RecipharmCobra Biologics Receives Patent for Antibiotic-Free Plasmid Maintenance System

September 8, 2010

RecipharmCobra Biologics Receives Patent for Antibiotic-Free Plasmid Maintenance System

RecipharmCobra Biologics (Keele, UK) has been granted European and US patents for oriSELECT, its antibody-free plasmid maintenance system.
Classically, genes of interest are inserted into plasmids with antibiotic resistance genes, and then inserted into E. coli with antibiotics present. Only E. coli cells that contain both the inserted genes and the antibiotic resistance genes will survive to produce plasmids or proteins. OriSELECT enables the production of plasmids or proteins in E. coli without the requirement for antibiotics in the medium or for plasmid selection and maintenance. This avoids some of the problems associated with the conventional selection method, including loss of the plasmid from the cell during cell division (which can reduce product yield), product contamination with antibiotics, and the potential transfer risk of antibiotic resistance genes to pathogens.
Although there are other antibiotic-free plasmid maintenance systems available, oriSELECT requires no selectable marker genes on the plasmid, thereby allowing smaller plasmids to be produced, and reducing the metabolic burden associated with selectable marker gene expression. The key to oriSELECT is that the pMB1 plasmid origin of replication, which is essential for plasmid replication, acts as the selectable marker. This means that no further modifications are needed to the plasmid, and existing plasmids can have their antibiotic resistance genes excised for use in the oriSELECT system.
The pMB1 origin of replication produces two RNA molecules, which regulate plasmid replication. The oriSELECT E. coli strain is modified such that an essential gene is controlled by one of these RNA molecules, and is switched off in the absence of a plasmid, preventing cell growth. Insertion of a plasmid enables the essential gene to be expressed, thus permitting cell growth and plasmid maintenance.