Mini-scale cultivation method enables expeditious plasmid production in Escherichia coli

  • The standard procedure in the lab for plasmid isolation usually involves a 2-mL, 16 h over-night cultivation in 15-mL bioreaction tubes in LB medium. This is time consuming, and not suitable for high-throughput applications. This study shows that it is possible to produce plasmid DNA (pDNA) in a 1.5-mL microcentrifuge tube with only 100 L cultivation volume in less than 7 h with a simple protocol. Compared with the standard LB cultivation for pDNA production reaching a final pDNA concentration range of 1.5-4 mu g mL(-1), a 6- to 10-fold increase in plasmid concentration (from 10 up to 25 mu g mL(-1) cultivation volume) is achieved using an optimized medium with an internal substrate delivery system (EnBase (R)). Different strains, plasmids, and the applicability of different inoculation tools (i.e. different starting ODs) were compared, demonstrating the robustness of the system. Additionally, dissolved oxygen was monitored in real time online, indicating that under optimized conditions oxygen limitation can be avoided. We developed aThe standard procedure in the lab for plasmid isolation usually involves a 2-mL, 16 h over-night cultivation in 15-mL bioreaction tubes in LB medium. This is time consuming, and not suitable for high-throughput applications. This study shows that it is possible to produce plasmid DNA (pDNA) in a 1.5-mL microcentrifuge tube with only 100 L cultivation volume in less than 7 h with a simple protocol. Compared with the standard LB cultivation for pDNA production reaching a final pDNA concentration range of 1.5-4 mu g mL(-1), a 6- to 10-fold increase in plasmid concentration (from 10 up to 25 mu g mL(-1) cultivation volume) is achieved using an optimized medium with an internal substrate delivery system (EnBase (R)). Different strains, plasmids, and the applicability of different inoculation tools (i.e. different starting ODs) were compared, demonstrating the robustness of the system. Additionally, dissolved oxygen was monitored in real time online, indicating that under optimized conditions oxygen limitation can be avoided. We developed a simple protocol with a significantly decreased procedure time, enabling simultaneous handling of more samples, while a consistent quality and a higher final pDNA concentration are ensured.show moreshow less

Export metadata

Additional Services

Share in Twitter Search Google Scholar Statistics
Metadaten
Author:Petra Grunzel, Maciej Pilarek, Doerte Steinbrueck, Antje Neubauer, Eva Brand, Michael Uwe KumkeORCiDGND, Peter Neubauer, Mirja Krause
DOI:https://doi.org/10.1002/biot.201300177
ISSN:1860-6768 (print)
ISSN:1860-7314 (online)
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=24130162
Parent Title (English):Biotechnology journal : systems & synthetic biology, nanobiotech, medicine
Publisher:Wiley-VCH
Place of publication:Weinheim
Document Type:Article
Language:English
Year of first Publication:2014
Year of Completion:2014
Release Date:2017/03/27
Tag:Escherichia coli; High-cell-density culture; Miniaturized cultivations; Optical oxygen sensor; Plasmid DNA production
Volume:9
Issue:1
Pagenumber:9
First Page:128
Last Page:136
Funder:German Federal Ministry of Education and Research (BMBF) [02PJ1150]
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Chemie
Peer Review:Referiert