AHL Extraction and Detection

Many bacteria in the proteobacteria group have been reported to produce AHL. Consequently, the AHL produced could affect the biological activity of MSB-28. Hence, MSB-28 was examined for the synthesis of AHL molecules. MSB-28 was grown overnight in LB medium (100 ml) buffered with 50 mM 3-[N-morpholino] propanesulfonic acid (MOPS) to pH 5.5 to prevent spontaneous degradation of AHLs (Yates et al., 2002). After that, CFS was extracted twice with equal volumes of acidified ethyl acetate (0.1% v/v glacial acetic acid). The resulting extracts were concentrated to dryness under vacuum and resuspended in a minimal amount of sterile milli-Q water. The presence of AHL molecules present in the extracts was analyzed by spotting the AHL extracts on TLC plates (TLC aluminum sheets 20 cm × 20 cm, Merck, Germany). Synthetic C₆-AHL was used as a control. TLC was developed with methanol and water at a ratio of 60: 40 v/v. Then the TLC plates were overlaid with 3 ml of top agar (0.7%, LB agar) seeded with C. violaceum CV026 (OD_600nm = 0.1). Further, the plates were kept at 30°C overnight or until the development of a violet color (Chen et al., 2013).