Cloning vectors ppt kiran b k final

CLONING VECTORS AND THEIR
APPLICATIONS
IN GENETIC ENGINEERING
A PRESENTATION BY
KIRAN B K
JUNIOR MSc
PALB 6296
DEPARTMENT OF PLANT PATHOLOGY
GKVK, BENGALURU
2

INTRODUCTION
CLONING :
• Cloning is the process of producing similar populations of genetically
identical individuals that occurs in nature.
• Cloning refers to processes used to create copies of DNA fragments
(molecular cloning), cells (cell cloning), or organisms.
VECTORS
• A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the
cell of a higher organism, that can be stably maintained in an organism, and into
which a foreign DNA fragment can be inserted(for cloning purposes).
3

In 1973, Stanley Cohen and Herbert Boyer described first successful
construction of recombinant vector.
Plasmid PSC101 – E.coli
4

Why TO Clone DNA?
• A particular gene can be isolated and its nucleotide sequence determined and
analyzed.
• Protein/enzyme/RNA function can be investigated
• Mutations can be identified, e.g. gene defects related to specific diseases
• Organisms can be ‘engineered’ for specific purposes, e.g. insulin production,
insect resistance, etc.
5

Cloning vectors and expression vectors:
• Cloning vectors are used to amplify DNA fragments, usually in E. coli.
They do not need a promotor to express the target sequence.
• Expressing vectors require specific promotors(prokaryotic, eukaryotic or RNA
promotors depending on what kind of cell you wish to express) the gene
product in.
6

Characteristics
• It should be able to replicate autonomously.
• Origin of replication.
• Selectable markers(often antibiotic resistance genes;)
• Reporter genes(these genes that allow successful clone to be easily identified. Such
feature present in cloning vectors is used in blue-white selection.)
• Restriction sites.
• Self replication, multiple copies.
• Replication origin site.
• Cloning site.
• Small size.(Larger plasmids are more difficult to characterize by restriction mapping and
replicate to lower copy numbers)
• Low molecular weight.
8

• No pathogenicity
• Easily isolated & purified.
• Easily isolated into host cell.
• Control elements – promoter, operator, ribosome binding site.
9

Vectors targeted host
• Plasmid
• Bacteriophages
• Cosmid
• Yeast Cloning Vectors
• Ti & Ri Plasmids
10
Bacteria, Streptomyces
Bacteria
Bacteria
Yeasts
Transformation of cloned
gene in higher plants.
TYPES OF CLONING VECTORS

1.PLASMID VECTOR
 Plasmid vector is a small, piece of circular
DNA found outside of the bacterial
chromosome.
 Capable of autonomous replication.
 Can transfer genes from one cell to other.
11

 Contains an origin of replication, allowing for replication independent of
host’s genome.
 Contains selective markers for the selection of cells containing a plasmid.
 Contains a Multiple Cloning Site (MCS).
 Easy to be isolated from the host cell.
 E.g pBR322, pUC19.
12

Plasmids have 3 key parts.
1. The origin of replication: used to indicate where DNA replication is to begin.
2. The selectable marker gene: used to distinguish cells containing the plasmid
from cells that don’t contain it.
3. The cloning site : a site in the plasmid where the DNA is inserted.
13

Varieties Of Plasmids Based On Functions:
(based on function there are five main classes:)
 Fertility / F- plasmids: They are capable of conjugation and result in the expression of
sex pilli.
 Resistance (R) plasmids: They contain genes that provide resistance against
antibiotics or poisons.
 Col plasmids: They contain genes that code for bacteriocins, proteins that can kill
other bacteria.
 Degradative plasmids: They enable the digestion of unusual substances, e.g. toluene
and salicylic acid.
 Virulence plasmids: They turn the bacterium into a pathogen.
14

15
pBR 322:
It contains:
• Selectable Markers:
• Ampicillin resistance gene.
• Tetracycline resistance gene.
• Col E I replication origin.
• Eco RI site(specific REN
recognition site.)
• 4.3kb size.
Structure of E.Coli plasmid cloning vector
pBR322
4362 bp

pUC 8:
– Popular E.coli cloning vector.
– Derivative of pBR322.
– Two parts derived:-
Ampicillin resistance gene.
ColEI – origin of replication.
– 2700 base pairs.
– lac Z gene derived from E.coli.
– Polylinker sequence having unique restriction sites lies in lac region.
16

pUC 18:
• Plasmid of Universty of California.
• J.Messing and J.Viera
• F¯ strain(does not contain fertility factor)
• 2686bp
• Antibiotic resistant gene for amphicillin
• Highly efficient Ori site’
• ∝ lac Z gene = codes for 𝛽 galactosidase
• MCS- It has 10-15 different sites for different restriction enzymes. And it is the
region where gene of interest is inserted.
17

Uses / IMPORTANCE of Plasmids :
• In genetic engineering: used to make copies of particular genes.
• Production of large amounts of proteins: This is a cheap and easy way of mass-
producing a gene or the protein it then codes for, for example, insulin or even
antibiotics.
• Molecular studies: of Plasmids are used in molecular studies of various organisms
i.e., in synthetic biology, medicine, ecology.
• Plasmids in Antibiotic Resistance: In addition plasmids carry antibiotic resistance
genes and their spread in pathogenic bacteria is of great medical importance.
• Gene therapy: Plasmid may also be used for gene transfer into human cells as
potential treatment in gene therapy so that it may express the protein that is lacking in
the cells.
19

ADVANTAGES AND DISADVANTAGES
ADVANTAGES:
• Readily isolated from cells
• Can be reintroduced into a bacterial cell
• Possess a single restriction site for 1 or more restriction enzyme
• MCS
• Small size (easy to manipulate and isolate).
• Circular (more stable).
20

• Replication independent of host cell.
• Several copies may be present (facilitates replication).
• Frequently have antibiotic resistance (detection easy).
• DISADVANTAGES
• Cannot accept large fragments(Sizes range from 0 – 10kb.)
21

BACTERIOPHAGE VECTOR
 Cloning Vector that uses a Bacteriophage as a means for making and storing
exact copies of segments of DNA.
 It infects bacteria.
 The bacteriophages used for cloning are ;
i. phage λ
ii. M13 phage.
22

Phage λ Vector
 Infect E.coli.
 Origin of replication.
 Size is 48,502 bp.
 High transformation efficiency, about 1000
times more efficient than the plasmid vector.
 Enterobacteria is important in the study of
specialized transduction
 cos site(cohesive end site)- site of cleavage of
phage DNA
23

Bacteriophage lamda
• Consists of an icosahedral head and flexible tail.
• Phage DNA packed inside the head
• Lambda DNA is a linear DNA duplex with cohesive single
strand extensions
• The single stranded extensions of the DNA are
COMPLEMENTARY to each other and consists of 12
nucleotides:Cos Sites
• Free end of the cos site has a 5’ phosphate group
24

Bacteriophage lambda vectors are commonly used for construction of genomic libraries
Advantages of this type of system vs plasmids like pBR322
• The phage genome is able to package efficiently with DNA inserts as large as 20 Kb.
(The packaged phage are highly infectious and infect E. coli at a much higher efficiency
than plasmid transformation methods.)
• Disadvantages:
• Lambda phage has narrow host range
26

Phage M13 Vector
• A gene for the lac repressor.
• The operator region of the lac Z gene.
• A lac promoter upstream of the lac Z gene
• A polylinker region
• Filamentous bacteriophage of Ecoli.
• Used for obtaining single stranded copies.
• Single stranded.
• Inside host cell become double stranded.
• Purpose: When the single-stranded DNA of a
fragment is required, a M 13 vector can be used
as a common cloning tool.
27

How M13 infects and reproduces
• infects through pili
• Protein coat is stripped and ss DNA is converted to double stranded replicative form
• DNA replicated by “rolling circle method”
• New particles assembled
• 200 particles per infected cell per generation
• M13 released without lysis
Uses
• Cloning
• ss DNA for probes, sequencing
• Phage display technology, M13 will produce foreign protein on surface as part of its
protein coat, can use to generate specific antibodies.
28

•
29
DNA Cloning Using Phages As Vectors

COSMIDS:
Combine parts of the lambda chromosome with parts of plasmids.
Contain the cos sites of λ and plasmid origin of replication.
Behave both as plasmids and as phages.
Cosmids can carry up to 50 kb of inserted DNA.
Structure of Cosmid
Origin of replication (ori).
Restriction sites for cleavage and insertion of foreign DNA.
Selectable marker from plasmid.
A cos site - a sequence yield cohesive end (12 bases).
Ampicillin resistance gene (amp).
30

• Cosmids are hybrids between a phage DNA molecule and a bacterial plasmid.
• It also needs a selectable marker, such as the ampicillin resistance gene, and a plasmid
origin of replication, as cosmids lack all the  genes and so do not produce plaques.
• Instead colonies are formed on selective media, just as with a plasmid vector.
32

Cloning By Using Cosmid Vectors
33

Uses Of Cosmids
34
1. Large DNA fragments
2. Used to establish gene libraries of lower & higher
organisms
3. Gene cloning through cosmid helps in the study of non-
sense sequence in the genome of organism.

• Phagemid
35
• A phagemid or phasmid is a plasmid that contains an
f1 origin of replication from an f1 phage.
• It can be used as a type of cloning vector in
combination with filamentous phage M13.
• A phagemid can be replicated as a plasmid, and also
be packaged as single stranded DNA in viral particles.

ARTIFICIAL CHROMOSOMEs
 Linear or Circular.
 1 0r 2 copies per cell.
 Different types –
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
P1 derived artificial chromosome (PAC)
Mammalian Artificial Chromosome (MAC)
Human Artificial Chromosome. (HAC)
 YAC – Cloning in yeast
 BAC & PAC – Bacteria
 MAC & HAC – Mammalian & Human cells.
36

Bacterial Artificial Chromosome
Bacterial artificial chromosome's usual insert size is 150-350 kbp.
Like other vectors, BACs contain
1. Origin (ori) sequence derived from an E. coli plasmid called the F factor.
2. Multiple cloning sites (restriction sites).
3. Selectable markers (antibiotic resistance)
37

 1st BAC Vector – PBAC108L.
 Cloning of large regions of eukaryotic genome.
 Origin of replication from bacterium Ecoli F -factor.
 BAC vectors are pBACe3.6, pBeloBAC11.
 Used in analysis of genomes.
• Host for BAC is mutant strain.
• Bacterial artificial chromosomes are sometimes introduced into their host cells by
electroporation, which consists of a brief treatment with high voltage electric
current that momentarily disrupts the cell membranes and facilitates entry of large
DNA molecules.
• Once in the cell, the BAC replicate like F plasmids.
38

YEAST ARTIFICIAL CHROMOSOME
 YAC are genetically engineered chromosomes derived from the DNA of the yeasts.
 Capable of carrying inserts of 100 - 1000 kbp.
 A YAC can be considered as a functional artificial chromosome since it includes
three specific DNA sequences:
 TEL: Telomere located at each chromosome end, protects the linear DNA from
degradation by nucleases.
 CEN: Centromere which is the attachment site for mitotic spindle fibers, "pulls"
one copy of each duplicated chromosome into each new daughter cell.
 ORI: Replication origin sequences which are specific DNA sequences.
40

Purpose:
 Cloning vehicles that propogate in eukaryotic cell hosts as eukaryotic
Chromosomes
 Clone very large inserts of DNA: 100 kb – 1000kb.
 Used to express eukaryotic proteins that require post-translational
modification.
 Used for detailed mapping of specific regions of the genome.
42

Human Artificial Chromosome(HAC)
• Synthetically produced vector DNA possessing the characteristics of human
chromosome.
• Discovered in 1997 by H.Williard.
Advantages:
1. Can carry Human genes that are too long
2. Can carry genes to be introduced into the cells via gene therapy
43

• Retroviral Vectors
44
• Retroviral vectors are used to introduce new or
altered genes into the genomes of human and
animal cells.
• Retroviruses are RNA viruses.
• The viral RNA is converted into DNA by the
viral reverse transcriptase and then is
efficiently integrated into the host genome.
• Any foreign or mutated host gene introduced
into the retroviral genome will be integrated
into the host chromosome and can reside there
practically indefinitely.
• Retroviral vectors are widely used to study
oncogenes and other human genes.

• Shuttle vectors:
45
Definition: They are the vectors that can shuttle
between more than one host, for example, one is E.
coli and the other is yeast.
Most of the vectors for use in eukaryotic cells are
constructed as shuttle vectors. E.coli
Yeast

Insect cell/Baculovirus
Definition: Baculovirus is an insect virus which can be
used for the overexpression of animal proteins in
insect cell culture.
Mechanism:
• Viral promoter: This viral gene has an extremely
active promoter.
• Insect cell culture: The same promoter can be used
to drive the over-expression of a foreign gene
engineered into the baculovirus genome.
Function: This method is being used increasingly for
large-scale culture of proteins of animal origin, since
the insect cells can produce many of the post-
translational modifications of animal proteins,
which a bacterial expression system cannot.
46
Baculovirus-infected SF21 cells

Screening procedure of cloning vector
48

Blue/White Selection
Only colonies
from bacteria that
have plasmid
IPTG + X-Gal
Overnight growth
Bacteria with
plasmid plus insert
Colonies with insert - white
Colonies w/o insert - blue
49

APPLICATIONS OF GENE CLONING IN
RESEARCH
1. Identifying the genes in a genome sequence
2. Determining the function of an unknown gene
3. To study the transcriptome and proteome
I. The transcriptome, which is the messenger RNA (mRNA)
content of a cell, and which reflects the overall pattern of gene
expression in that cell.
II. The proteome, which is the protein content of a cell and which
reflects its biochemical capability.
4. Studying protein–protein interactions
51

APPLICATIONS OF GENE CLONING IN
BIOTECHNOLOGY
• Production of Protein from Cloned Genes
• Production of recombinant pharmaceuticals in medicine. Eg.
Recombinant insulin
• Synthesis of human growth hormones in E. coli
• Recombinant vaccines eg. Vaccine for hepatitis B
• The gene addition approach to plant genetic Engineering in
agiculture field eg. Plants that make their own insecticides,
Herbicide resistant crops
52

Year Scientist /s Achievements
1928 Hilde Mangold First to perform somatic-cell nuclear transfer
using amphibian embryos – one of the first moves
towards cloning.
1950 Esther Lederberg Enterobacteria phage λ
1963 Hofschneider Bacteriophage M 13
1973 Herbet Boyer and Stanley Cohen First successful construction of recombinant vector
plasmid pSC101 in
E. coli
1977 Bolivar and Rodriguez. pBR322
1978 Collins and Hohn Cosmid
1982 Joachim Messing and co-workers pUC19
1983 Sanger Sequencing Bacteriophage M 13
1990s Nat Sternberg and colleagues PAC

Year Scientist /s Achievements
1996 Sir Ian Wilmut and Keith Campbell Dolly, a Finn-Dorset ewe was the first mammal to have
been successfully cloned from an adult somatic cell.
1997 Various institutions HACs were first constructed by adding alpha-satellite DNA
to telomeric and genomic DNA in human HT1080 cells.
2014 Jef Boeke of the Langone Medical Centre at
New York University
Synthesized one of the S. cerevisiae 16 yeast
chromosomes.

Agrobacterium tumefaciens—nature’s smallest
genetic engineer
• A. tumefaciens causes CROWN GALL DISEASE in many species
of dicotyledonous plants.
• Ccause a cancerous proliferation of the stem tissue in the
region of the crown.

Crown gall on blackberry caneCrown gall disease

Ti Plasmid
• This ability lies in Ti (tumor inducing) plasmid .
• This is a large (greater than 200 kb) plasmid .
• Carries numerous genes involved in the infective process.
• After infection, part of the molecule is integrated into the plant chromosomal DNA.
• This segment, called the T-DNA.
• Is between 15 & 30 kb in size, depending on the strain.
• It is maintained in a stable form in plant cell.
• And is passed on to daughter cells as an integral part of the chromosomes.

• T-DNA contains eight or so genes that are expressed in the
plant cell and are responsible for the cancerous properties of
the transformed cells.
• These genes also direct synthesis of unusual compounds,
called opines, that the bacteria use as nutrients.
• In short, A. tumefaciens genetically engineers the plant cell for
its own purposes.
Ti-plasmid gene
maps.

Using the Ti plasmid to introduce new genes into a
plant cell
• In practice this transfer is difficult mainly because the large size of the Ti plasmid
makes manipulation of the molecule very difficult.
• The main problem is, of course, that a unique restriction site is an impossibility with
a plasmid 200 kb in size.
• Novel strategies have to be developed for inserting new DNA into the plasmid.

The binary vector strategy
• Based on the observation that the T-DNA does not need to be
physically attached to the rest of the Ti plasmid.
• A two-plasmid system, with the T-DNA on a relatively small
molecule, and the rest of the plasmid in normal form, is just as
effective at transforming plant cells.
• In fact, some strains of A. tumefaciens, and related
agrobacteria, have natural binary plasmid systems.
• The T-DNA plasmid is small enough to have a unique restriction
site and to be manipulated using standard techniques.

The co-integration strategy
• Uses an entirely new plasmid, based on an E. coli vector, but carrying a small portion
of the T-DNA.
• The homology between the new molecule and the Ti plasmid means that if both are
present in the same A. tumefaciens cell, recombination can integrate the E. coli
plasmid into the T-DNA region.
• The gene to be cloned is therefore inserted into a unique restriction site on the
small E. coli plasmid, introduced into A. tumefaciens cells carrying a Ti plasmid, and
the natural recombination process left to integrate the new gene into the T-DNA.
• Infection of the plant leads to insertion of the new gene, along with the rest of the
T-DNA, into the plant chromosomes.

Transformation of plant cells by recombinant A. tumefaciens. (a) Infection of a wound: transformed plant cells are
present only in the crown gall. (b) Transformation of a cell suspension: all the cells in the resulting plant are
transformed.

• T-DNA contains gene for tumour.
• Has to be removed.
• Called Disarming.
• Since T-DNA is not needed for infection.
• Transformed plant cells are selected by plating onto agar
medium containing kanamycin.

The Ri plasmid
• Ri and Ti plasmds are simmilar,
• The main difference being that transfer of the T-DNA from an
Ri plasmid to a plant results not in a crown gall but in hairy
root disease,
• Ie., massive proliferation of a highly branched root system.
• This is used for obtaining large amounts of protein from
genes cloned in plants

• Ri plasmids are large (200 to greater than 800 kb) .
• Contain one or two regions of T-DNA and a vir (virulence)
region, all of which are necessary for hairy root formation.
• The Ri-plasmids are grouped into two main classes according
to the opines synthesized by hairy roots.

• First, agropine-type strains induce roots to synthesise
agropine, mannopine and the related acids.
• Second, mannopine-type strains induce roots to produce
mannopine and the corresponding acids.

Agrobacterium plasmid carries three genetic components that are
required for plant cell transformation.
• 1) T-DNA that is integrated into the plant cells, is a mobile DNA
element.
• 2)Virulence area (vir), which contains several vir genes.
(These genes do not enter the plant cell but, together with the
chromosomal DNA (two loci), cause the transfer of T-DNA.)

3) Border sequences (25 bp), resides in the Agrobacterium
chromosome.
(The mobility of T-DNA is largely determined by these sequences)

• The hairy roots are grown in vitro in bioreactors to study their
soil interaction with other pathogens like fungi and
nematodes.
• This technique has also led to the commercial production of
certain metabolic compounds that the plant is known to
secrete, especially in regard to the medicinal plants that are
difficult to cultivate in sufficient quantities by other means
• The root cultures are also used for genetic engineering.

Limitations of cloning with Agrobacterium plasmids
• Extensively used in dicots, but much more difficult to obtain the same
results with monocots.
• But yet eventually artificial techniques for achieving T-DNA transfer in
monocots were devised.
• Ease of recovery of plants varies with species.
• Hence gene gun technique is being used

Cloning vectors ppt kiran b k final

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Cloning vectors ppt kiran b k final

Similar to Cloning vectors ppt kiran b k final (20)

Recently uploaded

Recently uploaded (20)

Cloning vectors ppt kiran b k final

Editor's Notes