GENE CLONNING VECTORS
➢ A vector is a DNA molecule that has the ability to
replicate in an appropriate host cell, and into which the
DNA insert is integrated for cloning.
➢ Therefore a vector must have a origin of DNA
replication (Denoted as ori) that functions
efficiently in the concerned host cell.
➢ The vector is a vehicle or carrier which is used for
cloning foreign DNA in bacteria.
➢ Any extra- chromosomal small genome, e.g.
Plasmid, phage and virus may be used as vector.
Properties of a good vector
1) It should be able to replicate
autonomously that is independent of
the replication of host chromosome
2) It should be easy to isolate and purify
3) It should be easily introduced into the host
cells.
4) The vector should have suitable marker genes
that allow easy detection and/or selection of
the transformed host cell.
E.g. Genes for ampicillin and Tetracycline
resistance.
5. The cells transform with recombination
DNA should be identifiable or selectable
from those transformed by the unaltered
vector.
6. A vector should contain unique target sites
for as many restriction enzymes as
possible into which the DNA insert can be
integrated.
7. When expression of the DNA insert is
desired, the vector should contain suitable
regulatory elements like promoter,
operator, ribosome binding sites.
Types of Vectors
a. Cloning vectors
➢ The vectors used for propagation of DNA inserts in a
suitable host are called cloning vectors.
b. Expression vectors
➢ But when a vector is designed for the expression of i.e.
production of the protein specified by, the DNA insert,
it is known as expression vector.
➢ Such vectors contain at least the regulatory sequences
i.e. promoters, operators, ribosomal binding sites etc.
having optimum function in the chosen host.
➢ The gene carried by the expression vector is efficiently
transcribed and translocated by the host cell.
an idealized cloning vector
Plasmid vectors:
• Plasmids are self replicating double standard circular DNA
molecules which exist in cells as extra chromosomal units in the
bacteria. It is capable of independent replication and
transmission.
• Plasmids multiply at the same rate as that of bacterial cell
multiply at a rate independent of that of host cell.
• Even up to 1000 copies percell are obtained using
the plasmid as vectors.
• The length of the DNA segments to be cloned is
upto10-15 kilobase pairs.
• Naturally occurring plasmids are modified by in vitro
techniques.
Some common plasmid vectors
a. pBR – 322 : an ideal plasmid vector
• The name pBR denotes ‘p’ signifies plasmid BR is from
Boliver Rodriguez the scientist who developed pBR-
322 which is the most widely used plasmids.
• Size : 4.36 kb size
• Has selectable markers : antibiotic resistance
genes, tetr for tetracycline and ampr for
ampicillin
• Has several unique recognition sites for 12
different restriction endonuclease
enzymes located with in the tetr and ampr
genes
• Can take up a DNA insert of 10 kb length.
b. pUC vectors
❑ The name pUC is derived because it was developed in the
University of California (UC)
❑ This vector is a derivative of pBR322 ]
❑ Size: 2.7kb.
❑ It has all the essential parts of pBR322. E.g. the ampicillin resistant
genes and ori of replication.
❑ Second scorable marker is due to E. coli gene lac Z encoding ß-
galactosidase, which splits lactose into glucose and galactose.
❑ E. coil cells transformed by normal pUC vectors(non recombinant)
will be ampr and able to synthesize ß- galactosidase.
When an inducer of lac operon, a substrate for ß- galactosidase is
added in the media, it will be broken down by the enzymes to
give deep blue colonies.
Cells with recombinant pUC vectors – these cells
are ampr but unable to synthesize ß-galactosidase
and will give white colonies
because the substrate cannot be broken down.
Non recombinant -Blue colonies due to formation
of 5,5'-dibromo-4,4'-dichloro-indigo.
Recombinant-white colonies
Recombinants can therefore be selected by the
color of the colonies and there will be no need of
replica plating as is done incase of pBR322
Plasmids Size (Kb) Selective
markers
pBR322 <10 ampr , tetr
pBR324 9-22 ampr
pSC101 33-47 tcf
pRK 75-350 ampr
pUC 2.7 ampr and LacZ
Ti plasmid and Ri plasmid
✓ The Ti plasmid and Ri plasmid is a large conjugative plasmid or
mega-plasmid of about 200 kb.
✓ These are commonly used plasmids for transformation of plant cells
✓ A. tumefaciens has the Ti plasmid (200 kb) while A. rhizogenes has
the Ri plasmid have similar general features and can be interchanged
between the two species.
✓ These are plant pathogenic gram negative soil
bacteria to cause crown gall (A. tumefaciens) and hairy
root diseases of dicot plants.
✓ They infect plant cells near wounds, usually at the crown of roots at
the soil surface.
✓ These plasmids naturally transfer a part of their DNA, the T – DNA
into the host Plant genome, which makes Agrobacterium a natural
genetic engineer.
Ti Plasmid
Bacteriophage vectors
❖ Bacteriophages are viruses that infect bacteria.
❖ The phages are constructed from two basic components : capsid and nucleic acid genome.
❖ Size ~49 kb
❖ Phages can be both DNA and RNA phages(MS2)
❖ The DNA phages can be double stranded (T2, T4 and T6 ) or, single standard (X 174
and M-13) and RNA virus – MS2
❖ Several bacteriophages are used as cloning vectors, the most commonly used bacteriophage
vectors are: lambda phage, M-13 phages.
Advantages of phage vectors over plasmid vectors
❑They are more efficient than plasmids for cloning of large DNA
fragments.
❑ The length of the DNA segments is to be cloned is up to 25 kb.
❑ It is easier to screen a large number of phage plaques than bacterial colonies for the
identification of recombinant plaques / clones.
Lambda vectors
❖ The λ-genome (48.5Kb contains an origin of replication and
genes for head and tail, proteins and enzymes of DNA
replication, lysis and lysogeny, and single stranded protruding
cohesive ends of 12 bases at its 5' ends.
❖ These two cohesive ends are referred to as cos sites (sticky
or cohesive ends).
❖ These cohesive ends enables DNA to form a circular molecule
when it is injected into the E. coli cells.
❖ The central part (Nonessential segment-red and gam which
promotes lysogeny) of the λ-chromosome may be excised with
restriction enzyme and replace with foreign DNA.
Advantages of Lamda Vectors
✓Large size DNA fragment up to 25 kb can be
cloned as compared to plasmid vector which
can take up to 10kb
✓Large number of phage plaques can be easily
screened
✓They are more efficient than plasmids for
cloning of large DNA fragments
Identification of recombinant: Some posses
lacZ gene, some by palque morphology.
Phage M -13 vector
❑ They are derived from the 6.4 kb genome of the E. Coli filamentous
bacteriophage M13.
❑ This phage has a single stranded linear DNA genome in phase
particles which converts into double stranded circular DNA
molecule in the host cells.
❑ It contains origin of replication and a scorable marker gene lac-Z
that compliments the gal host giving blue colonies.
❑ On transformation only white or clear plaques are obtained,
thus permitting easy selection of recombinant plaques.
Cosmid vectors
❖ Cosmids are essentially plasmids that contain a minimum of
250bp of λ-DNA including cos site and sequences needed for
binding of and cleavage by terminase.
A typical cosmid has:
1) Origin of replication
2) Selectable markers from a plasmid ampr and tetr
3) Cloning sites (unique restriction sites) and Cos site (The
sequence yielding cohesive ends) of λ which are essential
for efficient packing of λ-DNA into virus particle which
infect host cells.
❖ Packaged cosmids infect host cells like λ -particle but inside
the host they replicate and propagate like plasmids.
The typical features of cosmids are as follows:
1. They are especially designed to clone large DNA fragments.
They can be used to clone DNA inserts up to 45kb.
2.They can be packaged into λ-particles which infect host cells,
which is many fold more efficient than plasmid transformation.
3.Selection of recombinant DNA is based on the procedure
applicable to the plasmid making up the cosmid.
4.Finally, these vectors are amplified and maintained in the same
manner as the contributing plasmid.
5. E.g.:- MUA3, pJC79 and pJB8.
Phagmid vectors
✓ Plasmids + Bacteriophage, a plasmid vector that contains the
original from the phage, in addition to that of plasmid.
✓ These are artificially constructed vectors containing the characters of
plasmids and bacteriophages.
✓ These consist of socrable and selectable markers for antibiotic
resistance. E.g. pUC-18, pUC-19
Yeast artificial chromosome vectors
These are linear vectors that behave like an Yeast chromosome
hence they are called yeast artificial chromosome vectors.
A typical YAC contains the following functional elements from yeast.
E.g. pYAC 3
1. An ARS sequence for replication
2. A CEN4 sequence – centromeric sequence ensure segregation
3. A TEL sequence (Telomeric sequence) at two endsfor protection
from exonuclease action.
4. One or two selectable marker genes E.g. TRP 1 and URA3 on left
and right arm, permits selection of transformants
5. SUP4, a selectable marker into which the DNA insert is integrated.
Transformed yeast with recombinant YAC molecule- red colony due
to absence of SUP4 gene, Non transformed- white colony
6. And necessary sequences from E. coli plasmid for selection and
propagation in E. coli.
YAC’s are used for cloning very large (1000-2000kb) DNA segments used for
mapping of complex Eukaryotic chromosomes.
Bacterial artificial chromosome (BAC):
➢ These are bacterial plasmids derived from the F plasmid. They are
capable of carrying up to 300 kb of DNA.
➢ Artificial chromosome are circular or linear vectors that are stably
maintained in, 1 to 2 copy per cell.
➢ These have the origin of replication ‘Ori S’ of E. coli F-factor
which allows a strict copy number control at 1 / 2 copies per
cell.
➢ The low copy number helps to maintain the DNA inserts
without any change arising from recombination between the
copies of DNA inserts and avoids any counter selection that
may arise due to over expression of cloned genes.
➢ These vectors are able to maintain in stable state and
extensively used in analysis of large genomes but the main
disadvantage of BAC vectors is some what laborious
construction of BAC libraries.
Shuttle vectors
❖These are designed to replicate in cells of two
different species. (These are plasmids capable of
propagating and transferring (Shuttling) genes
between two different organisms).
❖Therefore they contain two origins of replication, one
specific for each host species, as well as those genes
necessary for their replication and not provided by the
host cells.
❖These vectors are created by recombinant DNA technique.
❖Some of them can be grown in two different
prokaryotic species, while others can propagate in
prokaryotic species (E. coli) and a Eukaryotic one
(yeast, plants and animals).
Suttle Vectors Contd……
❖Since these vectors can be grown in one host
and then moved into another without any
extra manipulation they are called shuttle
vectors.
❖Shuttle vectors have been designed to
specifically satisfy the need i.e. for the initial
cloning of DNA inserts in E. coli and sub-
sequent functional tests in the species to
which the DNA inserts belong.
❖Most of the Eukaryotic vectors are in facts
shuttle vectors.
Cointegration and Binary vector system
Cointegration Vector system:
• The cointegration technique is based on in vivo
recombination of two plasmids.
• One plasmid carries desirable DNA sequence; the other
plasmid contains vir genes and the border repeats of T-
DNA.
• The recombination of these plasmids leads to large Ti
plasmid which then can be used to transform plants
Binary Vectors
• The binary vector system uses two separate plasmids:
mini-Ti plasmid to supply the disarmed T- DNA and
second one helper plasmid having vir genes.
• The mini-Ti plasmid bears the gene construct that
will be inserted into the plant genome, along with a
plant selectable marker between T-DNA border
sequences, so that both genes will be inserted as a
unit.
• The mini Ti plasmid also contains Origin of replication
for both E.coli and Agrobacterium and also marker
genes for bacteria.
Binary Vectors…..
• This plasmid when placed into an Agrobacterium
strain containing a plasmid with virulence functions,
the vir gene products are able to drive the transfer
of T-DNA into plant cells, even though T-DNA is
located on a separate DNA molecule.
• This is the most frequently used approach as mini Ti
plasmids are very easy to manipulate using standard
recombinant DNA techniques.
Binary Vectors
