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Robson, William Nigel
Languages: English
Types: Doctoral thesis
Subjects: QR
Genomic libraries of S. lavendulae and S. subrutilus\ud were constructed in S. lividans using the technique of\ud shotgun cloning. S. lividans is a genetically well\ud characterised recipient for heterologous DNA, but plasmid\ud deletions or the entry of plasmids with small inserts\ud occurred during transformation of the DNA into\ud S. lividans This was not due to a straightforward\ud restriction-modif ication system as this possibility was\ud checkea using the KC301 phage. Several gene libraries\ud were produced, using high and low copy number plasmids,\ud and the resultant transformants screened.\ud The nature of deoxynojirimycin (DNJ) and its lack of\ud microbial activity prevented use of a bioassay. However,\ud the inhibition of cc-glucosidase by DNJ was exploited by\ud development of a quantitative assay system f or DNJ and\ud nojirimycin (NOJ). The assay could not only detect DNJ\ud and NOJ-producing clones, but could also assess the titre\ud of DNJ and NOJ in culture broths. The assay was used to\ud demonstrate differential production of DNJ and NOJ by\ud selected StreRtOIFYces strains of cluster 61 (the\ud S-lavendulae species group). The assay also confirmed\ud the effectiveness of using microtitre plates as an\ud effective screening procedure. The microtitre screening\ud programme generated further data and statistical\ud treatment of the results delimited the number of isolates\ud for further examination. No DNJ-producing colony was\ud detected and examination of the size of the DNA inserts\ud showed almost all were too small to contain the DNJ gene\ud cluster.\ud Additionally, blocked DNJ production mutants were\ud characterised by the feeding of NOJ, one of the mutants\ud successfully converted NOJ to DNJ.
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