Lecture 3
Ti plasmid derived vector system
Ti Plasmid
Tumor-producing genes
Virulence region
Opine catabolism
ORI
T-DNAregion
DNA between L and R borders istransferred to plantas ssDNA;
T-DNA encoded genes can be substituted by target genes
Ti-plasmid based vectors
Binary systems Co-integrated vectors
Needs 2 vectors: Needs 3 vectors
Disarmed Ti plasmid with gene of interest(no vir genes)
Helper vectorfor infection(with vir genes)
Disarmed Ti plasmid capable for infection
Intermediate vector with T-region and gene of interest (transferred by conjugation)
Form co-integrated plasmidafter homologous recombination on T-DNA
Helper vectorfor transfer of intermediate plasmid into A.tum
Co-integrated vectors (hybrid ti-plasmids)
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
Right now rarely used
Ti plasmid vector systems are often working as binary vectors
Virulence region
T DNA region removed
ori for A. tum
Gene of interest
Plant selectable marker
Bacterial selectable marker
ori for A. tumefaciensori for E.coli
HELPER plasmid
Disarmed Tiplasmid
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