VECTORS:

TYPES AND
CHARACTERISTICS

E.g. PLASMIDS, PHAGES, HYBRID VECTORS,

ARTIFICIAL CHROMOSOMES
 CLONING VECTORS
• DNA molecules that can carry a foreign DNA fragment when
inserted into it

• Share four common properties:

1. Ability to promote autonomous replication.

2. Contain a genetic marker (usually dominant) for selection.
3. Unique restriction sites to facilitate cloning of insert DNA.
4. Minimum amount of nonessential DNA to optimize cloning.
 CHARACTERISTICS
 Self replication, multiple copies.
 Replication origin site.
 Cloning site.
 Selectable marker gene.
 Small size.
 Low molecular weight.
 Easily isolated & purified.
 Easily isolated into host cell.
 Control elements – promoter, operator, ribosome
binding site.
 TYPES
Two types :-
1) Cloning Vectors
Propagation or cloning of DNA insert inside a suitable host cells.
Examples: Plasmids, Phage or Virus
 Obtaining millions of copies.
Uses :-
Genomic library.
Preparing probes.
Genetic Engineering Experiments.
 Selection of cloning vector depends on :-
(a) Objective of cloning experiment
(b) Ease of working.
(c) Knowledge existing about the vector.
(d) Suitability.
(e) Reliability.
2) Expression Vectors

 Express the DNA insert producing specific protein.

 They have prokaryotic promoter.
 Ribosome binding site.
 Origin of replication.
 Antibiotic resistance gene.
 Expression vectors with strong promoters..
 Eukaryotic expression vectors.
 VECTOR TARGET HOST CELL

• Plasmid Bacteria, Streptomyces

• Bacteriophages Bacteria
• Cosmid Bacteria
• Yeast Cloning Vectors Yeasts
• Ti & Ri Plasmids Transformation of
cloned gene in higher
plants.
 PLASMID
 Extra chromosomal DNA molecules.
 Self replicating.
 Double stranded.
 Short sequence of DNA.
 Circular DNA molecules.
 Found in prokaryotes.
CHARACTERISTICS
• Minimum amount of DNA.
• suitable Genetic markers for identification .
• Unique restriction site.
• More restriction enzyme.
• Size range 1kg – 200kg.
• Origin of replication Relaxed replication control.
• Copy Number
• No pathogenicity
• Multiple Cloning Site
• Promoter Sequence
• Chang and Cohen first proved the use of plasmid as gene cloning
vectors.
• CHARACTERISTCS OF IDEAL PLASMID VECTORS
1. Small size: 1.2-3kb
2. Copy number
3. Genetic marker
4. Origin of replication
5. Unique Restriction Site
6. No pathogenicity
7. Multiple Cloning Site
8. Promoter Sequence
 THREE TYPES OF PLASMID
1. Fertility plasmids:- can perform conjugation.
2. Resistance plasmids:- contain genes that build a
resistance against antibiotics or poisons.
3. Col plasmids:- contain genes that code for proteins
that can kill bacteria.
NATURAL AND ARTIFICIAL PLAMIDS
• Plasmids isolated from bacteria & directly used for gene cloning
without any modification :-Natural Plasmid
• But not used in gene cloning due to large size,confer pathogenicity
etc
• Artificial/derived plasmids constructed by cutting out unwanted
portions from wild type plasmids.Eg:pBR322
• These are of much use in gene transfer
 EXAMPLES OF PLASMID VECTORS
 pBR322
 pBR327
 pBR325
 pBR328
 pUC8
 pUC9
 pUC12
 pUC13
 pGEM3Z
 PBR322
 Cloning Vector.
 15 copies.
 Reconstructed plasmid.
 Derived from Ecoli plasmid- ColE1.
PBR322 – 4362 base pairs
P – denotes Plasmid
B – Scientist Boliver
R - Rodriguez
322 – number given to distinguish.
 Ampilicin resistance gene derived from RSF2124.
 Tetracycline resistance gene from PSC101.
 Origin of replication from PMB1.
 Two selectable markers
ampr tetr
Structure of E.Coli plasmid cloning vector pBR322
 PBR327
 Derived from PBR322.
 30 – 40 copies.
 Expression plasmid.
 Ampilicin resistance gene.
 Tetracycline resistance gene.
 Deletion of nucleotide between 1427 & 2516.
 pUC8
 Popular Ecoli cloning vector.
 Derivative of pBR322.
 Two parts derived:-
 Ampicillin resistance gene.
 ColEI – origin of replication.
 2700 base pairs.
 lac Z gene derived from Ecoli.
 Polylinker sequence having unique restriction sites
lies in lac region.
How insertion takes place
Screening procedure of cloning vector

Blue/White selection.

replica plating technique.

 Blue/White Selection
Bacteria with plasmid
plus insert

IPTG + X-Gal

Overnight growth

Only colonies
Colonies with insert - white
from bacteria that
Colonies w/o insert - blue
have plasmid
Based on the enzymatic reaction of β-
galactosidase
Replica Plating Technique
 ADVANTAGES AND DISADVANTAGES

• ADVANTAGES:

1. Readily isolated from cells

2. Can be reintroduced into a bacterial cell
3. Possess a single restriction site for 1 or more restriction enzyme
4. MCS
5. Introduction of a linear molecule does not alter its replication

• DISADVANTAGES
1. Cannot accept large fragments
 PHAGE
 Cloning large DNA fragmance.
 Linear Phage molecule.
 Efficient than plasmid.
 Used in storage of recombinant DNA.
 Commonly used Ecoli phages :-
λ phage
M13 Phage
• Derivatives of phage have been developed as cloning
vectors since the early days of gene technology.
• The phage derivatives are considered to be the most
suitable cloning vehicles for cloning genomic
eukaryotic DNA because of the following advantages
over the plasmids.
 Thousands of phage plaques can be obtained in a
single petri dish.
 Selection by DNA-DNA hybridization is possible.
 In vitro packaging into empty phage head is possible
thus increasing phage infectivity
 Size selection of the packaged DNA is possible.
 Millions of independently cloned virus particle can be
constituted to form a gene library.
 BACTERIOPHAGE VECTORS

Lambda phage vector

Genome size is 48,502 bp.
Consists of an icosahedral head and flexible
tail.
High transformation efficiency.
1000 times more efficient than the plasmid
vector.
Origin of replication.
Genome linear in head.
bacteriophages • Bacteriophage is a genetically
complex but very extensively
studied virus of E.coli.
• The DNA isolated from virus
particles is a double stranded
linear molecule with short
complementary single
stranded projections of 12
nucleotides at its 5’ ends.
• These cohesive termini, also
referred to as cos sites, allow
the DNA to be circularized
after infection of the host cell.
 General structure of bacteriophage
• The genetic map of phage comprises approximately 40
genes which are organized in functional clusters.
• Genes coding for head and tail are proteins (genes A-J) are
on the left of the linear map.
• The central region contains genes, such as int, xis, exo etc.,
which are responsible for lysogenisation i.e the process
leading to the integration of viral DNA and other
recombination events.
• Much of this central region is not essential for lytic growth.
• Genes to the right of the central region comprise six
regulatory genes, two genes (O and P) which are essential
for DNA replication during lytic growth and two more
genes (S and R) which are required for the lysis of the
cellular membranes.
SHIVA'S
• In the phage DNA, larger central region is
not essential for phage growth and
replication.
• This region of phage can be deleted or
replaced without seriously impairing
the phage growth cycle.
• Using this non-essential region of phage,
several phage vector derivatives have
been constructed for efficient gene
cloning.

SHIVA'S
• Lambda Phage DNA can carry large DNA fragments and integrate
into host chromosome
• Wild type Lambda DNA can carry only small fragments;The
unwanted seq are cut out:- CONSTRUCTED LAMBDA DNA VECTOR
• It is of 2 types:

1. Insertion Vectors- 1 restriction site;fragments upto 18kb

2. Replacement Vectors-2 restriction sites;Removal of stuffer seq
 Types of phage vectors
• Wild type phage DNA itself cannot be used as a vector
since it contains too many restriction sites.
• Further, these sites are often located within the
essential regions for phage's growth and development.
• From these wild phages, derivatives with single target
sites and two target sites have been synthesized.
• Phage vectors which contain single site for the insertion
of foreign DNA have been designated as Insertional
vectors;
• vectors with two cleavage sites, which allow foreign
DNA to be substituted for the DNA sequences between
those sites, are known as replacement vectors.

SHIVA'S
 Insertional vectors
• A large segment of the non-essential
region has been deleted, and the two
arms ligated together.
• An insertion vector possesses at least
one unique restriction site into which
new DNA can be inserted.
• Two popular insertion vectors are:
• Egt10 : which can carry up to 8 kb of
new DNA, inserted into a unique EcoRI
site located in the cI gene.
• Insertional inactivation of this gene
means that recombinants are
distinguished as clear rather than
turbid plaques.
• EZAPII : insertion of up to 10 kb DNA
into any of 6 restriction sites within a
polylinker inactivates the lacZ′ gene
carried by the vector.
• Recombinants give clear rather than
blue plaques on X-gal agar.
SHIVA'S
 E.coli/ Replacement vectors
Examples: EMBL3 and  DASH.
A representative scheme for cloning:
Sho
1. The  vector DNA is cleaved with Long arm Replace. rt
BamH1 and the long (19 kb) and arm
short (9 kb) arms are purified; 48.5 kb
2. The target fragments are B B
prepared by digestion, also with
BamH1 or a compatible enzyme
(Sau3A);  20kb
3. The target fragments are treated
with alkaline phosphatase to
prevent them ligating to each
other;
B
4. The  arms and the target
fragments are ligated together at
relatively high concentration to
form long linear products.

SHIVA'
Can not infect
S Parking E.coli
 Packaging and infection
The Recombinants that can not be packaged: B
1. Ligated  ends which do not contain an insert;
2. The insert is much smaller or larger than the 20 kb;
3. The recombinants with two left or right arms.

Packaging: Replication concata-mers

in vivo

cleave individual  genomes

in vitro
A mixture of phage coat
proteins and Infection of E. coli
Packaging
the phage DNA-processing
enzymes9
phage 10 recombinants per
particles mg of vector DNA.
SHIVA'S
Formation of plaques
Plaques are the analogs of single bacterial colonies.
Formation:
The infected E.coli cells from a packaging reaction are
spread on an agar plate,
The plate has been pre-spread with uninfected cells,
which will grow to form a continuous lawn.
After incubation, phage-infected cells result in clear
areas, that are plaques, where cycles of lysis and re-
infection have prevented the cells from growing.
E.coli lawn
Plaques
Recombinant  DNA may be
purified:
• from phage particles isolated
from plaques or
• from the supernatant of a culture
SHIVA'S
 M13 BACTERIOPHAGE VECTOR
• A single stranded DNA contaning phage virus;Can infect F+ cells
• Consists of about 6402 b.p
• The Intergenic Seq (IS) of M13 modified so as to introduce
additional restriction site
• The gene LacZ is integrated and then introduced into the IS to get
M13 Vector
Bacteriophage M13
Genome features: Size is small (6.7 kb); Single-
stranded; Circular genome; DNA; Positive-sense.
Infection: M13 particles attach specifically to E.coli sex pili
(encoded by a plasmid called F factor), through a minor
coat protein (g3p). Binding of g3p induces a structural
change in the major capsid protein. This causes the
whole particle to shorten, injecting the viral DNA into
the host cell.
g6p g3p

end
g8p
RF
Host ini
enzymes
g9p
g7p
SHIVA'S
 Cloning in M13
Purpose: When the single-stranded DNA of a fragment is
required, a M 13 vector can be used as a common cloning tool.
Preparation of ssDNA:
1. Cloning: standard plasmid cloning method can be used to
incorporate recombinant DNA into M13 vectors;
2. Transformation: the M13 then infects sensitive E. coli cells;
3. Plating: the host cells grow to form the plaques;
4. Isolation: the ssDNA may then be isolated from phage particles
in the growth medium of the plate.
Screening: Blue-white screening using MCSs and lacZ' has been
engineered into M13 vectors.
Examples: The M13mpl8 and M13mp19, which are a pair of
vectors in which the MCS are in opposite orientations relative
to the M13 origin of replication.

SHIVA'S
.
DNA cloning using phages as vectors
 ADVANTAGES & DISADVANTAGES
• ADVANTAGES

1. Large DNA fragments upto 23kb can be carried

2. Efficiency of gene transfer is high
• DISADVANTAGES
1. Rec.DNA may enter lytic cycle
2. Lambda phage has narrow host range
 HYBRID VECTOR
 Component from both plasmid & phage chromosomes.
 Helper phage provided.
 Developed in 1978 by Barbara Hohn & John Collins.
 30 – 40 Kb
 Origin of replication, cloning site, marker gene, DNA
cos site.
 Smaller than plasmid.
 Use – construction of genomic libraries of eukaryotes.
 e.g. Cosmid
 cosmids
• Cosmids are hybrids between a phage DNA
molecule and a bacterial plasmid, and their
design centers on the fact that the enzymes that
package the  DNA molecule into the phage
protein coat need only the cos sites in order to
function.
• The in vitro packaging reaction works not only
with  genomes, but also with any molecule that
carries cos sites separated by 37–52 kb of DNA.
• A cosmid is basically a plasmid that carries a cos
site .
• It also needs a selectable marker, such as the
ampicillin resistance gene, and a plasmid origin
of replication, as cosmids lack all the  genes and
so do not produce plaques.
• Instead colonies are formed on selective media,
just as with a plasmid vector.
SHIVA'S
 COSMIDS
Combine parts of the lambda chromosome with parts of
plasmids.
Contain the cos sites of λ and plasmid origin of replication.
Behave both as plasmids and as phages.
Cosmids can carry up to 50 kb of inserted DNA.
Structure of Cosmid
Origin of replication (ori).
Restriction sites for cleavage and insertion of foreign DNA.
Selectable marker from plasmid.
A cos site - a sequence yield cohesive end (12 bases).
Ampicillin resistance gene (amp).
 ADVANTAGES AND DISADVANTAGES

• ADVANTAGES

1. Large DNA fragments

2. Used to establish gene libraries of lower &higher organisms
3. Gene cloning through cosmid helps in the study of non-sense seq
in the genome of organism
• DISADVANTAGES
1. The packaging enzyme fails to pack rec cosmid into phage heads
if 1 of cos-sites is missing;
 PHAGEMIDS
• A.k.a Phagemids
• A hybrid vector that has origin of repl from a a plasmid and a
lambda phage DNA
• It is constructed by inserting a linearised plasmid DNA into a
cleaved lambda DNA
 ADVANTAGES
• PHAGEMIDS can be maintained as plamids or phage particles in
E.coli
• The desired gene can be integrated into chromosomal DNA of E.coli
using phagemids
• Plasmid portion can be released free from a phagemid after
rec.phagemid is introduced into an E.coli Strain
• The phages having rec.phagemids can be stored easily for a long
time
 ARTIFICIAL CHROMOSOME
 Linear or Circular.
 1 0r 2 copies per cell.
 Different types –
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
P1 derived artificial chromosome (PAC)
Mammalian Artificial Chromosome (MAC)
Human Artificial Chromosome. (HAC)
 YAC – Cloning in yeast
 BAC & PAC – Bacteria
 MAC & HAC – Mammalian & Human cells.
 Yeast Artificial Chromosomes (YAC)

 YACs are artificial chromosomes that replicate in yeast

cells. They consist of Telomeres, which are ends of
chromosomes involved in the replication and stability of
linear DNA.
 Origin of replication sequences necessary for the
replication in yeast cells.
 A yeast centromere, which is a specialized chromosomal
region where spindle fibers attach during mitosis.
 A selectable marker for identification in yeast cells.
 Ampicillin resistance gene for selective amplification.
 Recognition sites for restriction enzymes.
SHIVA'S
 YEAST ARTIFICIAL CHROMOSOME(YAC)

Purpose:
• Cloning vehicles that propogate in eukaryotic cell hosts as
eukaryotic Chromosomes
• Clone very large inserts of DNA: 100 kb - 10 Mb

Features:
• Final chimeric DNA is a linear DNA molecule with telomeric ends:
Artificial Chromosome.
• Linear Plasmid Vector.

• Occurring two forms:-

Circular – grows in bacteria.
Linear – multiplies in yeast cells.
• Often have a selection for an insert

• YAC cloning vehicles often have a bacterial origin of DNA replication (ori)
and a selection marker for propogation of the YAC through bacteria.
• The YAC can use both yeast and bacteria as a host
The procedure for making YAC vectors is as
follows
1. The target DNA is partially
digested by a restriction
endonuclease, and the YAC vector
is cleaved by restriction enzymes.
2. The cleaved vector segments
are ligated with a digested DNA
fragment to form an artificial
chromosome.
3. Yeast cells are transformed to
make a large number of copies.
They are the largest of the cloning
vectors, with a cloning limit of
100-1000 kb, however they have
very low efficiency.
SHIVA'S
 pYAC3 - first YAC developed.
 It contains :-
ARS sequence – replication
CEN4 sequence – centromeric function
TRP1 & URA3 – 2 selectable markers
 Use – mapping complex eukaryotic chromosome .
 Yeast/YAC vectors
SnaBI
CEN4 is the centromere of
chromosome 4 of Yeast. The S
centromere will segregate the
daughter chromosomes.
ARS is autonomously replicating pYAC3
sequence, its function is as a
yeast origin of replication.
TRP1 and URA3 are yeast selectable
markers, one for each end, to
ensure the right reconstituted
YACs survive in the yeast cells. B B
TEL is the telomeric DNA sequence,
which is extended by the BamHI
telomerase enzyme inside the
yeast cell.
SUP4 is a gene, which is
insertionally inactivated, for a
red-white color test, like blue-
white screening in E. coli.
Function: YAC vectors can accept genomic DNA
fragments of more than 1 Mb, and hence can be
used to clone entire human genes.
SHIVA'S
SHIVA'S
 BACTERIAL ARTIFICIAL CHROMOSOME
1st BAC Vector – PBAC108L.
Cloning of large regions of eukaryotic
genome.
Origin of replication from bacterium Ecoli
F -factor.
BAC vectors are pBACe3.6, pBeloBAC11.
Used in analysis of genomes.
Host for BAC is mutant strain.
 BAC
• Can be cloned as a plasmid in a bacterial host,
and its natural stability generally permits
cloning of large pieces of insert DNA, i.e. up to
a few hundred kb of DNA.
• based on bacterial mini-F plasmids, which are
small pieces of episomal bacterial DNA that
give the bacteria the ability to initiate
conjugation with adjacent bacteria. They
have a cloning limit of 75-300 kb.
Bacterial Artificial Chromosomes (BAC)

SHIVA'S
 PAC
• PACs - P1-derived Artificial Chromosomes

• E. coli bacteriophage P1 is similar to phage lambda in

that it can exist in E. coli in a prophage state.

• Exists in the E. coli cell as a plasmid, NOT integrated

into the E. coli chromosome.
• P1 cloning vehicles have been constructed that permit
cloning of large DNA fragments- few hundred kb of DNA

• Cloning and propogation of the chimeric DNA as a P1

plasmid inside E. coli cells
 Human Artificial Chromosome(HAC)

• Synthetically produced vector DNA possessing the characteristics of

human chromosome.
• Discovered in 1997 by H.Williard

Advantages:

1. Can carry Human genes that are too long

2. Can carry genes to be introduced into the cells via gene therapy
 Other types

• Shuttle vectors
• Integration Vectors
• Transposons,Retroviruses,Adenoviruses,Bacul
o viruses
 CONCLUSION
• There are different types of cloning vectors used in Genetic
Engineering
• These include: Plasmids, Phages,Hybrid Vectors and Artificial
Chromosome
• The best vector is chosen for use according to the purpose of use
and according to how large/short the DNA fragment to be carried is
 REFERENCES
1. Kumaresan.V. Bitotechnology.2001.Saras Publications
2. Rastogi.S.C. Biotechnology:Principles and Applications. Narosa
Publishing House
3. Sasidhara.R. Animal Biotechnology.MJP Publications
4. Satyanarayana.U. Biotechnology . Books and Allied (P)Ltd.
5. http://en.wikipedia.org