Quorum Sensing

exposure to the bioassay organism. Although antibiotics are

required to maintain the plasmids in strains A136 (pCF218)

(pCF372) or KYC55 (pJZ372)(pJZ384)(pJZ410), these

strains are sufficiently stable to be grown without antibiotics

for the duration of the bioassay.

3. As a positive control in the C. violaceum bioassay, streak
   CVO26 (bioassay strain) andC. violaceum31532 (C6-HSL
   overproducer) in this same manner. ForA. tumefaciensbioas-
   say, first spread 50μl X-gal solution on the surface of the LB
   agar plate, and then streak the bioassay strain (A136 (pCF218)
   (pCF372) or KYC55 (pJZ372)(pJZ384)(pJZ410)) and KYC6
   (3-o-C8 HSL overproducer) in this same manner. For a nega-
   tive control, we simply streak the reporter strains (CVO26,
   A136 (pCF218)(PCF372) or KYC55 (PJZ372)(PJZ384)
   (PJZ410)) against itself (both as reporter and test) as these
   strains do not produce AHLs.

3.2.2 Soft Agar Overlay
Assay [33]

This approach gives more sensitivity than the plate-based assay

(Subheading3.2.1), and also enables the observation of potential

competition between the test organism and the bioassay organism

(C. violaceum), which may indicate production of an antibacterial

compound. This test typically takes 3 days:

1.Day One: Streak the test organism (seeNotes 5and 6 ) onto

suitable agar using the patterns shown (Fig.2a), and then grow

in incubator overnight. (For freshwater aquatic isolates, we

typically use R2A agar [55]).

2. As a positive control, streakP. aeruginosaonto LB agar in the
   same pattern and grow overnight at 37C. If desired, a nega-
   tive control would consist either of strain 12472 on the plate,
   or alternatively strain 31532 (C6-HSL overproducer).


3. Grow an overnight LB broth culture ofC. violaceum(30C
   with shaking).


4. If some other substance (example would be a plant leaf or other
   tissue) is to be examined, then place the object onto a plate of
   LB agar and immediately proceed tosteps 5and 6 (agar
   overlay).
   5.Day Two: Prepare and autoclave test tubes containing 5 ml LB
   soft agar. Store tubes in 50C water bath once they come out
   of the autoclave.


5. For each test plate (fromstep 1or 2 ), mix 5μl overnightC.
   violaceum12472 culture with 5 ml soft agar, vortex for 5 s, and
   then pour the mixture over plate. Incubate at 30C overnight.
   (It is important thatstep 6be done as quickly as possible asC.
   violaceumis susceptible to heat.)

16 Starla G. Thornhill and Robert J.C. McLean