supplemented with 50 μg/mL kanamycin and 10 μg/mL
chloramphenicol. The culture is grown for 16 h with aeration
(175 rpm) at 30C.- The culture fromstep 2is diluted to an optical density at
600 nm wavelength (OD 600 ) of 0.7 in 2 mL of AB medium
supplemented with 1.2 mM FeCl 3 , and incubated for 1–1.5 h
at 30C with shaking (175 rpm) to an OD 600 of 1.0–1.1 [29].
Then, the resulting culture is diluted 5000-fold in fresh AB
medium. - 25μL of the compounds to be tested freshly dissolved in AB
medium to the appropriate concentration are dispensed into
96-well microtiter plate. - 25μL of a freshly synthesized 20μM DPD solution (pH¼7)
and 25μL of 4 mM boric acid are also added to the test
compounds solutions into the 96-well microtiter plate (final
concentration of DPD in the well¼ 5 μM; final concentration
of boric acid in the well¼1 mM) (seeNote 4). - 25μL of the bacterial culture prepared instep 3are dispensed
into the wells of the microtiter plate prepared insteps 4and 5.
The microtiter plate is covered with a nontoxic plate sealer and
incubated at 30C with shaking for 4–6 h. - Light production is measured every 30 min by using a lumin-
ometer microplate reader (Fig.5).
Fig. 4Docking conformations of 42 hits and AI-2 within the binding site ofV.
harveyiLuxPQ (LuxP:green ribbonsand LuxQ:orange ribbons)
In silicoIdentification of AI-2 Inhibitors 359