VECTORS
INTRODUCTION
A Cloning vector is a DNA molecule in which
foreign DNA can be inserted or integrated and
which is further capable of replicating within
host cell to produce multiple clones of
recombinant DNA.
Examples : Plasmids , Phage or virus
CLASSIFICATION OF VECTORS
On the basis of our aim with gene of interest:
1.Cloning vectors
2.Expression vectors
On the basis of host cell used:
1.Vectors for bacteria
2.Vectors for yeast
3.Vectors for animals
4.Vectors for plants
On the basis of cellular nature of vectors of host cell:
1.Prokaryotic vectors
2.Eukaryotic vectors
CHARACTERISTIC FEATURES
OF AN IDEAL VECTORS
◊ It should be able to replicate autonomously.
◊ It should be easy to isolate and purity.
◊ Transformation of host with the vector should be easy.
◊ It should have suitable marker genes that allow easy
detection.
◊ It should have the ability to integrate either itself or
the DNA insert it caries into the genome of host cell.
◊ It should contains unique target sites.
◊ It should contain at least suitable control elements,
eg.promoter,operator,and ribosome binding sites.
GENERAL CONSTRUCTION
OF VECTORS
▫ A DNA molecule should posses the following two
essential characteristics to act as cloning vector.
▫ Origin of replication : It is required for autonomous
replication of plasmid using the host replication
machinery.
▫ Selectable markers : 4 Types
Antibiotics resistance marker genes
Herbicides tolerant marker genes (Hb)
Metabolic/auxotrophic marker genes
Screenable marker genes
PLASMID
S
Most common vector
Small circular dsdna molecules
lacking protein coat
Eg;pUc ,pBR322
pBR322
o This was the first widely used plasmid
vector.
o pR322 has a relatively small size of
4.363bp.
o Also this vector has a reasonably high
copy no ( ～ 15 copies per cell).
Nomenclature of pR322 :
p-Plasmid
BR- Boliver and Rodriguez-
The two researchers who
developed it.
322-No.of developed in the same
Construction of pBR322 :
1. Origin of replication
2. Selectable markers-It carries 2 antibiotic resistance
genes
(Ampicillin and Tetracycline)
3. Cloning sites.
Uses of pBR322 :
It is widely used as cloning vector.
It is wdely used as a model system for study of
prokaryotic transcription and translation.
BACTERIOPHAGES
Bacteriophage is a genetically complex but
very extensively studied virus of E.coli.
The DNA of phage, is a linear duplex
molecule of 48502 bp ( ～ 49kb) in length.
The DNA isolated from virus particles is a
double stranded linear molecule with short
complementary single stranded projections
of 12 nucleotides at its 5’ends.
These cohesive termini, also referred to as
COS sites, allow the DNA to be circularized
after infection of the host cell.
GENERAL STRUCTURE OF
BACTERIOPHAGE
The genetic map of phage comprises approximately 40 genes
which are organized in functional clusters.
Genes coding for head and tail are proteins (genes A) are on
the left of the linear map.
The central region contains genes, such an int, xis, exo etc.,
which are responsible for lysogenisation i.e. the process
leading to the integration of viral DNA and other recombination
events.
Much of this central region is not essential for lytic growth.
Genes to the right of the central region comprise six regulatory
genes, two genes (O and P) which are essential for DNA
replication during lytic growth and two more genes (S and R)
which are required for the lysis of the cellular membranes.
PHAGES
Derivatives of phage have been developed as cloning vectors
since the early days of gene technology.
The phage derivatives are considered to be the most suitable
cloning genomic eukaryotic DNA because of the following
advantages over the plasmids.
Thousands of phage plaques can be obtained in a single petri dish.
Selection by DNA-DNA hybridization is possible.
In vitro packaging into empty phage head is possible thus increasing
phage infectivity.
Size selection of the packaged DNA is possible.
Millions of independently cloned virus particle can be constituted to
form a gene library.
In the phage DNA, larger control region in not essential for
phage growth and replication.
This region of phage can be deleted or replaced without
seriously impairing the phage growth cycle.
Using this non essential region of phage, several phage
vector derivatives have been constructed for efficient gene
cloning.
TYPES OF PHAGE VECTORS
Wild type phage DNA itself cannot be used as a vector
since it contains too many restriction sites.
Further, these sites are often located within the essential
regions for phage’s growth and development.
From these wild phages, derivatives with single target sites
and two target sites have been synthesized.
Phage vectors which contain single site for the insertion of
foreign DNA have been designated as Insertional vectors.
Vectors with two cleavage sites, which allow foreign DNA to
be substituted for the DNA sequences between those sites,
are known as replacement vectors.
COSMIDS
Cosmids are hydrids between a phage DNA
molecule and a bacterial plasmid, and their
design centers on the fact that the enzymes that
package the λDNA molecule into the phage
protein cost need only the cos sites in order to
function.
The in vitro packaging reaction works not only
withλgenomes, but also with any molecule that
carries cos sites separated by 37-52 kb of DNA.
A cosmid is basically a plasmid that carries a cos
site.
It also needs a selectable marker, such as the
ampicillin resistance gene, and a plasmid origin
of replication, as cosmids lack all the λgenes and
so do not produce plaques.
Instead clonies are formed on selective media,
just as with a plasmid vector.
PHAGEMID VECTORS
Phagemid (phage from M13 bacteriophage and mid
from plasmid).It contains 1500bp.
Construction of phagemid :
1. Origin of replication
2. Lac-Z gene
3. Multiple cloning sites
4. Origin of replication
5. resistant gene
Uses of phagemid :
Used as cloning vectors, sequence in vectors,
expression vectors
Advantages of phagemid :
It can e used to provide single or double stranded
material without any recloning
BAC
BACs are based on bacterial
mini-F plasmids, which are
small pieces of episomal
bacterial DNA that bacteria the
ability to initiate conjugation
with adjacent bacteria. They
have a cloning limit of 75-300
kb.
YAC
YAC s are artificial chromosomes that replicate in yeast cells.
They consist of Telomeres, which are ends of chromosomes
involved in the replication and stability of linear DNA.
Origin of replication sequences necessary for the replication
in yeast cells.
A yeast centromers, which is a specialized chromosomal
region where spindle fibers attach during mitosis.
A selectable marker for identification in yeast cells.
Ampicillin resistance gene for selective amplification.
Recognition sites for restriction enzymes.
SHUTTLE VECTORS
Definitions : They are the vectors that can
shuttle between more than one host, for
example, one is E. coli and the other is
yeast.
Structure and function : Most of the vectors
for use in eukaryotic cells are constructed as
shuttle vectors.
In E. coli.:
This means that they can survive and
have the genes(ori and amp) required
for replication and selection in E. coli.
In the desired eukaryotic cells:
They can also survive in the desired
host cells, and let the target insert
sequences take effects.
Thank
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