<i>E. coli </i>plasmid DNA production

摘要

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.

主权项

1. A method for extraction of plasmid DNA from gram negative bacterial cells, comprising the steps of:
a. introducing at least one gene encoding at least one peptidoglycan hydrolase into said bacterial cells, each bacterial cell having cytoplasm, a cytoplasmic membrane, an outer bacterial membrane, and an inner bacterial membrane; b. growing said bacterial cells in the presence of said plasmid; c. directing production of said peptidoglycan hydrolase from the introduced gene into the cytoplasm of the cells; and d. permeabilizing the cytoplasmic membrane at a slightly acid pH in the range of pH 4.5-6.0, whereby said method extracts said plasmid DNA from bacterial cells without complete cellular lysis.