Strategies for high titre plasmid DNA production in Escherichia coli DH5α

Abstract The production of plasmid pEGFP-N1 in Escherichia coli DH5α was optimised. A strategy evaluating different media components separately was not successful (OD  600 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, OD 600 20) was observed. By switching to a fed-batch procedure with linear feeding these values increased to 20 mg/L plasmid (OD 600 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 °C), low dO 2 ) initial batch phase with an adaptive feeding strategy in the fed-batch phase (37 °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 OD 600 of 80.