5 bromo 4 chloro 3 in dolyl β d galactopyranoside x gal

Saliva metagenomic DNA was partially digested using HindIII, ligated into pCC1BAC and transformed into E. coli EPI300 as described previously (Seville et al., 2009 (link)).
After transformation, cells were recovered in SOC media (New England Biolabs^®), cultured on LA containing chloramphenicol, 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Promega^©) and 40 μg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal; Promega^©) for 16 h. White clones were cultured in LB with chloramphenicol in individual wells of 96-well plates at 37°C for 16 h. The cultures were then stored at -80°C in 20% glycerol.

Agrobacterium tumefaciens KYC55 (pJZ372)(pJZ384)(pJZ410) (Zhu et al., 2003 (link)) and Chromobacterium violaceum CV026 (McClean et al., 1997 (link)) were kindly offered by Zhigang Zhou (Chinese Academy of Agricultural Sciences, Beijing, China) and used for the detection of AHLs as previous described (Ravn et al., 2001 (link)) A. tumefaciens KYC55 was grown in LB medium supplemented with 1 μg/mL tetracycline,100 μg/mL tetracycline, and 100 μg/mL spectinomycin. C. violaceum CV026 was gown in LB medium supplemented with 20 μg/mL kanamycin. N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL), N-(3-oxohexanoyl)-D-homoserine lactone (3-oxo-C8-HSL), and N-(3-hydroxydodecanoyl)-DL-homoserine lactone (3-OH-C8-HSL) were purchased from Sigma-Aldrich (St. Louis, MO, United States). 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) was bought from Promega (Madison, WI, United States). All other chemicals were analytical grade.