Effect of Plasmid Copy Number and lac Operator Sequence on Antibiotic-Free Plasmid Selection by Operator-Repressor Titration in Escherichia coliCranenburgh R.M. · Lewis K.S. · Hanak J.A.J.
Cobra Biomanufacturing Plc, The Science Park, Keele, Staffs., UK
The Escherichia coli strain DH1lacdapD enables plasmid selection and maintenance that is free from antibiotics and selectable marker genes. This is achieved by using only the lac operator sequence as a selectable element. This strain is currently used to generate high copy number plasmids with no antibiotic resistance genes for use as DNA vaccines and for expression of recombinant proteins. Until now these have been limited to pUC-based plasmids containing a high copy number pMB1-derived origin of replication, and the principle lacO1 and auxiliary lacO3 operators. In this study we have shown that this system can also be used to select and maintain pBR322-based plasmids with the lower copy number pMB1 origin of replication, and that lacO1 alone or a palindromic version of lacO1 can provide a sufficient level of repressor titration for plasmid selection. This is advantageous for recombinant protein production, where low copy number plasmids are often used and plasmid maintenance is important. The degree of repressor titration due to these plasmids was measured using the natural lactose operon in E. coli DH1 as a model.
© 2004 S. Karger AG, Basel
Rocky M. Cranenburgh
Cobra Biomanufacturing Plc
The Science Park
Keele ST5 5SP (UK)
Tel. +44 1782 714181, Fax +44 1782 799817, E-Mail email@example.com
Number of Print Pages : 7
Number of Figures : 4, Number of Tables : 1, Number of References : 29
Journal of Molecular Microbiology and Biotechnology
Vol. 7, No. 4, Year 2004 (Cover Date: Released September 2004)
Journal Editor: Saier, M.H., Jr. (La Jolla, Calif.)
ISSN: 1464–1801 (print), 1660–2412 (Online)
For additional information: http://www.karger.com/mmb