sonication, make sure the microtip is surface sterilized with 70%
(vol/vol) ethanol.- Sonicate the cell suspension in ice 20 times with 10 s bursts at
200 W, with a 10 s cooling interval. Sterilize the surface of
microtip with 70% (vol/vol) ethanol before sonicating another
bacterial sample (seeNote 8). - Centrifuge the clear cell lysate at 13,000gfor 10 min at 4C.
Transfer the supernatant to a clean 1.5-ml tube, being careful
not to disturb the cell debris. - Collect cell content of Th120 by filtering the supernatant
through a 0.22μm filter. Keep it on ice (seeNote 9).
3.2.2 Determine Whether
the QQ Activity of Th120 is
Due to Lactonase Activity
- Transfer 250μl of whole culture, supernatant, and cell content
of Th120 to clean 1.5-ml tubes, respectively. - Prepare heat-treated samples by boiling samples in water bath
capable of reaching temperatures of 100C for 5 min. - Transfer another 250μl of whole culture, supernatant, and cell
content of Th120 to clean 1.5-ml tubes, respectively. - Prepare proteinase K-treated samples by adding 2.5 μlof
20 mg/ml proteinase K (final concentration of approximate
200 μg/ml), and incubate samples at 37C for 3 h. Run a
control without adding proteinase K but incubating the sample
at 37C for 3 h to exclude possible effects of temperature on
the reduction of QQ activity. - In order to determine whether QQ activity is due to enzymatic
degradation, add 178μl of each sample into a clean 1.5-ml
Fig. 2The normalizedβ-galactosidase activities of the 25 QQ bacterial strains. Each strain was compared with
negative control using unpairedt-test(n¼3; two-tailedpvalue; p<0.0001, p<0.001, p¼0.0143).
Reproduced with permission from Copyright©2013, Rights Managed by Nature Publishing Group [9]
302 Kaihao Tang and Xiao-Hua Zhang