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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___________________________________________________________________________________Chapter 2: Materials <strong>and</strong> MethodsSection 2.1: General Microbiological Methods __________________________522.1.1 Act<strong>in</strong>obacteria Cultures ________________________________________522.1.2 Ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g Cultures (solid media) _______________________________53Section 2.2: Primer Design For Act<strong>in</strong>obacteria-Specific SecondaryMetabolite <strong>Biosynthetic</strong> <strong>Genes</strong>______________________________532.2.1 In silico Analysis <strong>of</strong> Nucleotide Sequences __________________________532.2.1.1 Retrieval <strong>of</strong> Nucleotide Sequences from databases__________________542.2.1.2 PILEUP Multiple Sequence Alignment (MSA) Program _____________552.2.1.3 PRETTY Consensus Sequence Program __________________________552.2.1.4 Database Similarity <strong>of</strong> Primer Sequence us<strong>in</strong>g the FASTA program ____552.2.1.5 Calculat<strong>in</strong>g Anneal<strong>in</strong>g Temperatures <strong>of</strong> Primers____________________552.2.1.6 In silico PCR experiments us<strong>in</strong>g AMPLIFY simulation program______562.2.1.7 Selection <strong>of</strong> primers for screen<strong>in</strong>g_______________________________ 56Section 2.3: Molecular Biology Methodologies __________________________572.3.1 Extraction <strong>of</strong> Bacterial DNA ____________________________________572.3.2 PCR Reaction Conditions _______________________________________572.3.2.1 General PCR Conditions for Designed Non-degenerate Primers _______582.3.2.2 Degenerate PCR Conditions for Type I PKS_______________________582.3.2.3 PCR Controls _______________________________________________592.3.2.4 Agarose Electrophoresis <strong>of</strong> PCR Products ________________________60Section 2.4: Analysis <strong>of</strong> sequenced amplified PCR products _______________60Section 2.5: Phylogenetic Analysis ____________________________________61Section 2.6: Extraction <strong>and</strong> Process<strong>in</strong>g <strong>of</strong> Act<strong>in</strong>obacteria Culture Extracts __612.6.1 Act<strong>in</strong>obacteria agar metabolite extraction __________________________622.6.1.1 Small scale act<strong>in</strong>obacteria fermentation metabolite extraction <strong>and</strong>process<strong>in</strong>g _________________________________________________632.6.2 Large scale production <strong>and</strong> recovery <strong>of</strong> antimicrobial metabolites ______632.6.3 Concentration <strong>of</strong> Extract _______________________________________64Section 2.7: Bioassays <strong>of</strong> Secondary Metabolites ________________________642.7.1 Plug Type Bioassay____________________________________________642.7.2 Well Type Bioassay ___________________________________________652.7.3 Bioautography________________________________________________65Section 2.8: Fermentation <strong>of</strong> Secondary Metabolites _____________________652.8.1 Small Scale Submerged Shake-Flask Fermentations __________________652.8.2 Small Scale Solid State Fermentations (SSF)________________________662.8.3 Liquid Fermentations Supplemented with Ref<strong>in</strong>ed Oils _______________ 67Section 2.9: Physicochemical Characterisation Methods Used toElucidate Semi-Purified Fermented Extract __________________672.9.1 Th<strong>in</strong> Layer Chromatography (TLC) _______________________________682.9.2 Ultraviolet-Spectrophotometry ___________________________________68___________________________________________________________________________________
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