Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 2: MATERIALS & METHODSVermiculite NT 1.2 4.5 ml Trace Salts b5.0 ml LF42 cPerlite NT 1.2 4.5 ml Trace Salts bWhole Grain OatsWhole Grain RyeSoaked in sterilewater overnight,maceratedSoaked in sterilewater overnight,macerated5.0 ml LF42 c10 a 4.5 ml Trace Salts b5.0 ml LF42 c10 a 4.5 ml Trace Salts b5.0 ml LF42 cPsyllium Husk NT 10 4.5 ml Trace Salts b5.0 ml LF42 cLSA Mix* NT 10 4.5 ml Trace Salts b5.0 ml LF42 c*LSA = Linseed, Sunflower Kernels, and Almonds^ NT = not treateda wet weightb Trace Salts Solution added: 0.1g Sodium Tartrate., 0.01g ZnSO 4 .5H 2 O., 0.01gFeSO 4 .7H 2 O., 0.1g KH 2 HPO 4., 0.1g MgSO 4 .7H 2 O., 0.2g Yeast Extract made up to1000 ml with distilled water and sterilised.c LF42 Solution: 5g Yeast Extract., 5g Peptone., 5g Soyaflour., 40 ml glycerolsolution., 2g Soluble Starch., 2g CaCO 3 ., 5g NaCl made up to 1000 ml with distilledwater and sterilised.d maceration was carried out using a hand held kitchen blender.2.8.3 Liquid Fermentations Supplemented with Refined OilsThe two liquid fermentation media IM25 and 153m, which had been shown to beuseful for the production of antimicrobial compounds were further evaluated with theaddition of refined oils. 1.5 g of each refined oil was added to 50 ml of fermentationmedia IM25 or 153m to achieve a final concentration of 3 % w/v. Inoculum of theactinobacterial environmental isolates A0347, A2702 and A3675 was obtained fromYME plates which were grown for 7 days. The duration of the fermentation wascarried out over 10 days, at 27 o C, at the speed 120 rpm on a Innova 2300 platformshaker (New Brunswick Scientific). Following the end of the fermentation thesamples were processed and assayed as in section 2.7.Section 2.9: Physicochemical Characterisation Methods Usedto Elucidate Semi-Purified Fermented ExtractChemical characterisation methods at early stages of the purification process includedanalytical techniques such as Thin Layer Chromatography (TLC) and Ultra-violet-___________________________________________________________________________________67