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A method for plasmid copy number determination in recombinant Streptomyces

Wrigley-Jones, CA; Richards, H; Thomas, CR; Ward, JM; (1992) A method for plasmid copy number determination in recombinant Streptomyces. Journal of Microbiological Methods , 16 (1) pp. 69-80. 10.1016/0167-7012(92)90026-Z.

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Abstract

A method for the measurement of plasmid copy number in Streptomyces mycelia is described. It is based on the preparation (on a mini-scale) of the total DNA present in the mycelium followed by gel electrophoresis with ethidium bromide, photography and densitometric scanning of photographic negatives. The method is suitable for pIJ101-derived multi-copy plasmids and is shown to have comparable accuracy to similar methods developed for unicellular host organisms. A novel 'copy number' reconstitution approach was employed to define the sensitivity of the method. We recommend the optimisation of plasmid topoisomer separation since we have found evidence for transient changes in their relative proportions for certain Streptomyces plasmids. Various plasmid topoisomers present in the extracts were identified by progressive DNasel nicking of pure plasmid DNA. © 1992.

Type: Article
Title: A method for plasmid copy number determination in recombinant Streptomyces
DOI: 10.1016/0167-7012(92)90026-Z
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: http://discovery.ucl.ac.uk/id/eprint/10025593
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