Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 2: MATERIALS & METHODS2.7.2 Well Type BioassayScreening was also performed using the conventional well type bioassay, 6-mm wellswere extruded from BA plates seeded with test cultures. 30 μls aliquot of resuspendedextract, fermentation broth supernatant or mycelial extract were pipetted into thewells. The plates were then incubated at 37 o C and zones of inhibition recorded after 2– 4 days.2.7.3 BioautographyThin Layer Chromatography (TLC) plates were assayed to identify active bands. TLCplates were placed face down on bioassay agar plates seeded with a test culture for 30minutes to allow transfer of metabolites. The pattern of the bands were traced ontotracing paper, so as to establish the position of the active bands. Following incubationthe TLC plate was removed and the plates incubated for 2-4 days at 37 o C and zones ofinhibition recorded. 0.1% Tetrazolium Blue Chloride (SIGMA# T-4375) was sprayedover the top of the agar so as to clearly visualize these zones.Section 2.8: Fermentation of Secondary MetabolitesPreliminary screening for antimicrobial activities in liquid media was conducted todetermine (1) which conditions were optimal for fermentative expression of bioactivemetabolites, (2) to make a comparison of solid and liquid fermentations and (3) todetermine optimal fermentation conditions for selected environmental isolates.2.8.1 Small Scale Submerged Shake-Flask FermentationsActinobacterial cultures were grown on PDA or YME plates for 7-10 days and usedas inoculum into 50 ml of IM22 inoculum medium. Following 3 days of fermentationat 27 o C, 120 rpm on a Orbital shaker (Ratek, Australia) or Innova 2300 platformshaker (New Brunswick Scientific). 2 ml of inoculum medium IM22 was transferredto baffeled 250 ml erylemeyer flasks containing 50 ml of selected production mediaas shown in table 28. Fermentations were conducted over a 10 day period, withsamples taken at days 2, 5, 7 and 10 for biological activity assessment.___________________________________________________________________________________65