Download Vectors - Rajshahi University

Dr. A.K. Saha
Professor
Department of Zoology
University of Rajshahi
Vector is an agent that can carry a DNA fragment into
a host cell. If it is used for reproducing the DNA
fragment, it is called a “cloning vector.” If it is used
for expressing certain gene in the DNA fragment, it is
called an “expression vector”.
Commonly used vectors include Plasmid, Lambda
phage, Cosmid and Yeast artificial chromosome
(YAC).
The cosmid can be defined as the hybrid derived from
plasmids which contain cos site of phage λ (cosmid =
cos site + plasmid).
A cosmid, first described by Collins and Hohn in 1978,
is a type of hybrid plasmid (often used as a cloning
vector) that contains cos sequences, DNA sequences
originally from the Lambda phage.
Characteristics of Cosmid
Cosmids are able to contain 37 to 52 kbp of DNA,
while normal plasmids are able to carry only 1-20 kbp
They can replicate as plasmids if they have a suitable
origin of replication.
They frequently also contain a gene for selection
such as antibiotic resistance.
Those cells which did not take up the cosmid would
be unable to grow.
Special Features
1. Origin of replication.
2. A marker gene coding for antibiotic resistance.
3. A special cleavage site for the insertion of
foreign DNA and
4. Small in size.
Polylinker
pUC19
2686 bp
ORI
ampr
Figure. A typical plasmid vector. It contains a polylinker site, an
ampicillin resistance gene (ampr) and a replication origin (ORI).
Yeast Artificial Chromosome (YAC)
The YAC vector is capable of carrying a large DNA
fragment (up to 2 Mb), but its transformation efficiency
is very low.
CEN= Centromeres
TEL= Telomeres
ARS=Autonomous
replicating
sequence
Figure. Cloning by the yeast artificial chromosome (YAC) vector.
Centromers (CEN), telomeres (TEL) and autonomous
replicating sequence (ARS) for proliferation in the host cell.
ampr for selective amplification and markers such as TRP1
and URA3 for identifying cells containing the YAC vector.
Recognition sites of restriction enzymes (e.g., EcoRI and
BamHI).
Procedure :
1. The target DNA is partially digested by EcoRI and the
YAC vector is cleaved by EcoRI and BamHI.
2. Ligate the cleaved vector segments with a digested DNA
fragment to form an artificial chromosome.
3. Transform yeast cells to make a large number of copies.
Phages are viruses that can infect bacteria. The major
advantage of the phage vector is its high transformation
efficiency, about 1000 times more efficient than the plasmid
vector.
Plasmids
Plasmids are extrachromosomal circular DNA molecules
found in most bacterial species and in some species of
eukaryotes.
Many important bacterial genes are not part of the main
chromosome but are on separate circles of DNA called
plasmids.
A plasmid is a molecule which can be stably inherited
without being linked to the chromosome.
Plasmids were originally defined as ‘‘extrachromosomal,
hereditary determinants’’(Lederberg,1952). They are
generally covalently closed circular (CCC) molecules of
double-stranded deoxyribonucleic acid (DNA) that very in
length from 1 kbp to 700 kbp. And occur in the majority of
bacterial cells.
Fig. The structure of pBR322 showing the unique cleavage sites.
CHARACTERSTICS OF PLASMID
Plasmids are important in medicine and in agriculture
because they confer antibiotic resistance on pathogens of
animals and man, and because they can code for toxins
and other proteins which increase the virulence of these
pathogens.
Nitrogen Fixation:
Plasmids enable species of Rhizobium to fix nitrogen in the
nodules of leguminous plants.
Antibiotic Production:
Plasmid also code for antibiotics which can be used to
control pathogenic bacteria.
Biodegradation:
Plasmids genes code for a wide range of metabolic
activities and enable bacteria to degrade compounds
which would accumulates as pollutants.
Recombinant DNA:
Plasmid can be isolated quite easily from bacterial cells.
New genes from other species can be inserted into
isolated plasmids and the modified plasmid then can be
put back into its normal host cell. Such “recombinant
DNAs” are transcribed, and translated into potentially
useful products.
Figure: Two super-coiled plasmid DNA molecules
TRANSFER OF PLASMID DNA
The Plasmid transfer process can be divided into
four stages:
1. Formation of specific donar -recipient pairs
(effective contact)
2. Preparation for DNA transfer (mobilization)
3. DNA transfer
4. Formation of a replicative functional plasmid in
the recipient (replication)
Figure: Plasmid transfer by conjugation between bacterial cells.
The donor and recipient cells attach to each other by a pilus, an
appendage present on the surface of the donor cell. A copy of
the plasmid is then passed through the pilus to the recipient cell.
TYPES OF PLASMIDS
Stringent or Low copy number plasmid:
Which may range from 1 or 2.
Relaxed or high copy number plasmids:
Range from 10-100.
Many types of plasmids are found in a variety
of E. coli strains but three main types- F, R, and
Col plasmids have been studied.
1. F, the sex plasmid: Ability to transfer chromosomal
genes (that is, genes not carried on the plasmid) and the
ability to transfer F itself to a cell lacking the plasmid.
2. R, the drug-resistance plasmid: Resistance to one or
more antibiotics and often the ability to transfer the
resistance to cells lacking R.
3. Col, the colcigenic plasmid: Ability to synthesize
colicins- that is, proteins capable of killing closely related
bacterial strains that lack the Col plasmid.
On the basis of Co-existence:
1. Compatible plasmid: Bacteria often contain two or more
different plasmid which can co-exist, and are said to be
compatible
2. Incompatible plasmid: Can not co-exist together.
A
(a)
Plasmids
Cell division
Bacterial chromosome
Plasmids
Bacterial chromosome
Chromosome carrying
integrated plasmid
Cell division
B
(b)
B
Figure: Replication strategies for (a) a non-integrative plasmid,
and (b) an episome
Chromosomal
DNA
F- plasmid
Donor
Pilus
Chromosomal
DNA
Recipient
DNA polymerase
F- plasmid
Copying of transferred strand in
recipient
F- plasmid
Pilus
Old Donor
New Donor
Figure: A model for transfer of F plasmid DNA from an F+
cell by a looped rolling-circle mechanism
(a)
(b)
(c)
A new copy of F, generated by replication,
is transferred to a recipient cell
A copy of F remains
in the donor cell cell
A
B
Plasmid replication
Cell division and
random
segregation
daughter cells
Cell division and
partitioning into
plasmid daughter
cells
A. For-high-copy number plasmids
B. For-low-copy number plasmids
Differences between chromosomal and plasmid DNA
Chromosomal DNA is much larger than the DNA of
plasmids.
Chromosomal DNA extracted from
obtained as broken, linear molecules.
cells
is
Most plasmid DNA is extracted in a covalently
closed, circular form.
Plasmids as Tools for Molecular Biology
They are used as a vectors to clone DNA. A variety of
different enzymes can be used to insert pieces of DNA,
from animals, plants or prokaryotes, into plasmids.
Circular molecules consisting partly of plasmid DNA and
partly of inserted DNA can then be put back into a
suitable bacterium.
The plasmids replicate during bacterial growth so that
many copies of the cloned DNA can easily be obtained.
The Agrobacterium Plasmid Ti
This plasmid has recently become very important in plant
breeding because specific genes can be inserted into Ti
plasmid by recombinant DNA techniques, and sometimes
these genes can become integrated into plant
chromosome, thereby permanently changing the genotype
and phenotype of the plant. New plant varieties having
desirable and economically valuable characteristics
derived from unrelated species can be developed in this
way.
The Agrobacterium Plasmid Ti
Role of Ti plasmid
Role of Ti plasmid
Transgenic plant by Ti plasmid