Team:East Chapel Hill/Results

Testing for the Effect of Fluoride on CHOP on E. coli Growth at Different Concentrations of Chloramphenicol

June 23rd, 2017

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 differential growth of CHOP on plates with and without fluoride. This allowed us to determine 35ug/mL as a reasonable concentration of chloramphenicol for future experiments.

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

July 22nd, 2017

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

July 22nd, 2017

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 1mM fluoride.

In this experiment, we show the role of pH contributes to fluoride toxicity. When we grew ΔcrcB on pH 8 in 1mM fluoride we saw robust growth regardless of the presence of Wild Type because fluoride is not entering the bacteria. On non-buffered plates our results mirrored our previous results, ΔcrcB can grow on high concentrations of Fluoride only after the Wild-Type grows. When we buffered the plates with MES pH 6.5 we observed even more delayed growth of ΔcrcB compared to the unbuffered plates when grown next to Wild-Type, and this delay was dependent on the concentration of buffer. This results show the the Wild-Type are increasing the pH of the plate as they grow, thereby providing protection to the ΔcrcB on toxic concentrations of Fluoride.

A Colormetric Assay Using Phenol Red to Confirm Wild Type’s Effect on pH

July 29th, 2017

To confirm our hypothesis that Wild Type E. coli are increasing the pH of the agar plates, we prepared a 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 is able to alter the pH of the plate visibly turning it red. Accordingly, once the region of the plate where ∆crcB is plated turns red, the E. coli can grow.

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

July 31st, 2017

CHOP and ∆crcB (E. coli with the Fluoride Channel deleted) were added to 50µg/mL 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 screen for fluoride riboswitches with higher responsiveness or select new fluoride riboswitches from engineered libraries.
    • High-throughput screening of potential fluoride riboswitches
    • Using CHOP, putative fluoride riboswitches can be characterized by inserting new promoter riboswitch pairs into CHOP and using serial dilutions on plates with varying levels of fluoride to determine the responsiveness to fluoride.
    • CHOP can be used to screen many thousands of riboswitches at the same time, by plating bacteria on plates with various levels of fluoride and sequencing clones that survive. This approach could also be used for libraries
    • CHOP can be used to screen other transcriptional riboswitches to help identify which ligands bind and determine the responsiveness.
  • In the future we hope to use fluoride riboswitch technologies for sequestration, bioremediation, and detection of fluoride
    • To sequester fluoride we hope to identify fluoride riboswitches with a higher affinity to fluoride and attach multiple copies of the riboswitches to nanoparticles
    • To bioremediate fluoride we hope to control enyzmes that metabolize fluoride (fluorinase) with the fluoride riboswitch
    • To detect fluoride we hope to identify a set of fluoride riboswitches with different responsiveness to fluoride. These riboswitches will regulate a reporter, like a fluorescent protein of luciferase, so there will be a visible signal if fluoride is present. This will allow people to determine the concentration of fluoride in drinking water