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VECTORS IN
BIOTECHNOLGY
Presented By- Baisali Dora
Sonali Pati
+3 1st Yr. – Botany Hons.
Under The Guidance of
Faculties of Dept. of Botany,
V. Dev (Auto.) College, Jeypore
Dated- 23/ Feb/2015
DEFINATION
These are carrier or vehicular DNA molecules,
which carry gene of interest. A vector when
combined with the gene of interest; a recombinant
DNA molecule is obtained.
The aim of using vector in RDT is one of the
following
To obtain multiple copies of the gene of interest. In this
case we use cloning vectors or
To obtain the product of gene of interest . In this case
we use expression vectors
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CHARECTERISTIC FEATURES OF AN IDEAL
VECTORS
It should be able to replicate autonomously.
It should be easy to isolate and purify.
Transformation of host with the vector should beeasy.
It should have suitable marker genes that alloweasy detection.
It should have the ability to integrate either itself orthe DNA insert it caries into the genome of host cell.
It should contains unique target sites.
It should contain at least suitable control elements,e.g. promoter, operator, and ribosome binding sites
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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
machinary
Selectable markers- 4 types
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Selectable markers-
Antibiotics resistance marker genes
Herbicides tolerant marker genes (Hbr)
Metabolic/auxotrophic marker genes
Screenable marker genes
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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
o On the basis of cellular nature of vectors of host cell
1 . Prokaryotic vectors
2. Eukaryotic vectors 6
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Plasmid based and bacteriophage vectors are most
common prokaryotic vectors
The prokaryotic vectors include:- plasmid derived
vectors, bacteriophage derived vectors, phagemid
vectors, plasmid vectors and fosmid vectors
Plasmid vectors
These are the most common vectors for the
prokaryotic host cell.
Plasmids are small, circular, double stranded DNA
molecules lacking protein coat exist naturally in the
cytoplasm of many strains.
Ex. pBR322, pUC vector
Prokaryotic vectors
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PBR322:
This was the first widely used plasmid vector .
pBR322 has a relatively small size of 4.363bp.
Also this vector has a reasonably high copy number
(~15 copies per cell).
Nomenclature of pBR322:
p- Plasmid
BR- Boliver and Rodriguez-
The two researchers who developed it.
322- No. of developed in the same laboratory.
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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 widely used as a model system for study of prokaryotictranscription and translation.
Advantages of pBR322:
Due to its small size (~4.4kb) enables easy purification andmanipulation.
2 selectable markers ( Amp and Tet.) allow easy selection ofrecombinant DNA.
It can be amplified up to 1000- 3000 copies.
Disadvantages of pBR322:
It has very high mobility .
There is a limitation in the size of genes of interest that it canaccommodate. 9
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II. PUC VECTORS:
pUC are obtained by modifying the pBR322 vectors .
These are smaller than pBR322 of being only ~2.7 kb.
It produce 500-600 copies.
Nomenclature of pUC vectors:
P- plasmid
UC- University of California
Construction of pUC:
1. Origin of replication
2. Selectable markers
3. Lac-z gene having MCS(Multiple Cloning Sites)
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Uses of pUC vectors:
pUC vectors can be used both as cloning vector
and expression vector.
Advantages of pUC vectors:
1. Produces high copy number of 500-600 copies
per cell.
2. Easy and single step selection.
Disadvantages of pUC vector:
It cannot accommodate a gene of interest larger
than 15kb.
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III. FOSMID VECTORS
These are similar to cosmid vectors ,
but are based on bacterial F-Plasmids.
Fosmids are 40 kb of random genomic DNA .
IV. BACTERIOPHAGE DERIVED VECTORS
Bacteriophages/ phages are commonly known viruses that infect
bacteria .
Like viruses, phages are very simple in structures consisting merely of
DNA.
Ex. Lambda(λ) phages vectors, Bacteriophages m13 vectors
Lambda(λ) phages vectors .
These are widely used vectors for cloning of very large pieces of
genes .
Lambda is a typical example of head and tail phage. The lambda
molecules is 49kb in size. 12
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Types of lambda(λ) vectors:
1. Lambda insertion vectors
2. Lambda replacement vector
Advantages of lambda(λ) vectors :
I. Storage of phage particles is comparatively much easier
than that of plasmid based vector.
II. The self-life of phage particles is infinite .
III. Transfection of E. coli is much easier with phage
particles.
Disadvantages of lambda(λ) vectors :
I. It is difficult to isolate.
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V. COSMID VECTORS
It is the most sophisticated type of Lambda based vector. Cosmid arehybrid between phage DNA molecule and vector plasmid.
Constructions of Cosmid Vectros:
Cosmid is basically a plasmid that carries a cos site.
It contain selectable markers such as Ampicillin resistant(Ampr) geneand a origin of replication.
Cosmid lacks all the lambda genes, so it doesn’t produce plaques.
Uses of Cosmid Vectros :
Used for construction of genomic libraries of eukaryotes.
Advantages of Cosmid Vectros :
Used to clone gene of interest up to 40kb.
Easy screening method is found
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VI. PHAGEMID VECTORS
Phagemid (phage from M13 bacteriophage and mid from plasmid).
It contains 1500 bp.
Constructions of Phagemid :
1. Origin of replication
2. Lac-Z gene
3. Multiple cloning sites
4. Origin of replication
5. Ampr resistant gene
Uses of Phagemid:
Used as cloning vectors, sequence in vectors, expression vectors
Advantages of Phagemid :
It can be used to provide single or double stranded material without
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VI. PHASMID VECTOR
These are truly plasmid with phage genes.
These are linear duplex DNA whose ends are
lambda segments that contains all the genes.
Both the lambda and plasmid replication function
are intact.
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Yeast, animal and plant vectors are all considered
as eukaryotic vectors.
Yeast vectors :
Yeast plays very important role in brewing and
bread making .
It can be used in pharmaceuticals from cloned
genes.
EUKARYOTIC VECTORS
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General construction Yeast Vectors:
1. All of them contains unique target sites for no. of
restriction endonucleases.
2. High copy number.
3. Employ markers.
Types of Yeast Vectors :
It can be divided into 3 types:
a. Yeast cloning vector.
b. Yeast expression vectors.
c. Yeast artificial chromosomes (YAC)
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ANIMAL VECTORS
1. Baculovirus Vectors:
It infects insects.
This virus is rod shaped with a large double
stranded genome.
The gene of Interest is expressed during the
infection and very high yields of protein, can be
achieved by the time lysis.
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2. Bovine Papilloma Virus Vector:
BPV causes warts in cattle.
BPV has a capsid protein surrounding a circular
double stranded DNA of size 79 kb.
69% of this genome is imp. for viral function, 31%
of genome can be replaced by any foreign DNA
sequence.
Ex- p3.7LDL
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3. SV 40 Virus Based Vector:
It is a spherical virus contains double stranded
circular DNA of size 5.2 kb.
The viral protein contains 3 viral coded proteins; out
of which large T-Protein is imp. for viral DNA
replication.
Types of SV 40:
SV 40 Passive Transforming Vector
SV 40 Transducing Vectors
SV 40 Plasmid Vectors
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PLANT VECTORS
These vectors are used to produce Genetically
Modified(GM) crops.
Shuttle Vectors:
Can Multi[ply into 2 different unrelated species.
Contains 2 ori sites for replication
Types of Shuttle Vectors:
Eukaryotic- Prokaryotic Shuttle vectors
Prokaryotic- Prokaryotic Shuttle vectors
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ARTIFICIAL CHROMOSOMES
Synthetically designed DNA molecules of known
structures; which are assembled in vitro from
specific DNA sequences that acts like a natural
chromosomes.
Mainly contain 3 components-
Centromere
2 telomeres
Origin(Ori) of Replication
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Types of Artificial chromosomes:
1. Yeast Artificial Chromosomes (YAC):
Uses:
Help in production Gene Libraries
Can be used to study various aspects of chromosomestructure and behavior during Meiosis.
2. Bacterial Artificial Chromosomes(BAC):
Cloning Vectors based on F factor of E. coli plasmid.
Uses:
Help in studying Neurological disease viz. Alzheimer’sDisease in Mice
In case of Aneuploidy, it is associated with Down’s Syndrome.
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3. Human Artificial Chromosomes(HAC):
47 artificial mini chromosome
Uses:
Used in Expression Studies as Gene Transfer
Vector
4. P1 Derived Artificial Chromosomes(PAC):
Derived from DNA of P1 bacteriophage
Uses:
Used to clone DNA fragments of E. coli cells 25
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