title: Enhanced plasmid production in miniaturized high-cell-density cultures of Escherichia coli supported with perfluorinated oxygen carrier.
abstract: A simple method for plasmid minipreps in closed 1.5 mL microcentrifuge tubes using a cultivation medium with internal substrate delivery (EnBase(®)) in combination with a two-phase perfluorodecalin (PFD) system supplying additional oxygen to the E. coli culture is described. The procedure can simply be performed on a thermoshaker using only 50 μL cultivation volume. Twenty and twenty-five percent higher cell densities and plasmid concentration, respectively, were obtained with the additional oxygen delivery system when compared to cultures without PFD. Compared to standard 2 mL LB cultures ninefold higher cell densities and eightfold higher plasmid concentrations were achieved for the smaller culture volume. The μL-scale cultures can be directly utilized in further plasmid purification without any centrifugation step or the subsequent removal of the supernatant. This simplifies the routine procedure considerably. Furthermore, the new method is very robust considering the time of cultivation. Highest plasmid concentrations were already obtained after only 6 h of cultivation, but the plasmid concentration remained high (87 % of the maximum) even until 8 h of cultivation. Aside from the advantage of this method for the daily routine, we believe that it could also be applied to automated high-throughput processes.
authors.affiliation: Chair of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Berlin, Germany. pilarek@ichip.pw.edu.pl
authors.forename: Maciej
authors.initials: M
authors.lastname: Pilarek
authors.forename: Eva
authors.initials: E
authors.lastname: Brand
authors.forename: Friederike
authors.initials: F
authors.lastname: Hillig
authors.forename: Mirja
authors.initials: M
authors.lastname: Krause
authors.forename: Peter
authors.initials: P
authors.lastname: Neubauer
mesh_terms: D019149:Bioreactors; D001709:Biotechnology; D003470:Culture Media / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005285:Fermentation; D005466:Fluorocarbons / Q000737:chemistry*; D007218:Industrial Microbiology; D010100:Oxygen / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism*; D013053:Spectrophotometry; D013997:Time Factors
chemical_list: D003470:Culture Media; D005466:Fluorocarbons; C008805:perfluorodecalin; D004247:DNA; D010100:Oxygen