degradation, and prediction of QQ enzymes from whole genome
sequencing data. The discovery of more QQ enzymes will contrib-
ute to our knowledge about their diversity and may provide poten-
tial as therapeutic agents.2 Materials
2.1 High-Throughput
Screening for QQ
Bacteria
1.A. tumefaciensA136 (pCF218) (pCF372) [11](seeNote 1).- Marine Broth 2216 (MB; Becton Dickinson).
- Luria-Bertani (LB) agar: 10 g/l tryptone, 5 g/l yeast extract,
10 g/l NaCl, 15 g/l agar. Sterilize by autoclaving. - AT minimal glucose medium:
(a) 20AT salts: 40 g/l (NH 4 ) 2 SO 4 , 3.2 g/l MgSO 4 ·7H 2 O,
200 mg/l CaCl 2 , 100 mg/l FeSO 4 ·7H 2 O, and 40 mg/l
MnSO 4 ·7H 2 O in distilled water, autoclave-sterilized, and
stored at 4C.
(b) 20 AT buffer: 214 g/l KH 2 PO 4 in distilled water,
adjusted with KOH to pH 6.7, autoclave-sterilized, and
stored at 4C.
(c) 50% (wt/vol) glucose: filter-sterilized and stored at
20 C.
(d) AT minimal glucose medium (1 l): 50 ml of 20AT salts,
50 ml of 20AT buffer, and 10 ml of 50% (wt/vol)
glucose combined with sterile deionized H 2 O to a total
volume of 1 l. - 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal)
stock solution: 20 mg/ml X-gal dissolved in dimethylforma-
mide (DMF), stored at 20 C. - 1,4-Piperazinediethanesulfonic acid (PIPES) stock solution:
1 M PIPES dissolved in distilled water, pH 6.7, filter-sterilized,
and stored at 20 C.
7.N-Hexanoyl-L-homoserine lactone (C6-HSL) stock solution:
1 mM C6-HSL dissolved in dimethyl sulfoxide (DMSO),
stored at 20 C(seeNote 2). - Antibiotic stock solutions: 50 mg/ml spectinomycin and
4.5 mg/ml tetracycline dissolved in distilled water, filter-
sterilized, and stored at 20 C. - Sterile 96-well plates.
- Sterile 0.22μm filter.
- Microplate absorbance reader.
Identifying Microbial Quorum Quenching Enzymes 299