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