4.2.1. Quorum Sensing Inhibition Test with Bio-Reporter Chromobacterium violaceum (CV026) and Agrobacterium tumefaciens (KYC55)

Thin Layer chromatography (TLC) preparation: All derivatives were dissolved in 100% acetonitrile to a concentration of 40 mM. Then placed 20 μL on the same spot on the TLC (Merk, Darmstadt, Germany) and let the solvent evaporate before use. CV026: few colonies were transferred to liquid LB (Difco Lenox medium, Le Pont se Claix, France) for overnight incubation at 30 °C in a 45 degree in orbital shaker (Gerhardt, Königswinter, Germany). At the O.D. 0.8 3 mL of the starter was added to preheated soft LB agar containing 500 nM of 3-oxo-C6 (K3007, Sigma-Aldrich, Saint louis, MO, USA). The mixture was then loaded on the TLC in a glass plate for O.N. incubation in 30 °C. The next day, the inhibition zone was measured, while QS was taking place that produced a bacterial purple pigment, and when QS is inhibited, no pigment is produced. KYC55 (pJZ372) (pJZ384) (pJZ410): 20 µL from the frozen stock into 25 mL AT media (AT buffer ×20:1.57 M KH₂PO₄, pH = 7.3. AT salts mix ×20:300 mM (NH₄)₂SO₄, 13 mM MgSO₄, 1.4 mM CaCl₂, 0.4 mM FeSO₄7H₂O and 0.26 mM MnSO₄H₂O. AT media:50 mL × 20 AT-buffer, 50 mL × 20 AT salts, 10 mL 50% (w/v) glucose and add 890 mL to a final volume of 1 L). For O.N. at 30 °C in an orbital shaker, into preheated water agar with 25 mL AT buffer and 60 μg/mL Xgal and 5-nM 3-oxo-c8 (O1764, Sigma-Aldrich, Saint louis, MO, USA), 25 mL of the starter was added. Then, that mixture was loaded on the TLC in a glass plate for O.N. incubation in 30 °C. The next day, the inhibition zone was measured. The bacteria produce blue pigment during QS and when QS is inhibited no pigment is produced. KYC55 (pJZ372) (pJZ384) (pJZ410) preservation in tetracycline (1 μg/mL), spectinomycin (100 μg/mL) and gentamicin (100 μg/mL) (all antibiotics from Sigma Aldrich, Saint louis, MO, USA) in AT media O.N. then centrifuged and suspend in 15% glycerol frozen and maintained at −80 °C [31,36].