Quorum Sensing

3.1 QS Monitor
Assays

1. Inoculate the two monitor strains (LasB-GFP or RhlA-GFP) in
   2  10 ml ABT media supplemented with 0.5% (wt/vol)
   glucose and 0.5% (wt/vol) casamino acids.


2. Incubate cultures overnight at 30C with shaking at 180 rpm.


3. Add 150μl AB-medium containing 0.5% (wt/vol) glucose and
   0.5% (wt/vol) casamino acid to all wells in a black 96 wells
   microtiter dish.


4. Add 150μl of test sample (possible QSI) to the first row of
   microtiter dish (seeNote 2).


5. Make a twofold serial dilution from row 1–11. No test sample/
   QSI are added to row number 12, which works as a reference in
   order to confirm the growth of the monitor strain and compare
   the fluorescence.


6. The overnight culture (monitor strain) is diluted in 0.9% (wt/
   vol) NaCl to an optical density at 450 nm (OD 450 ) of 0.2.


7. Add 150μl of diluted overnight culture to all wells in order to
   make a total volume of 300μl in each well.


8. The microtiter dish is placed in the multi label plate reader and
   measurements are started. The fluorescence (GFP-expression)
   is measured with an excitation and emission wavelength at
   485 nm and 535 nm, respectively, and growth of the bacteria
   is determined by measuring OD 450. Both GFP expression and
   growth are measured every 15 min for 15 h. The temperature is
   set at 34C.

3.2 In Vitro
Continuous-Culture
Flow Cell System

1. The flow cells are assembled minimum 24 h prior to use by
   gluing (use silicone) a 2450 mm glass cover slip onto the top
   of the flow cell (Fig.2b).


2. Assemble the rest of the system as shown in Fig.2a.


3. The following description is based on a 16 channel pump.
   Sterilize the whole system using 1 l of a 0.5% (vol/vol)
   NaClO solution in sterilized Milli-Q water. The pump is set
   at 90 rpm to fill the whole system. When the bubble traps are
   filled, the lids are put on and the pump is set at 12 rpm. Sterilize
   for approximately 2 h. Make sure that nothing is leaking,
   otherwise use silicone to stop it. Remember to wear gloves
   and glasses when working with NaClO (seeNote 3).


4. Empty the system at 90 rpm.


5. Wash the system with 21 l of sterilized Milli-Q water the
   same way as with the NaClO. Set the pump at 50 rpm when the
   system is filled. The system is emptied after 1 l and filled up
   again with the new flask (seeNote 3).


6. After the last wash, the system is emptied and filled up with
   ABT minimal medium. Place the system at 37C overnight (see
   Note 3).

280 Tim Holm Jakobsen et al.