TI VECTOR

(CIRCULAR)
Molecular Biotechnology – Group 4
Lecturer: Assoc. Prof. Nguyen Phuong Thao
 MEMBER OF GROUP
STT Name ID Contribute

1 Lê Thị Thảo Mi BTBTIU17035

2 Nguyễn Ngọc Thảo Uyên BTBTIU20244

3 Phạm Nguyễn Minh Trí BTBTIU20283

4 Trương Anh Thư BTBTIU19121

5 Nguyễn Thị Thu Linh BTBTIU20282

6 Đỗ Bùi Khánh My BTBTIU20192

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 TABLE OF CONTENTS
I II III IV
Structure Future
Introduction and Applications
Prospects
Mechanism

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 I. INTRODUCTION
Vector - small DNA molecule that carries foreign DNA
molecules into the host cell (termed as cloning vehicle or
cloning DNA)
Example: Cosmid, Bacteriophage, etc
Plasmid - the extrachromosomal DNA structure that carries
foreign DNA molecules
Example: pBR322, pUC18, pUC19, etc
Ti plasmid (Tumor-inducing plasmid) is a part of Ti vector

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 I. INTRODUCTION
Ti plasmid found in Agrobacterium tumefaciens which
causes crown ball disease in certain dicot plant species.
Agrobacterium tumefaciens can transform normal cells
into tumor cells by inserting a DNA piece
Discovered in the 1970s
It is widely used as a cloning vector to deliver desirable
genes to the host plant

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MAIN CHARACTERISTICS OF TI PLASMID
Size of the plasmid is ~ 250kbp - contains one or more
T-DNA region
The desired gene can be inserted into Ti plasmid -> loses
its pathogenic ability
It contains virulence genes
It can be modified as per the requirement to insert the
desired genes

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MAIN CHARACTERISTICS OF TI PLASMID
Based on the differences in the T-DNA region, there are two
main types of Ti plasmids: Nopaline, Octopine
Nopaline Octopine
It has a continuous It is subdivided into two
region of T-DNA, which is regions which are 13 kb and
approximately 25 kb in 8 kb long.
length. It produces an opine known
It produces an opine as Octopine (C9H18N4O4).
known as Nopaline It was first isolated from
(C11H20N4O6). Octopus octopodia in 1927.
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II. STRUCTURE AND MECHANISM
1 2

Structure of Ti Principle and

plasmid Mechanism

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 1.TI PLASMID STRUCTUE

1 Transfer DNA (T-DNA) region

2 Virulence (vir) region

3 Opine catabolism region

4 Origin (ori) of replication

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 TRANSFER DNA (T-DNA) REGION

T-DNA borders:
+ Blanked by left right border and right border
+ Right border play a important role in transfer and intergration
of T-DNA
LB, RB: left and right borders (direct repeat)
auxA + auxB: enzymes that produce auxin
cyt: enzyme that produces cytokinin
Ocs: octopine synthase, produces octopine
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 Operon Function
THE VIRULENCE
VirA
Transports acetosyringone into bacterium, activates virG
post-translationally (by phosphorylation)
(VIR) REGION
VirG Promotes transcription of other vir genes

Endonuclease/Integrase that cuts T-DNA at the borders

VirD2
but only on one strand On the Ti plasmid.
Transfer the T-DNA into
VirE2 Can form channels in membranes plant chromosome.
virA,B,C,D,E,F,G - 7
VirE1 Chaperone for virE2
complementation groups.
Operon of 11 proteins, gets T-DNA through bacterial
VirB
membranes

VirD2 and virE2 Also have NLSs, gets T-DNA to the nucleus of plant cell
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OTHER STRUCTURE OF TI PLASMID
Opine catabolism Origin of
region replication
Ori region that is
This regions code for
responsible for the
protein involved in the
origin of DNA
uptake and
replication which
meatabolism of opines
permits the Ti plasmid
to be stably
maintained in
A.tumefaciens
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Two types of Ti plasmid-derived vectors are used for
genetic transformation of plants by agrobacterium

1 2
Co-integrate
Binary vector
vector

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 CO-INTEGRATE VECTOR
The normal co-integrated plasmid which is
assembled by in vitro manipulation contains:
Vir genes
The left and right T-DNA borders
Exogenous DNA sequence between the two
T-DNA borders
Plant and bacterial selectable markers

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 BINARY VECTOR
Gene of interest (foregin gene)
LB: contains the promoter and enhancer sequences
T-DNA
RB: contains the terminator sequence
Disamered Bacteria selectable
plasmid marker Plant selectable marker
Ori (E.coli and Agrobacterium)

BINARY VECTOR
Ori
Helper
plasmid
Virulence gene

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 Principle of Ti
vector
Agrobacterium uses Ti plasmid
to transfer DNA to wounded
plants, activating virulence
genes and Vir proteins for
precise genetic engineering.

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T-DNA TRANSFER AND INTEGRATION
1. Signal recognition by Agrobacterium
Agrobacterium perceive signals such as sugar and phenolic
compounds which are released from plants
2. Attachment to plant cells
Two step processes:
+ i) initial attachment polysaccharide
+ ii) mesh of cellulose fiber is produced by bacteria.
Virulence genes are involved in the attachment of bacterial cells
to the plants cells.

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T-DNA TRANSFER AND INTEGRATION
3. Production of virulence proteins
VirA senses phenolics ans subsequently phosphorylating and
thereby activating VirG. VirG then induces expression of all the
vir genes.
4. Production of T-DNA strand
VirD1/virD2 complex recognises the LB and RB. virD2 produces
single-stranded nicks in DNA. Then virD2 attached to ssDNA. virC
may assist this process.

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T-DNA TRANSFER AND INTEGRATION
5. Transfer of T-DNA out of Agrobacterium
The ss T-DNA/VirD2 complex in association with vir G is exported
from the bacterial cell. Vir B products form the transport
apparatus.
6. Transfer of T-DNA into plant cells and integration
T-DNA and Vir proteins cross plasma membrane channels to
facilitate nuclear localization in plants.
VirE2 protects T-DNA from nucleases and ensures correct
conformation, integrating into host chromosome.

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 T-DNA TRANSFER AND INTEGRATION

Figure: Major
steps of the
Agrobacterium
tumefaciens-
mediated plant
transformation
process.

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III. APPLICATION
1 2
Advantages and
Use of Ti vector Disadvantages of
(circular) using Ti vector
(circular)

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 USE OF TI VECTOR (CIRCULAR)
Producing transgenic plants.
With the use of restriction enzymes, a particular gene of
interest can be inserted into the plasmid and transformed.
It is used for the development of stress-tolerant plant
varieties.
Ti plasmids are also responsible for inducing amino acids
and sugar-phosphate derivatives known as opines.

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Figure: Agrobacterium-Mediated gene transfer to plant
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 TI PLASMID DERIVED VECTOR SYSTEM
*The use of wild type Ti plasmid as a vector presents the problems
1) Presence of oncogenes in T-DNA.
2) The Ti plasmid is too large to manipulate easily in vitro.
3) A general lack of unique cloning sites with in the T-DNA.
4) Opine production in transformed plant cell lowers the plant yield.
5) Ti plasmids cannot replicate in E. coli this limits their utility as E. coli.

*These problems have been solved by:

1) Deleting the oncorenes from the T-DNA (Disarming).
2) Two strategies evolved to overcome this manipulation difficulty.

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 2. ADVANTAGES AND DISADVANTAGES
OF USING TI VECTOR (CIRCULAR)
ADVANTAGES DISADVANTAGES

Natural gene delivery Restricted host range

Stable integration The removal of phytohormones

High transformation efficiency Inability to transport genes

into organelles

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IV. FUTURE PROSPECTS
1 2
Future
Conclusion
Prospects

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 1. CONCLUSION
Ti vector technology is a powerful tool for plant genetic
engineering
Virulence genes are responsible for the transfer of T-DNA
into the host cell and integration of T-DNA with the host
genome
T-DNA has three types of genes: Auxin, Cytokine,
Octopines synthetase.
Despite many advantages, wild-type Ti plasmids cannot be
directly used as vectors.

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 2. FUTURE PROSPECTS

Stable transgene expression in plants: need to understand

and control the factors that affect transgene expression
stability.

Plastid genetic transformation by Agrobacterium: There are

challenges in redirecting T-DNA from the nucleus to plastids,
despite the existence of NLS sequences in VirD2 and VirE2
proteins.

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 2. FUTURE PROSPECTS

Genetic transformation of human and animal cells:

Agrobacterium-mediated transformation suggests the exciting
possibility of using Agrobacterium, or Agrobacterium-like
processes, for human and animal gene therapy.

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 REFERENCES
Schell, J., & Van Montagu, M. (1977). The Ti-plasmid of Hiei, Y., Komari, T., & Kubo, T. (1997). Transformation of
Agrobacterium tumefaciens, a natural vector for the rice mediated by Agrobacterium tumefaciens. Plant
introduction of nif genes in plants?. Basic life molecular biology, 35, 205-218.
sciences, 9, 159–179. Gordon, J. E., & Christie, P. J. (2014). The Agrobacterium
Admin. (2022, August 9). Difference between Plasmid and Ti Plasmids. Microbiology spectrum, 2(6),
Vector. BYJUS. https://byjus.com/biology/difference- 10.1128/microbiolspec.PLAS-0010-2013.
between-plasmid-and-vector/ Weaver, J., Goklany, S., Rizvi, N., Cram, E. J., & Lee-
Admin. (2023, July 26). Plasmid: Definition, Structure, Parsons, Procedure or Protocol of Gene Transfer:
C. W. (2014). Optimizing the transient fast
Procedure or Protocol of Gene Transfer:
vector, PBR322, TI Plasmid. BYJUS. agro-mediated seedling transformation (FAST) method in
https://byjus.com/neet/plasmid/ Catharanthus roseus seedlings. Plant cell reports, 33,
Kulkarni, N. A. (2023, August 3). Ti plasmid- Definition, 89-97.
Structure, Types, Applications. Microbe Notes. Hwang, H. H., Yu, M., & Lai, E. M. (2017). Agrobacterium-
https://microbenotes.com/ti-plasmid-structure-types- mediated plant transformation: biology and
applications/#structure-of-ti-plasmid applications. The Arabidopsis Book, 15.
Adriana Gallego, Ph.D. An Overview of Stable and
Transient Transformation

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 QUIZ TIME
Let's Put Your Knowledge to The Test!
 QUESTION:
How many main structures of Ti plasmid?

2 4

3 5

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 TI PLASMID STRUCTUE

1 Transfer DNA (T-DNA) region

2 Virulence (vir) region

3 Opine catabolism region

4 Origin (ori) of replication

 QUESTION:
How many types of Ti plasmid?

1 3

2 4

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Two types of Ti plasmid-derived vectors are used for
genetic transformation of plants by agrobacterium

1 2
Co-integrate
Binary vector
vector
THANK YOU!
 DOES ANYONE
HAVE A QUESTION?