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Title: Plasmid vector stability in Streptomyces lividans fermentations
Author: Wrigley-Jones, Carys A.
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1991
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Plasmid instability is frequently cited as a common problem in both laboratory and pilot-scale fermentations of recombinant microorganisms. Streptomyces are increasingly being explored as hosts for the production of useful cloned gene products since they are already employed industrially for antibiotic production. A method was developed to facilitate the study of plasmid copy number and structural stability in Streptomyces lividans cultures. The protocol involves total deoxyribonucleic acid (DNA) extraction followed by gel electrophoresis and scanning densitometry. The protocol has been successfully applied to follow transient changes in plasmid copy number of the multi-copy Streptomyces vectors pIJ303 and pIJ702. The stability of the pIJ702-derived recombinant plasmids pMT605 and pMT608 was also studied to a lesser extent. Experiments were conducted using shake-flask cultures and 5L working volume stirred vessels. Plasmid vector stability was tested by varying medium composition, growth temperature and levels of dissloved oxygen tension. The effects, upon plasmid copy number of the drug novobiocin and simulated Inoculum development protocols were also examined. Also the effect of different enzyme induction strategies upon tyrosinase expression encoded by pIJ702 was investigated. In general, comparable plasmid copy numbers were obtained from both shaken cultures and 5L fermentations. A dramatic increase in plasmid copy number was measured during initial rapid growth followed by a decline during stationary phase. Maximum plasmid copy number was always obtained in early stationary phase. Under low dissolved oxygen tension or high temperature conditions, recombinant cultures retained plasmid molecules. The yield of DNA under such conditions was low, but plasmid copy number remained stable. The relative quantities of plasmid forms (topolsomers) was found to change at various stages during growth. Supercolled forms were often seen to Increase as the culture ages. No structural instability was detected. These host-vector systems were generally stable but copious acid production by the host was found to be undesirable.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available