4.5μg/ml tetracycline and incubate at 28C, 170 rpm for 12 h
(seeNote 5).- Prepare the A136 X-gal assay solution by inoculating the cul-
ture of A136 (OD 600 2) in AT minimal glucose medium
(supplemented with 50μg/ml spectinomycin and 4.5μg/ml
tetracycline) with an inoculum size of 1% and mixed with X-gal
to a final concentration of 250μg/ml. Generally, 20 ml of the
A136 X-gal assay solution are sufficient for assay in a whole 96-
well plate (seeNote 5). - After incubation (step 3), centrifuge the reaction mixture at
13,000gat 4C for 10 min and transfer 100μl of superna-
tant to a clean 1.5-ml tube. Incubate the supernatant at 95C
for 3 min (seeNote 6). Centrifuge the mixture at 13,000g
for 10 min. Transfer 10μl of supernatant to a well of sterile 96-
well plate, being careful not to disturb the cell pellet. Add
190 μl of the A136 X-gal assay solution and mix gently by
pipetting. Incubate the mixtures at 28C for 12 h under static
condition. - Detect the absorbance at the wavelengths of 492 and 630 nm
with microplate absorbance reader. Calculate the values of
OD 492 and OD 630 by subtracting original values of blank
control from that of samples. - Calculate the normalizedβ-galactosidase activity using the for-
mula: 00 :: 267653 ODOD^492630 ODOD 492630 - Compare samples with negative control. Select samples that
cause more than 40% reduction in the normalizedβ-galactosi-
dase activities for further tests (seeNote 7). The normalizedβ-
galactosidase activities of 25 QQ bacterial strains in our previ-
ous work are shown in Fig.2.
3.2 Further
Characterization of QQ
Activities of Selected
Bacteria
The marine bacteriumM. oleariaTh120 is used as an example in
the subsequent Sections [10].3.2.1 Collect Whole
Culture, Supernatant, and
Cell Content of M. olearia
Th120
- Inoculate Th120 in 5 ml of MB, and incubate the culture at
28 C, 170 rpm for 24 h. Run at least two technical replicates. - Transfer 1.5 ml of bacterial culture into two distinct 2-ml
tubes. Keep the remaining 2 ml of the bacterial culture on ice. - Centrifuge the culture at 5000gfor 10 min at 4C.
- Collect the cell-free supernatant of Th120 by filtration through
a 0.22μm filter. Keep it on ice. - Wash the harvested cells three times with ice-cold MB, and
resuspend in 3 ml of ice-cold MB. Keep it on ice until sonica-
tion using a sonicator equipped with a microtip. Before
Identifying Microbial Quorum Quenching Enzymes 301