Quorum Sensing

independent transposition events and not siblings. As a con-

trol, and to check the quality of the plates that will be used the

following day, streak both your donor and recipient cultures on

LB plates supplemented with 100μg/ml Km or 25μg/ml Nal.

The donor should not grow on LB Nal and the recipient

should not grow on LB Km.

4. The following day, pipet 5 ml of LB onto each of the mating
   plates. Scrape the bacteria from the surface and remove 1 ml of
   the LB and bacteria mixture into a 1.5 ml microcentrifuge
   tube. You should now have between 10 and 50 microcentri-
   fuge tubes of mated bacteria. From each tube, plate 100μl onto
   each of the ten selective plates (seeNote 12). In our case, the
   selective plates are LB agar containing 100μg/ml Km and
   25 μg/ml Nal. Incubate overnight at 37C.


5. The following day, each selective plate should have hundreds
   of colonies, each ideally representing a recipient strain
   containing a single-transposon insertion (see Note 13 for
   troubleshooting).


6. Repeat this procedure until the required number of mutants is
   achieved (seeNote 14).

3.3.2 Screening and
Characterization of Fusions

1. To screen large numbers of fusions for AHL responsiveness, we
   typically use 96-well plates. The wells are pre-filled with a
   growth medium that is permissive for the expression and activ-
   ity of the LuxR homolog of interest (seeNote 15). Alternating
   rows contain one or more AHLs or solvent control.


2. Using toothpicks or pipet tips, stab each isolated colony and
   stab it into the 96-well microtiter plate, first into the solvent
   control well and then the AHL-containing well (to prevent
   carryover of AHLs). The toothpick will easily carry enough
   bacteria to inoculate both wells and does not need to be re-
   stabbed into the original colony between wells.


3. After the bacteria have achieved stationary phase, or at multiple
   time points during growth, depending on your reporter, com-
   pare the fusion activity of the adjacent wells to identify those
   that are AHL responsive. In our case, we used a spectropho-
   tometer/luminometer to measure luciferase activity after 9 h of
   growth.


4. Fusions that appear to be AHL responsive in the well of the 96-
   well plate need to be streaked to isolate single colonies and
   retested.


5. The location of the transposon insertion point in the genome
   must be determined. There are a variety of methods to do this,
   including direct sequencing of genomic DNA, inverse PCR,
   and cloning of the transposon.

154 Vittorio Venturi et al.