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Biotechnology and Applied Biochemistry (2003) 38, (1–7) (Printed in Great Britain)

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Impact of plasmid size on cellular oxygen demand in Escherichia coli

Alison Kay*¹, Ronan O'Kennedy*², John Ward† and Eli Keshavarz-Moore*³

*The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, U.K., and †Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, U.K.

Key words: chemostat, gene therapy, plasmid DNA, plasmid size, recombinant protein production.

Abbreviations used: DNS, dinitrosalicylic acid; IPTG, isopropyl b-D-thiogalactoside; LBM agar, modified Luriana–Bertrani agar; OUR, oxygen uptake rate; X-gal, 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside.

¹Present address: Eli Lilly and Company, Fleming Road, Speke, Liverpool L24 9LN, U.K.

²Present address: GlaxoSmithKline, South Eden Road, Beckenham, Kent BR3 3BS, U.K.

³To whom correspondence should be addressed (e-mail e.keshavarz-moore@ucl.ac.uk).

Escherichia coli DH5a harbouring either pBGS18, a 4.4 kb pUC-based plasmid, or pQR150, a 20 kb derivative of pBGS18, were grown in glucose-limited chemostat culture to investigate the effects of plasmid size and recombinant protein production on oxygen demand. Under non-induced conditions, where no recombinant protein was expressed, the cellular oxygen demand of cells harbouring pBGS18 and pQR150 was not significantly different, mean specific oxygen uptake rate (OUR) being 0.75±0.24 and 0.78±0.32 mmol/h per mg of plasmid respectively. Under isopropyl b-D-thiogalactoside-induced conditions, where recombinant protein was expressed, cells harbouring pBGS18 demonstrated a statistically insignificant 37% increase in mean specific OUR while those harbouring pQR150 demonstrated a statistically significant 415% increase. It is concluded that plasmid size does not significantly affect oxygen demand and that increasing oxygen demand observed with increasing plasmid size is due to production of recombinant protein.

Received 30 January 2003/7 April 2003; accepted 11 April 2003

Published as Immediate Publication 11 April 2003, DOI 10.1042/BA20030022

© 2003 Portland Press Ltd