Detection and Expression of Biosynthetic Genes in Actinobacteria ...
Detection and Expression of Biosynthetic Genes in Actinobacteria ...
Detection and Expression of Biosynthetic Genes in Actinobacteria ...
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___________________________________________________________________________________2.9.3 Reverse-Phase High Performance Liquid Chromatography (RP-HPLC)___682.9.4 HPLC/UV Mass Spectrometry (MS) ______________________________692.9.5 Literature Search<strong>in</strong>g <strong>of</strong> Natural Product databases____________________69Chapter 3: ResultsSection 3.1: Development <strong>of</strong> a Polymerase Cha<strong>in</strong> Reaction-Based Screen<strong>in</strong>gAssay for the <strong>Detection</strong> <strong>of</strong> SMBG _________________________ 703.1.1 Primer design <strong>and</strong> PCR screen<strong>in</strong>g assay for act<strong>in</strong>obacterial type II PKSsynthases ____________________________________________________ 703.1.2 PCR Screen<strong>in</strong>g Assay for Modular Polyketide Synthases _____________ 823.1.3 PCR Screen<strong>in</strong>g Assay for dDTP-Glucose Synthase __________________ 883.1.4 PCR Screen<strong>in</strong>g Assay for Isopenicill<strong>in</strong> N Synthase Gene _____________ 92Section 3.2: Chemical Screen<strong>in</strong>g for Secondary Metabolites <strong>in</strong>Act<strong>in</strong>obacteria _________________________________________ 973.2.1 Bioassays <strong>of</strong> Environmental Act<strong>in</strong>obacteria cultures __________________ 973.2.1.1 Plug Type Antimicrobial Assays ______________________________ 983.2.1.2 Well Type Antimicrobial Assays <strong>of</strong> Metabolites Extracted from Agar _ 983.2.2 Small Scale Liquid Fermentations <strong>of</strong> Antimicrobial Produc<strong>in</strong>gAct<strong>in</strong>obacteria________________________________________________ 983.2.2.1 Well-Type Antimicrobial Assays <strong>of</strong> Metabolites Extracted fromLiquid Fermentation Media __________________________________ 983.2.3 Improv<strong>in</strong>g Antimicrobial Metabolite Production <strong>in</strong> Low Yield<strong>in</strong>gAct<strong>in</strong>obacteria Cultures _______________________________________ 993.2.3.1 Solid-Substrate Fermentations (SSF)___________________________ 993.2.3.2 Liquid Fermentations Supplemented with Ref<strong>in</strong>ed Oils ____________ 1023.2.4 Th<strong>in</strong> Layer Chromatography (TLC) <strong>of</strong> Antimicrobial Metabolites fromFermented Act<strong>in</strong>obacteria Cultures ______________________________ 1033.2.4.1 Liquid Fermentations_______________________________________ 1043.2.5 Bioautography <strong>of</strong> Antimicrobial Metabolites _______________________ 1053.2.6 UV-Vis Spectroscopy <strong>of</strong> Semi-Purified Antimicrobial Organic Extract __ 1073.2.7 Reverse-Phase High Performance Liquid Chromatography <strong>of</strong>Semi-Purified Antimicrobial Organic Extracts______________________ 1093.2.8 Determ<strong>in</strong>ation <strong>of</strong> antibacterial activity <strong>of</strong> HPLC fractions from OrganicExtracts ____________________________________________________ 1103.2.9 Electrospray Ionisation High Performance Liquid-Chromatography Mass-Spectrometry (ES –HPLC-MS) <strong>of</strong> Organic Extracts _________________ 1103.2.10 Literature search <strong>of</strong> natural product database <strong>of</strong> organic extract physicochemicocharacteristics ________________________________________ 111___________________________________________________________________________________
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