The production of plasmid pEGFP-N1 in Escherichia coli DH5 alpha was optimised. A strategy evaluating different media components separately was not successful (OD < 2.5, low plasmid titres), a statistical approach via a Plackett Burman design (11 parameters) allowed some improvement (7 mg/L plasmid, OD600 8.5). Generally, high biomass did not correlate with high plasmid titres. When conditions were transferred to the bioreactor (batch operation) little improvement in plasmid titres (10 mg/L plasmid, OD600 20) was observed. By switching to a fed-batch procedure with linear feeding these values increased to 20 mg/L plasmid (OD600 50). By using an adaptive feeding strategy, plasmid titres could be increased to 50 mg/L. Finally, by combining a growth controlled (reduced temperature (35 degrees C, low dO(2)) initial batch phase with an adaptive feeding strategy in the fed-batch phase (37 degrees C, glucose-/dO(2)-limitation) we were reproducibly able to produce up to 250 mg/L of plasmid DNA in cultures that reached a final OD600 of 80. (C) 2007 Elsevier Ltd. All rights reserved.