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US 2016/0355827 A1Published application
E. coli plasmid dna production
AssigneeNature Technology Corporation
InventorsJAMES A. WILLIAMS, CLAGUE P. HODGSON, AARON E. CARNES
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Abstract
General methods and strains of bacteria are described that dramatically simplify and streamline plasmid DNA production. In one preferred embodiment, endolysin mediated plasmid extraction combined with flocculation mediated removal of cell debris and host nucleic acids achieves increased yield and purity with simplified downstream purification and reduced waste streams, thus reducing production costs.
Claims8
-33. (canceled)
A method for production of plasmid DNA in E. coli cells,
- comprising the steps of:
- a. introducing at least one gene encoding at least one peptidoglycan hydrolase into said E. coli cells, each E. coli cell having cytoplasm, a cytoplasmic membrane, and an outer bacterial membrane;
- b. growing said E. coli cells in the presence of said plasmid;
- c. directing the production of said peptidoglycan hydrolase from the introduced gene into the cytoplasm of the cells;
- d. permeabilizing the cytoplasmic membrane at a slightly acid pH in the range of pH4.5-6.0 to extract said plasmid DNA from said E. coli cells without complete cellular lysis;
- e. adding a flocculating agent in order to flocculate cell debris and bacterial genomic DNA into flocculant precipitates;
- f. removing flocculant precipitates to create a clarified plasmid DNA solution;
- and
- g. purifying said plasmid DNA from said clarified plasmid DNA solution.
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Dependencies are inferred from the claim text.