Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 2: MATERIALS & METHODSisolated, the sequence was translated into the corresponding amino acid sequenceusing the Translate program (Wisconsin Package, 10.0, GCG via WebAngis).Verification of the correct translated protein was performed by conducting a FASTAsequence similarity search. In the case where a reverse primer was used forsequencing, the nucleotide sequence was reversed and complemented using theReverse program (Wisconsin Package, 10.0, GCG via WebAngis) and the resultingsequence was evaluated as previously described. The nucleotide chromatograms werevisualised using the software package CHROMAS (Version 1.45).Section 2.5 Phylogenetic AnalysisThe nucleotide and protein sequences incorporated in the construction of thephylogenetic trees were retrieved from the two databases GenBank(http://www.ncbi.nlm.nih.gov/Genbank/) and EMBL (http://www.ebi.ac.uk/embl/).The sequences were uploaded in FASTA format. The nucleotide sequences obtainedfrom the sequenced SMBG’s in this study were converted into FASTA format, thisconversion is necessary for further processing by sequence analysis programs.Sequences were aligned across specific regions which included only the secondarymetabolite biosynthetic gene under investigation, these alignments were performedusing the ClustalW alignment program (http://www.ebi.ac.uk/Tools/clustalw2/)(Thompson et al., 1994). The output of the ClustalW multiple sequence alignmentwas incorporated into the program TreeView (http://taxonomy.zoology.gla.ac.uk/rod/treeview) which was used to generate phylogenetic trees from it. The imagingapplication program Windows Paint was used to edit and print the phylogenic trees.Section 2.6Extraction and Processing of ActinobacteriaCulture ExtractsA general extraction procedure adopted in this study (Figure 18) involved extractingmetabolites from the whole broth with methanol (MeOH) or ethyl acetate (EtAc). Thebroth supernatant fraction following centrifugal separation was also retained foranalysis. Following solvent extraction the fractions were tested in well-type assays todetermine if they contained biological activity. Fractions containing bioactivity werefurther analysed using bioautography to determine the active component. Inactive_____________________________________________________________________61