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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BERVANAKIS, G.Chapter 2: MATERIALS & METHODSTable 19: Cerylid environmental act<strong>in</strong>obacterial isolatesIsolateGenus*Location <strong>of</strong> Isolate Obta<strong>in</strong>edNumber1. A0371 Streptomyces Cow paddock, VIC2. A2010 Streptomyces Walsh River Burke Rd, QLD3. A2360 Streptomyces Walker Hill, QLD4. A0350 Streptomyces Sediment, Townsville Port, QLD5 .A2226 Streptomyces Shadow soil over granite, QLD6. A2381 Streptomyces Lamb range Douglas creek, QLD7. A1215 Streptomyces Koomboolomba, QLD8. A2056 Streptomyces Open eucalypt forest, QLD9. A3675 Streptomyces Under native grass, VIC10. A0096 Streptomyces Track, N <strong>of</strong> Rocky Pt. Rd. NT11. A1113 Streptomyces Plant material <strong>and</strong> wood chips, VIC12. A1488 Modestobacter Ol<strong>in</strong>da-D<strong>and</strong>enongs, VIC13. A1664 Geodermatophilus Cape York, QLD14. A3020 Asiosporangium Cape Ferguson (sediment)15. A3023 Verrucosispora Cape Ferguson (sediment)16. A2702 Micromonospora Eucalyptus woodl<strong>and</strong>, QLD17. A2376 Micromonospora Open eucalyptus forest, QLD18. A3014 Micromonospora Mt Fox, QLD19. A0347 Couchioplanes Australian Antarctic Territory, Antartica20. A3771 Couchioplanes Port Melbourne, VIC21. A1990 Nocardioides Tall eucalyptus forest, QLD22. A2834 Nocardioides Eucalyptus pauciflora tablel<strong>and</strong>s, QLD* The Cerylid environmental act<strong>in</strong>obacterial isolates were assigned to appropriate genusgroups follow<strong>in</strong>g 16SrDNA sequenc<strong>in</strong>g carried out by Cerylid Biosciences.2.1.2 Ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g Cultures (solid medium): Type stra<strong>in</strong>s <strong>and</strong> environmentalcultures were ma<strong>in</strong>ta<strong>in</strong>ed for long, medium <strong>and</strong> short term storage. In the case <strong>of</strong> longterm storage two loopfuls <strong>of</strong> agar cultures were immersed <strong>in</strong> a sterile 20% v/vglycerol solution <strong>and</strong> cryopreserved <strong>in</strong> a –80 o C freezer (Well<strong>in</strong>gton & Williams,1978), <strong>in</strong> the medium term (up to 3 months) cultures were ma<strong>in</strong>ta<strong>in</strong>ed on Yeast-MaltExtract (YME) Agar slants conta<strong>in</strong><strong>in</strong>g (g/L) Yeast Extract 4, Malt Extract 10, Glucose4, Agar 16, pH 7.2 <strong>and</strong> held at 27 o C, for the short term (2-4 weeks) cultures werema<strong>in</strong>ta<strong>in</strong>ed on their respective agar plates <strong>and</strong> held at 27 o C.Section 2.2: Primer Design For Act<strong>in</strong>obacteria-SpecificSecondary Metabolite <strong>Biosynthetic</strong> <strong>Genes</strong>2.2.1 In Silico Analysis <strong>of</strong> Nucleotide SequencesComputational analysis <strong>of</strong> nucleotide sequences <strong>in</strong>volved us<strong>in</strong>g WebANGIS(www.angis.org.au). This s<strong>of</strong>tware package was utilised for extract<strong>in</strong>g <strong>in</strong>formationfrom sequence databases <strong>and</strong> conduct<strong>in</strong>g a large number <strong>of</strong> procedures <strong>and</strong> genomeanalysis tasks outl<strong>in</strong>e below. A stepwise procedure was followed <strong>in</strong> the design <strong>of</strong>_____________________________________________________________________53
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