Embed Size (px)
Citation preview
Dr. A.K. SahaProfessor
Department of ZoologyUniversity 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.
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.
Special Features
Figure. A typical plasmid vector. It contains a polylinker site, an ampicillin resistance gene (ampr) and a replication origin (ORI).
Polylinker
pUC192686 bp
ORIampr
The YAC vector is capable of carrying a large DNA fragment (up to 2 Mb), but its transformation efficiency is very low.
Yeast Artificial Chromosome (YAC)
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 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.
Plasmids
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
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)
The Plasmid transfer process can be divided into four stages:
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)
Bacterial chromosome
Plasmids
Cell division
Bacterial chromosome
Plasmids
Cell division
Chromosome carrying integrated plasmid
(b)
Figure: Replication strategies for (a) a non-integrative plasmid, and (b) an episome
B
Bacterial chromosome
Plasmids
Cell division
Bacterial chromosome
Plasmids
Cell division
Chromosome carrying integrated plasmid
B
A
Donor Recipient
Pilus
Pilus
DNA polymerase
Copying of transferred strand in recipient
Chromosomal DNA
Chromosomal DNAF- plasmid
F- plasmid
New DonorOld Donor
F- plasmid
Figure: A model for transfer of F plasmid DNA from an F+ cell by a looped rolling-circle mechanism
A new copy of F, generated by replication, is transferred to a recipient cell
A copy of F remains in the donor cell cell
(c)
(a) (b)
A. For-high-copy number plasmids
B. For-low-copy number plasmids
BA
Plasmid replication
Cell division and random
segregation daughter cells
Cell division and partitioning into plasmid daughter
cells
Chromosomal DNA is much larger than the DNA of
plasmids.
Chromosomal DNA extracted from cells is
obtained as broken, linear molecules.
Most plasmid DNA is extracted in a covalently
closed, circular form.
Differences between chromosomal and plasmid DNA
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
Transgenic plant by Ti plasmid
