To begin, obtain the Lysogeny broth or LB medium and add the antibiotics to the medium. Aliquot five milliliters of the medium into sterile test tubes with caps. Inoculate the media with Escherichia coli strains expressing plasmids taken from freezer stocks and incubate the culture in a shaker overnight.
Next, prepare a 10 millimolar thiophene sulfonamide solution in DMSO and vortex to mix thoroughly. Back-dilute the cultures 1 to 100 times in 10 milliliters of media taken in 15 milliliter conical tubes. Vortex briefly to mix.
Add cultures to the appropriate wells of a 96-well plate. After mixing, prepare a dilution series from rows A to E, transferring 50 microliters of the solution each time. Cover the plate with microporous tape and incubate it.
After removing the tape, using a plate reader, read the optical density at 600 nanometers GFP and mCherry. Finally, record and plot the data as normalized fluorescence per cell. Data for 3-thiophene sulfonamide compounds suggested that compounds 1A and 3B were each inhibitory to LuxR/HapR to different extents, whereas 2B did not have any activity.
