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

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.
Author Maciej Pilarek (FCPE / DBBE)
Maciej Pilarek,,
- Department of Biotechnology and Bioprocess Engineering
, Eva Brand - [Technical University of Berlin]
Eva Brand,,
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, Friederike Hillig - [Technical University of Berlin]
Friederike Hillig,,
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, Mirja Krause - [Technical University of Berlin]
Mirja Krause,,
-
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, Peter Neubauer - [Technical University of Berlin (TU Berlin)]
Peter Neubauer,,
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- Technische Universität Berlin
Journal seriesBioprocess and Biosystems Engineering, ISSN 1615-7591
Issue year2013
Vol36
No8
Pages1079-1086
Publication size in sheets0.5
ASJC Classification2700 General Medicine; 1502 Bioengineering; 1305 Biotechnology
DOIDOI:10.1007/s00449-012-0861-7
URL http://link.springer.com/article/10.1007/s00449-012-0861-7
Languageen angielski
File
Pilarek M. (i in.) - Enhanced plasmid production....pdf 287.32 KB
Score (nominal)30
Score sourcejournalList
ScoreMinisterial score = 25.0, 06-08-2020, ArticleFromJournal
Ministerial score (2013-2016) = 30.0, 06-08-2020, ArticleFromJournal
Publication indicators WoS Citations = 20; Scopus Citations = 20; GS Citations = 27.0; Scopus SNIP (Source Normalised Impact per Paper): 2013 = 1.076; WoS Impact Factor: 2013 = 1.823 (2) - 2013=2.012 (5)
Citation count*27 (2020-09-14)
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* presented citation count is obtained through Internet information analysis and it is close to the number calculated by the Publish or Perish system.
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