title: Effect of plasmid copy number and lac operator sequence on antibiotic-free plasmid selection by operator-repressor titration in Escherichia coli.
abstract: 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 lacO(1) and auxiliary lacO(3) 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 lacO(1) alone or a palindromic version of lacO(1) 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.
authors.affiliation: Cobra Biomanufacturing Plc, The Science Park, Keele, Staffs., UK. rocky.cranenburgh@cobrabio.com
authors.forename: Rocky M
authors.initials: RM
authors.lastname: Cranenburgh
authors.forename: Kathryn S
authors.initials: KS
authors.lastname: Lewis
authors.forename: Julian A J
authors.initials: JA
authors.lastname: Hanak
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics / Q000502:physiology*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics* / Q000502:physiology; D005798:Genes, Bacterial; D007763:Lac Operon; D009875:Operator Regions, Genetic; D010957:Plasmids; D018741:Replication Origin; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012641:Selection, Genetic; D014169:Transformation, Bacterial; D001616:beta-Galactosidase / Q000032:analysis
chemical_list: D012097:Repressor Proteins; D000217:Acyltransferases; C040777:succinyl-CoA-tetrahydrodipicolinate N-succinyltransferase; D001616:beta-Galactosidase