the effectivity and affinity of the riboswitches to them.
• 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.
• A fluorinase gene can be utilized in the fluoride riboswitch to bio-remediate the excess fluoride in water. The excess fluoride in water can be incorporated into the 5’ position of the adenosine sugar in the bacteria’s DNA after the fluorinase enzyme undergoes a chemical reaction.