Biotech 208
1. VECTOR
A vector is a DNA molecule that has the ability to replicate autonomously in the host cell and into
which DNA fragment to be cloned is inserted. It is usually a piece of DNA that carries a sequence of
DNA or other genetic material and introduces it into a new cell. Vectors act as vehicles to transfer
genetic material from one cell to the other for different purposes like multiplying, expressing, or
isolation.
2. PROPERTIES OF AN IDEAL VECTOR
1. It should be replicate autonomously.
2. A vector should be less than 10 kb in size.
3. It should be easily isolated and purify.
4. It should be easily introduced into the host cell.
5. It should have suitable marker genes.
6. Vector should consist of a unique target sites and recognition sites for various restriction
enzymes.
7. It should have the capability to incorporate either itself or the DNA insert in the Genome of the
host cell.
8. The cells transformed with such vector molecules that contain the DNA insert should be
identifiable from those transformed by the vector molecules only.
9. When expression of the DNA insert is desired, the vector should contain at least suitable control
elements e.g. promoters and operators.
3. CLONING AND EXPRESSION VECTORS
A vector is a DNA molecule that is used to carry a foreign DNA into the host cell. It has the
ability to self-replicate and integrate into the host cell. Cloning vectors are generally used to
obtain multiple copies of desired foreign gene.
Expression vector is a type of vector which not only introduces a gene of interest into the host
cell but also aids in the analysis of the foreign gene via relevant protein product expression. It is
type of vector which is used to obtain or analyses the gene product, which may be RNA or
protein of the inserted desired gene.
4. E. coli PLASMIDS
There are many types of cloning vectors, but the most commonly used ones are genetically
engineered plasmids. Cloning is generally first performed using Escherichia coli, and cloning
vectors in E. coli include plasmids, bacteriophages (such as phage λ), cosmids, and bacterial
artificial chromosomes (BACs).
i. F plasmid or Fertility: F plasmid contains specifically the tra gene and facilitates the transfer of
plasmids by conjugation.
ii. R plasmid or Resistance: This type of plasmids contains one of the multiple antibiotic
resistance genes. R plasmid plays an important role as in the development of antibiotic resistance
in bacterial pathogens.
iii. Col Plasmid: Col plasmids are responsible for the production of colicin and commonly found in
some strains of E. coli. This is a type of toxin which is generally released in the environment.
5. PLASMID VECTRORS
Plasmids are the extra-chromosomal, self-replicating, and double stranded closed and circular
DNA molecules present in the bacterial cell. Extrachromosomal means that they are separate
from the bacterial chromosome. Plasmid carries an origin of replication. It is most widely used
for gene cloning.
i. pBR322
The pBR 322 vector is commonly used plasmid cloning vector in E. coli. In this, B and R stands
for Bolivar and Rodriguez respectively.
It is 4363 base pairs long.
It possesses high copy number.
It carries two sets of antibiotic resistance gene: Ampicillin and Tetracycline. These antibiotic
resistance genes are used as selectable markers.
It contains only unique recognition sites for 12 different restriction enzymes including Pst I, Pvu
I, BamHI and Sal I
ORI: origin of replication
ROP: It codes for proteins, which are involved in the process of replication of plasmid
ii. pUC18/19
pUC19 is 2686 bp in length.
It is a small plasmid with a high copy number.
pUC19 plasmid is similar to pBR322 plasmid in structure.
Origin of replication – The origin of replication of the pUC19 plasmid is derived from pMB1.
It contains the lacz gene and has multiple cloning sites (MCS).
Multiple Cloning Sites – There is a short sequence which contains sites for various restriction
enzymes. This increases the number of potential restriction sites available, enabling the
production of the desired fragment for cloning.
Selectable markers – The pUC19 plasmid contains an Ampicillin resistance gene which can be
used to screen the recombinants. The plasmid also contains the E. coli gene lacZ.
The Blue-White screening method is used for pUC19 vectors. The recombinant cells, which
contain newly inserted DNA fragments, lack the lacZ gene activity and hence appear white on
the agar plates.
6. BACTERIOPHAGE VECTORS
Bacteriophages are viruses that attack bacteria. They reproduce by infecting bacterial cells. On
entering the host cell, the phage adopts one of two life cycles: lytic growth or lysogeny. In lytic
growth, approx. 100 new virions are synthesized and packaged before lysing the host cell,
releasing the progeny phage to infect new hosts. In lysogeny, the phage genome undergoes
recombination into the host chromosome, where it is replicated and inherited along with the host
DNA. The bacteriophages used for cloning are the λ phage and M13 phage.
i. Lambda phage vectors
A lambda phage is a bacteriophage that infects E. Coli cells. TThe bacteriophage λ genome
comprises 48,502 bp. Lambda Phage also plays a vital role in recombinant DNA technology as a
cloning vector, capable of carrying up to 25 kilobases of foreign DNA to a host cell. This makes
it a very efficient tool for introducing foreign DNA into cells.
Structure of bacteriophage
This phage, like any virus, readily undergoes two types of life cycles - the lytic cycle and the
lysogenic cycle. In the lytic cell, the phage hijacks the machinery of the cell to replicate the DNA
and produce viral proteins. These viral molecules are then packaged into new viral particles,
which eventually increase in size and burst the cell, thereby allowing the newly-synthesized
virions to escape into the outside environment. In the lysogenic cycle, the phage takes a more
inactive and calm approach. It simply integrates its viral DNA into the host cell's own genome.
When the cell divides, it replicates its own DNA along with the viral DNA, which means all
subsequent offspring cells will have the viral DNA. Under the right environmental conditions,
the virus can turn to the lytic cell cycle. So how do we use phages as vectors? We can extract the
phage's DNA and remove a piece of their DNA and replace it with the DNA fragment to be
cloned. As long as the recombinant DNA produced via this splicing process is still about the
same size as the original, it can be successfully inserted back into the lambda phage. Once inside
the lambda phage, the phage can infect E. Coli cells, which will in turn begin replicating the
DNA fragment of interest.
ii. M13 vectors
M13 is a filamentous bacteriophage which infects E. coli host. M13 was developed into a useful
cloning vector by inserting the following elements into the genome:
Gene for the lac repressor (lacI) protein to allow regulation of the lac promoter
Operator-proximal region of the lac Z gene
Promoter upstream of the lac Z gene
A polylinker (multiple cloning site) region inserted into the lacZ gene
7. COSMID VECTORS
It is a type of hybrid plasmid. It contains lambda phage cos sequence.
Cosmids = cos sites + plasmid.
Genomic size of cosmids is about 30 to 52 kb.
Cos sequences are ~200 base pairs long and essential for packaging.
8. PHAGEMID VECTORS
A plasmid vector which contains an origin of replication from a phage, in addition to that of the
plasmid, is termed as Phagemids. pBlue script SK (+/-) is a type of phagemid vector of 2,958 bp
derived from the pUC19.