Lecture 3

1. Lecture 3 Ti plasmid derived vector system

2. Ti Plasmid T-DNA region DNA between L and R borders is transferred to plant as ssDNA; T-DNA encoded genes can be substituted by target genes Tumor- producing genes Opine catabolism Virulence region ORI

6. Ti-plasmid based vectors Binary systems Co-integrated vectors Needs 3 vectors Needs 2 vectors: Disarmed Ti plasmid capable for infection Disarmed Ti plasmid with gene of interest (no vir genes) Helper vector for infection (with vir genes) Form co-integrated plasmid after homologous recombination on T-DNA Intermediate vector with T-region and gene of interest (transferred by conjugation) Helper vector for transfer of intermediate plasmid into A.tum

9. Co-integrated vectors (hybrid ti-plasmids) Right now rarely used DISADVANTAGES: 1) Long homologies required between the Ti plasmid and the E. coli plasmids (pBR322 based Intermediate vectors) making them difficult to engineer and use 2) Relatively inefficient gene transfer compared to the binary vecto

12. Ti plasmid vector systems are often working as binary vectors T DNA region removed Gene of interest Plant selectable marker HELPER plasmid Disarmed Ti plasmid Virulence region Bacterial selectable marker ori for A. tum ori for A. tumefaciens ori for E.coli DISADVANTAGE: Depending on the orientation, plasmids with two different origins of replication may be unstable in E. coli ADVANTAGE: small vectors are used, which increases transfer efficiency from E. coli to Agrobacterium. No intermolecular recombination is needed