Enhanced plasmid production in miniaturized high-cell-density cultures of Escherichia coli supported with perfluorinated
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ORIGINAL PAPER
Enhanced plasmid production in miniaturized high-cell-density cultures of Escherichia coli supported with perfluorinated oxygen carrier Maciej Pilarek • Eva Brand • Friederike Hillig Mirja Krause • Peter Neubauer
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Received: 24 September 2012 / Accepted: 9 November 2012 / Published online: 23 November 2012 Ó The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract A simple method for plasmid minipreps in closed 1.5 mL microcentrifuge tubes using a cultivation medium with internal substrate delivery (EnBaseÒ) 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 lL 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 lL-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.
M. Pilarek E. Brand F. Hillig M. Krause P. Neubauer Chair of Bioprocess Engineering, Department of Biotechnology, Technische Universita¨t Berlin, Ackerstrasse 71-76, ACK24, 13355 Berlin, Germany M. Pilarek (&) Biotechnology and Bioprocess Engineering Division, Faculty of Chemical and Process Engineering, Warsaw University of Technology, Waryn´skiego 1, 00-645 Warsaw, Poland e-mail: [email protected]
Keywords High titre plasmid DNA production Escherichia coli Miniaturized fed-batch culture Liquid oxygen carrier Perfluorochemical (perfluorocarbon)
Introduction Successful high-yield isolation of high-quality plasmid DNA is determined by the bacterial cell culture conditions. The aim of all preparative plasmid production cultures should be the selection of best culture conditions to maximize the yield of plasmids in their covalently closed circular DNA (CCC DNA) form. For plasmid purification, well-designed kits are available from many commercial producers. Thus, it is remarkable that the standard cultivation procedure which typically uses 2 mL of LB medium overnight as the standard procedure for miniprep cultures has not been critically evaluated to date in view of robustness and quality control. This is mainly due to the wide need to apply a very simplistic method for plasmid p
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