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Lecture 3. Ti plasmid derived vector system. 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. Ti-plasmid based vectors.

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Lecture 3

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Lecture 3 l.jpg

Lecture 3

Ti plasmid derived vector system


Ti plasmid l.jpg

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


Ti plasmid based vectors l.jpg

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


Co integrated vectors hybrid ti plasmids l.jpg

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


Ti plasmid vector systems are often working as binary vectors l.jpg

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


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