Ti plasmid derived vector system

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
3
(No Transcript)
4
(No Transcript)
5
(No Transcript)
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
7
(No Transcript)
8
(No Transcript)
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
10
(No Transcript)
11
(No Transcript)
12
Ti plasmid vector systems are often working as
binary vectors
T DNA region removed
Gene of interest
Plant selectable marker
HELPER plasmid
Virulence region
Disarmed Ti plasmid
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
13
(No Transcript)