A non-antibiotic marker for amplification of plant transformation vectors in E. coli

Abstract

Concern over perceived risks from the presence of antibiotic resistance genes in transgenic plants is leading to selection strategies which do not use antibiotic resistance genes as selectable markers. The concern regarding the presence of antibiotic resistance genes has focused particularly on the bla gene for ampicillin resistance, which is used as a bacterial marker when pUC-based plasmids are amplified prior to microprojectile bombardment. The rtl operon, which permits the use of ribitol as the sole carbon source, is present in Escherichia coli strain C but absent in all the K-12 laboratory strains commonly used for molecular biology. A ClaI fragment containing the kinase, dehydrogenase and transporter components of the rtl operon was isolated from strain C and subsequently used to replace bla from two cloning vectors, pBluescript and pMECA, to create pBluescript-R and pMECA-R. E. coli K-12 strain DH10B transformed with either plasmid acquired the ability to grow on ribitol as the sole carbon source, as long as pMECA-R was present within the bacterial cell. Plasmid yields were evaluated and found to be comparable to those obtained from bacteria growing on LB medium supplemented with ampicillin. Hence, the use of genes from the rtl operon might be an acceptable alternative to the use of bla.