Team:East Chapel Hill/Measurement


Using CHOP to measure the responsiveness of fluoride riboswitches

The fluoride riboswitch regulated chloramphenicol acetyltransferase operon (CHOP) can be used to measure the responsiveness of fluoride riboswitches to fluoride. This can be determined by growing ΔcrcB E. coli transformed with CHOP on chloramphenicol plates with vary amounts of fluoride. In order to use the system, one must clone an E. coli promoter and their fluoride riboswitch of interest into the CHOP system with gibson (see design for more details). Next the user needs to test for the optimal chloramphenicol concentration to use for the switch. This is the concentration of chloramphenicol that show the biggest difference in E. coli growth between plates with fluoride and plates without fluoride. For the B. cereus fluoride riboswitch we found that 35μg/mL had the best dynamic range between plates with no fluoride and plates with fluoride (Figure 1).

Figure 1) Testing for the optimal dynamic range of the B. cereus fluoride riboswitch.

Once an optimal concentration of fluoride is found the next step is growing serial dilutions (Figure 2A) or spreading E. coli on the plate (Figure 2B). For the B. cereus fluoride riboswitch it responds at fluoride concentrations above 75μM.

Figure 2A) Testing the responsiveness of the B. cereus fluoride riboswitch with serial dilutions, B) and with plating.

Using CHOP to qualitatively measure fluoride concentration in water

CHOP can also be used to qualitatively determine the fluoride concentration in water. For example, if one were to make agar plates using water with an unknown fluoride concentration and grow ΔcrcB E. coli containing CHOP with the B. cereus riboswitch and see growth, then that would indicate that the fluoride concentration is above 75μM. In the future, we envision developing a suite of fluoride riboswitches with different responsiveness to fluoride to develop systems that can more precisely measure fluoride. Also we imagine regulating other reporter genes besides chloramphenicol acetyltransferase that would give a different readout than bacterial growth.