0.25μg/μl solution of SCB1 or 2–3μl of a previously validated
methanol extract and let dry for 1 min.- Incubate at 30C incubator for 24 h. Check plates every 12 h
for 3 days monitoring growth using a conventional scanner
(Fig.3)(seeNote 10). A confluent lawn should be visible in
the growth control after incubation at 30C for 24 h. - To estimate the concentration of GBLs in the methanol extract
prepare serial dilutions of the same and perform the bioassay by
adding 1μl of each prepared sample. Estimate the concentra-
tion of GBLs by multiplying the dilution factor of the mini-
mum concentration that show growth of the reporter strain, in
the presence of 5μg/ml kanamycin after 3 days of incubation
at 30C, by 0.025μg/μl(seeNote 11). This quantification
method has also been used in previous studies [19].
4 Notes
- The ethyl acetate used for extraction should be previously
tested in the same bioassay that the extracted sample will be
subjected to before using it for extracting the butyrolactones,
as it might have background bioactivity. - Depending on the soya flour used sporulation may take longer.
WhenS. coelicolorsporulates, the culture will acquire a grey
color. Attempting to harvest them before the shift to grey will
result in no spores being collected or a very low spore
concentration. - To prevent contamination of the spore stock, use previously
sterilized tweezers to pick up the cotton pad. - Keep spore stocks frozen ( 20 C), and when thawing keep at
low temperature (4C) and on ice. - The number of viable spores in the spore stock can be deter-
mined using a serial dilution (up to 10^10 ) on Lysogeny Broth
(LB) agar plates and incubation at 37C for 20–24 h. - The sensitivity of both bioassays towards SCB1 has been
reported to be the same, being the optimal amount for the
positive control of 1 μl of a 0.25μg/μl solution of SCB1
[12, 13]. Note that SCB1 is not commercially available,
although its chemical synthesis has been described in [12]. - In the antibiotic bioassay, add the methanol extract within 8 h
of plating. Otherwise, no induction of Red or Act will be
seen [12]. - For the bioassays, the added volume of methanol extract onto
bioassay plates should never exceed 3μl. If larger volumes are
necessary, because of the very low expected concentration, add
Butyrolactones inStreptomyces 125