- Add 10^7 Streptomycesspores/plate, diluted with sterile water in
100 μl total volume, and spread evenly across the entire plate
using a sterile cotton bud to generate confluent lawns. Grow
for about 20 h (when using SMMS medium) or until the onset
of Red production (when using other types of medium). - Cut the agar into pieces of about 25–50 mm in diameter, using
a clean razor blade, and transfer into a 500-ml conical flask.
Cutting the agar into too small pieces might cause difficulties in
step 5. - Add enough ethyl acetate into the flask containing the pieces of
agar to just cover the entire amount of agar, and gently swirl
and allow resting for 5 min at room temperature. - Carefully transfer the ethyl acetate into a round-bottom flask
(using pipettes if required), avoiding the transfer of any piece of
agar, and remove solvent by rotary evaporation at room
temperature. - Resuspend the remaining brown oil in 5 ml of 100% methanol
and transfer to three 2-ml plastic tubes. Bring the solution to
dryness using a speed vacuum concentrator and resuspend in a
total volume of 50–150μl of 100% methanol per each tube,
making sure that the entire remaining brown oil dissolves, by
gentle vortexing for 5–10 s. Pool all the samples in one tube.
3.2 Extraction
of Butyrolactones
from Liquid Medium
The following protocol is intended for 50 ml ofS. coelicolorM145
culture. Where higher yields of GBLs are required, add more
cultures or scale-up accordingly.- Add 10^10 spores (seeNote 5) to 50 ml of SMM medium in a
250-ml sterile conical flask containing a stainless steel spring to
avoid cells to clump and provide good aeration [17]. - Incubate at 30C until early stationary phase. Monitor growth
every 2 h after the initial 12 h of inoculation by measuring the
optical density at 450 nm using a spectrophotometer. - Transfer culture to a 50 ml plastic tube, centrifuge the cells at
10,000g, and transfer the supernatant to a 500-ml conical
flask. Add 50 ml of ethyl acetate to the supernatant and mix
vigorously. Transfer mixture to a separation funnel and allow
for both phases to separate (the lower one will be the aqueous
phase and the upper one the ethyl acetate). - Transfer the aqueous phase to a beaker and the upper organic
phase into a clean round-bottom flask. Transfer the aqueous
phase to the separation funnel again, add 50 ml of ethyl acetate,
and shake vigorously. Discard the aqueous phase and pool the
organic phases. - Dry the organic phase by adding two spatulas of MgSO 4
(anhydrous)/50 ml of organic layer and vortex vigorously.
122 Marc Biarnes-Carrera et al.