Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 2: MATERIALS & METHODSvisible (UV-Vis) spectrophotometry which provided useful indications in establishinga purification process.2.9.1 Thin Layer Chromatography (TLC)TLC was used to separate and isolate active constituents from fermentation brothmixtures. 20 μl of microbial extract was spotted onto an aluminium backed pre-coatedsilica gel plate (60 F 254 20 x 20 cm, 0.2 mm layer thickness plates Merck, Cat. No.5554) and air dried using a handheld hairdryer; Plates were eluted with a solventsystem consisting of Ethyl Acetate: Methanol (90:10) with a drop of 28% Ammoniasolution. TLC plates were run for 10-15 min. In an airtight glass tank, pre-saturatedwith solvent vapors, containing the eluting solvent system. Bands were visualisedunder 254 nm and 365 nm wavelength UV light and their respective R f values calculated.2.9.2 Ultraviolet-Visible SpectrophotometryAn evaluation of the UV-Vis spectrum of the microbial extracts at different levels ofpurity were carried out. To determine the chromophores present in the metabolites.Optical measurements were performed on a UV-Visible SpectrophotometerSHIMADZU Model UV-160A. The spectrum was scanned over the desiredwavelength range of 200 nm to 600 nm .2.9.3 Reverse-Phase High Performance Liquid Chromatography (RP-HPLC): In the current study, organic extracts were subjected to RP-HPLC (Table 30)and peaks were further analysed by recording their UV absorbance across thewavelengths 200 nm to 400 nm . In addition, eluting fractions were collected at 0.25 minintervals and subjected to antimicrobial assays. Both the RP-HPLC and antimicrobialassays was carried out in the laboratory of the industry partner Cerylid Biosciences.Table 30: RP-HPLC conditions and parametersConditions Specification and ParametersSystemColumnWATERS 2960Xterra MS (WATERS) C 18 Reverse phase 4.6 x 50 mm,2.5 μm diam.Solvents A: 0.1% formic acid in waterB: 0.1% formic acid in acetonitrileGradient Profile 0 to 100% solvent A in B 17 minsFlow RateDetectionSample volume100% solvent B 20 mins1 ml.min -1Photodiode array (PDA), Elutions monitored at 200 nm to 400 nm10 μl___________________________________________________________________________________68