Quorum Sensing

2.3 Detection
of Butyrolactones
by Means of Antibiotic
Bioassay

1. SFM medium [17]: 20 g/l Agar (Difco), 20 g/l mannitol,
   20 g/l soya flour. Autoclave the medium.


2. Spore stock ofS. coelicolorM145 strain. Evenly spread one
   single colony ofS. coelicolorM145 on an SFM plate (25 ml of
   SFM/100 mm diameter plate) using a sterile cotton bud,
   followed by incubation at 30C for 4–5 days (seeNote 2).
   Add 1 ml of 20% (v/v) glycerol to the plates containing spores
   and gently rub the spores off the surface of the plate with a
   sterile cotton pad (seeNote 3). Carefully, collect the spores
   with a 5-ml syringe and transfer to a 2-ml sterile screw-cap
   tube. Store at 20 C(seeNotes 4and 5 ).


3. SMMS (seeSubheading2.1,item 2).


4. Methanol extract containing GBLs.


5. Methanol, HPLC grade.


6. Previously validated GBL extract or 0.25μg/μlofS. coelicolor
   GBL SCB1 [13](seeNote 6).


7. Laminar flow hood.


8. 30C incubator.


9. Petri dishes.

2.4 Detection
of Butyrolactones
by Means of
Kanamycin Bioassay

1. Spore stocks of LW18 or LW94, prepared as previously
   described for M145 spore stock (Subheading2.3,item 2).


2. DNAgar [17]: Add 9.2 g of Difco Nutrient Agar into 400-ml
   of deionized H 2 O in a 500-ml bottle and autoclave.


3. Kanamycin.


4. Methanol extract containing GBLs.


5. Methanol, HPLC grade.


6. Previously validated GBL extract or 0.25μg/μlofS. coelicolor
   GBL SCB1 [13](seeNote 6).


7. Laminar flow hood.


8. 30C Incubator.


9. Petri dishes.

3 Methods

3.1 Extraction
of Butyrolactones
from Solid Medium

GBLs are best extracted in batches of 10–20 plates, as more plates

would be complicated to handle and fewer plates might not yield

sufficient GBLs for later bioassay and characterization steps.

1. Prepare SMMS plates (25 ml/100 mm diameter plate). Rich
   media can also be used, but GBLs are less stable in these,
   potentially reducing the extraction yields.

Butyrolactones inStreptomyces 121