CLONING VECTORS

Vectors
Vector is an agent that can carry a DNA fragment into a host cell in
which it is capable of replication.

Vectors are ship for carrying the target DNA into a host cell.
Cloning vector –
• used for obtaining millions of copies of cloned DNA segment.

• Used for creating genomic library or preparing the probes or

genetic engineering experiments or other basic studies.

Expression vector –
• allows expression of cloned gene, to give the product (protein).
This can be achieved through the use of promoters and expression
cassettes and regulatory genes.

• Used for transformation to generate trangenic plants, animals or

microbes where cloned gene expresses to give the product.
Properties of a good vector:

 It should be autonomously replicating i.e. it should have

ori region.
 It should contain at least one selectable marker e. g.
gene for antibiotic resistance (tetR for tetracycline
resistance).
 It should have unique restriction enzyme site (only one
site for one RE) for different REs (preferably in one of the
marker genes) to insert foreign DNA.
 It should be preferably small in size for easy handling.
 It should contain specific control systems like promoters,
terminators, ribosome binding sites etc so that the
cloned DNA should express properly.
TYPES

Vectors are of different types depending on

the host. These are as follows:
1. Bacterial vectors
2. Yeast vectors
3. Plant vectors
4. Animal vectors
Bacterial vectors
E.coli is the most commonly used bacterium for gene
cloning though other bacteria such as Bacillus are also
used.

Vectors for cloning in E.coli

A number of vectors are used for cloning in E. coli.

These are categorized as plasmids, phages, cosmids,

phagemids and bacterial artificial chromosomes.
Bacterial plasmid
 PLASMIDS

Plasmids are classified

1. By their ability to be transferred to other bacteria

Conjugative
The sexual transfer of plasmids to another bacterium through a pilus.
those plasmids possess the 25 genes required for transfer.

Non-conjugative
Non-conjugative plasmids don’t initiate conjugation.
2. By function

1. Resistance-(R) plasmids,
contain gene (s) that can build resistance against
one or several antibiotics or poisons.

2. Col-plasmids,
contain genes coding for colicines, proteins that
can kill other bacteria.

3. Degradative plasmids
able to digest unusual substances, e.g., toluene or
salicylic acid.

4. Virulence plasmids
turn a bacterium into a pathogen.
Conjugative plasmids

The sexual transfer of plasmids to another bacterium through a pilus.

Those plasmids, F plasmids, possess the 25 genes required for transfer.

Plasmid forms: 1.covalently closed circles –both strands of DNA intact

2. open circles-only one of the two strand is intact
3. Linear.
4.Supercoiled circles.
Plasmid
vectors
 Plasmids are autonomously replicating circular, double stranded
DNA molecules found in bacteria.
 They have their own origin of replication (ori region) and can
replicate independently of the host chromosome.
 The size of plasmids ranges from a few kb to 200 kb. Plasmid
vectors are often used for cloning DNA segments of small size
(upto 10 kilobases).
 Single copy plasmid - maintained as single copy per cell
 Multicopy plasmid - maintained as 10-20 copies per cell
 Plasmids under relaxed control of replication – over 1000 copies
per cell – used as cloning vectors.
 In each bacterial cell about 20-25 plasmids are maintained under
normal growth condition.
 Some of the commonly used plasmid vectors are described
below:
pBR32
2
The first plasmid vector that has been constructed
artificially is pBR322.
 It is named after the scientists Bolivar and Rodriguiz
who constructed it in 1977.
 It is 4362bp in size and most widely used cloning
vector.
 It has an origin of replication derived from a colicin-
resistance plasmid (ColE1).
This origin allows a fairly high copy number, about 100
copies of the plasmid per cell.
 Plasmid pBR322 carries two selectable markers viz.
genes for resistance to ampicillin (Apr) and tetracycline
(Tcr ).
Figure pBR322
The map shows the positions of the ampicillin-resistance gene (amp R),
the tetracycline-resistance gene (tet R), the origin of replication (ori) and
the recognition sequences for seven restriction endonucleases.
Streptomyces caespitosus
Figure 4.18 Recombinant selection with pBR322
pUC
 A series of small plasmids (about 2.7 kb) have been developed (by
Messings and co-workers in1983) at the University of California and
hence the name pUC e.g. pUC7, 8,9,12,13, 18 and 19 etc.

 These are high copy number plasmids that carry an ampicillin

resistance gene and an origin of replication, both from pBR322.

 They also have a multiple cloning site (MCS) – a sequence of DNA

that carries unique sites for many REs.

 The MCS contains a portion of lacZ gene that codes for the enzyme
β-galactosidase.

 When such plasmids are introduced into E. coli, the colonies are
blue on plates containing X-gal (5-bromo-4-chloro-3-indolyl-β-d-
galactopyranoside, the substrate for β- galactosidase) and IPTG
(isopropyl thiogalactoside, an inducer).
 Recombinants and non-recombinants can therefore be
distinguished simply by plating the transformed cells onto agar
containing ampicillin and X-gal.

 All colonies that grow on this medium are made up of

transformed cells because only transformants are ampicillin
resistant.

 Blue colonies contain cells with functional β-galactosidase

enzymes and hence with undisrupted lacZ′ genes these colonies
are therefore non-recombinants.

 The white colonies comprise cells without β-galactosidase activity

and hence with disrupted lacZ′ genes; these are recombinants.

 Thus, cells containing recombinant plasmids form white (not blue)

colonies.
Recombinant selection with pUC8
 Must have:
1) Ori
2) A dominant selectable
Marker
3) Cleavage sites for
cloning
4) (high copy no.)

The plasmid cloning

vector pUC19. This
plasmid has an origin of
replication (ori), an
ampR selectable
marker, and a polylinker
located within part of
the -galactosidase
gene lacZ+.
Phage
vectors
 Bacteriophages or phages are viruses that specifically infect
bacteria.

 The phage particle attaches to the outer surface of bacterium and

injects its DNA into the cell.

 The phage DNA is then replicated inside the host and its genes are
expressed to make phage capsid proteins and new phage particles
are assembled and released from the bacterium.

 Phage vectors can accommodate more DNA (upto 25 kb) than

plasmids and are often used for preparation of genomic libraries.

 They also have higher transformation efficiency as compared to

plasmids.
 The main reason for seeking a different type of vector was the
inability of plasmids such as pBR322 and pUC8 to handle DNA
fragments greater than about 10 kb in size.

 The first attempts to develop vectors able to handle larger

fragments of DNA centered on bacteriophage λ.

 Two bacteriophages namely, Lambda (λ) and M13 have been

commonly used for construction of vectors for cloning in E. coli.

 The phage can have two modes of life cycles i.e. lytic and
lysogenic.

 During lytic cycle, it replicates independently in the host cell and

produces a large number of phage particles which are released by
lysis of the host. Alternatively, it can take up lysogenic growth,
meaning that it integrates its DNA into the bacterial chromosome
and multiplies along with it.
 (N, cro, cI genes)
Map of the λ chromosome of wild type .

The genes are not essential for phage

growth and can be deleted or replaced
without seriously impairing the
infectious growth cycle
The lysogenic
infection cycle of
bacteriophage λ

The special feature

of the lysogenic
cycle is the
insertion of the
phage genome into
the bacterium's
chromosomal
DNA, where it can
remain quiescent
for many
generations.
integration host factor
Lambda (λ) phage
vectors
 Lambda is a temperate bacteriophage with a genome size of 48.5
kb. Its entire DNA sequence is known.

 The lambda genome is a linear, double-stranded molecule with

single-stranded, complementary ends. These ends can hybridize
with each other (and do so when the DNA is within an infected
cell) and are thus termed cohesive (cos) sites.

 The λ genome is 48.5 kb, of which some 15 kb or so is ‘optional’

in that it contains genes that are only needed for integration of the
phage DNA into the E. coli chromosome.

 These segments can therefore be deleted without impairing the

ability of the phage to infect bacteria and direct synthesis of new λ
particles by the lytic cycle.
The λ genome is linear, but the two natural ends of the molecule
have 12-nucleotide single-stranded overhangs, called cos sites,
which have complementary sequences and so can base-pair to one
another.
Insertional vectors have one unique restriction site for a
particular restriction enzyme and can accommodate 6-7 kb DNA.

Examples of insertional vectors are λgt10, λgt11 and λZAP II.

 On the other hand, replacement vectors have two cleavage sites

for a restriction enzyme and can accommodate up to 25 kb DNA.

When vector is cut with a restriction endonuclease, a stuffer

fragment is removed and replaced with a foreign DNA.

Some examples of replacement vectors are EMBL3, EMBL3A,

EMBL4, λDASH, λFIX, GEM11 and GEM12.
Cloning vectors based on bacteriophage λ
(A) In the λ genome, the genes are arranged into functional groups. For example, the region marked
as ‘protein coat’ comprises 21 genes coding for proteins that are either components of the
phage capsid or are required for capsid assembly, and ‘cell lysis’ comprises four genes involved
in lysis of the bacterium at the end of the lytic phase of the infection cycle. The regions of the
genome that can be deleted without impairing the ability of the phage to follow the lytic cycle
are indicated in green. (B) The differences between a λ insertion vector and a λ replacement
vector.
Bacteriophage lambda can be reconstituted in a test tube by simply
mixing phage DNA with a mixture of phage proteins, an infective
viral particle with the DNA inside the phage head can be produced.
This process is called in vitro packaging.

This feature allows only the recombinants to be packaged inside the

phage head.

 In addition, some lambda phage vectors have a stuffer fragment

that carries the β-galactosidase gene.

When it is removed or when foreign DNA is cloned within the gene,

β-galactosidase activity may be abolished. The accompanying loss of
activity may be used to select recombinant clones.
Cloning with a λ insertion vector
The linear form of the vector is shown at the top of the diagram. Treatment with the appropriate restriction
endonuclease produces the left and right arms, both of which have one blunt end and one end with the 12-
nucleotide overhang of the cos site. The DNA to be cloned is blunt ended and so is inserted between the two
arms during the ligation step. These arms also ligate to one another via their cos sites, forming a concatamer.
Some parts of the concatamer comprise left arm-insert DNA-right arm and, assuming this combination is 37–52
kb in length, will be enclosed inside the capsid by the in vitro packaging mix. Parts of the concatamer made up of
left arm ligated directly to right arm, without new DNA, are too short to be packaged.
Bacteriophage infection is visualized as a plaque on a lawn of bacteria
DNA cloning with single stranded DNA
vectors

These coliphages are developed as cloning vectors

This phage particles have dimensions 900 nm × 9 nm contain a

single-stranded circular DNA molecule (6407 (M13) or 6408 (fd)
nucleotides long).

they have many advantages over other vectors

M13, f1 and fd are filamentous coliphages containing a circular

single-stranded DNA molecule.
The biology of the filamentous coliphages

The phages only infect enteric bacteria harboring F-Pili

the end of the F pilus is the adsorption site .

infected cells continue to grow and divide, and extrude virus

particles.

Up to 100 phage particles may be released into the medium per
cell per generation

Replication of phage DNA does not result in host-cell lysis.

M13 Phage
vectors
M13 is a filamentous bacteriophage of E. coli and contains a
single stranded circular DNA of 7.2 kb.

 A series of vectors (M13 mp series) have been developed from

this phage.

These vectors have a polylinker with unique restriction enzyme

sites in lac Z gene that complements host (e.g. JM 103 or JM 104).

 Screening of recombinants is done based on formation of

blue/white plaques.
Cloning vectors and their insert capacities
Vector system Host cell Insert capacity (kb)

Plasmid E. coli 0.1-10

Bacteriophage l E. coli 10-20

Cosmid E. coli 35-45

Bacteriophage P1 E. coli 80-100

BAC (bacterial E. coli 50-300

artificial
chromosome)
P1 bacteriophage- E. coli 100-300
derived AC
YAC Yeast 100-2,000
Human AC Cultured human >2,000
cells
A typical cosmid
pJB8 is 5.4 kb in size and carries the ampicillin-resistance
gene (amp R), a segment of λ DNA containing the cos site,
and an Escherichia coli origin of replication (ori).
cosmid
vector
 The cosmid vector is a combination of the plasmid and
bacteriophage lambda.

 It is small (5-7 kb) circular DNA containing

 an origin for DNA replication (ori),
 selectable markers and restriction sites from plasmid plus
 a sequence from lambda needed for packaging the DNA (cos site).

 Cosmids may be used to clone large DNA molecules of up to 45

kb.

 They also have high transformation efficiency.

 Some examples of cosmid vectors include pJB, PWE and SuperCos

series.
Vectors for cloning in yeast
 The discovery of a 2μm plasmid in most strains of Saccharomyces
cerevisiae led to the development of cloning vectors in yeast.

 The 2μm plasmid is 6 kb in size. It is present in 50-100 copies per

cell.

 A number of shuttle vectors based on 2μm plasmid and bacterial

plasmids have been constructed which can replicate either in
E.coli or yeast.

 Yeast plasmid vectors are of four types, yeast episomal plasmids

(YEps), yeast integrative plasmids (YIps) yeast replicative plasmids
(YRps) and yeast centromeric plasmids (Ycps).

 In addition to plasmid vectors, yeast artificial chromosomes

(YACs) are also used as vectors for cloning large pieces of DNA.
Yeast episomal plasmids (YEps)
 Some YEps contain the entire 2μm plasmid; others include just the
2μm origin of replication.

 An example of latter type is YEp13. It is a shuttle vector and can be

replicated both in E.coli and yeast.

 It contains 2μm origin of replication, yeast gene leu2 as selectable

marker and entire sequence of pBR322.

 The leu2 gene codes for an enzyme involved in biosynthesis of

amino acid leucine.

 YEps may replicate autonomously or integrate in one of the yeast

chromosomes by homologous recombination.

 They have high transformation frequency of 10,000 to 100,000

transformants/μg DNA.
Yeast integrative plasmids (YIps)

 These are basically bacterial plasmids carrying a yeast gene.

 YIp5 is an example of yeast integrative plasmid. It has ura3 gene

inserted in pBR322. Orotidine 5'-phosphate decarboxylase

 The gene ura3 codes for an enzyme involved in biosynthesis of

pyrimidine nucleotides and acts as selectable marker.

 The plasmid cannot replicate autonomously as it lacks 2μm origin

of replication and survives by integrating in yeast chromosomal
DNA.

 They have very low transformation frequency, less than 100

transformants/μg DNA.
Yeast Artificial Chromosomes (YACs)

YACs are artificial chromosomes that replicate in yeast cells. Main

features of these vectors are:
1. Autonomously replicating sequence (ARS) necessary for the
replication in yeast cells.
2. Telomeres (TEL), which are ends of chromosomes involved in the
replication and stability of chromosomes.
3. A yeast centromere (CEN), required for proper segregation of
chromosomes
4. Selectable markers that allow the easy isolation of yeast cells
that have taken up the artificial chromosome.
5. Unique RE sites.

YACs are capable of carrying a large DNA fragment (up to 3000 kb),
but their transformation efficiency is very low.
Phosphoribosylanthranilate
isomerase, an enzyme that
catalyzes the third step in
Sup4
tryptophan biosynthesis
Figure 4.25Working with a YAC
(A) The cloning vector pYAC3. (B) To clone with pYAC3, the circular vector is
digested with BamHI and SnaBI. BamHI restriction removes the stuffer fragment
held between the two telomeres in the circular molecule. SnaBI cuts within the
SUP4 gene and provides the site into which new DNA will be inserted. Ligation of
the two vector arms with new DNA produces the structure shown at the bottom.
This structure carries functional copies of the TRP1 and URA3 selectable
markers. The host strain has inactivated copies of these genes, which means that
it requires tryptophan and uracil as nutrients. After transformation, cells are
plated onto a minimal medium, lacking tryptophan and uracil. Only cells that
contain the vector, and so can synthesize tryptophan and uracil, are able to
survive on this medium and produce colonies. Note that if a vector comprises
two right arms, or two left arms, then it will not give rise to colonies because the
transformed cells will still require one of the nutrients. The presence of insert
DNA in the cloned vector molecules is checked by testing for inactivation of
SUP4. This is done by a color test: on the appropriate medium, colonies
containing recombinant vectors (i.e. with an insert) are white; non-recombinants
(vector but no insert) are red.
 Retroviruses
(including Lentivirus, HIV and MMLV based vectors)

•Single stranded RNA genome

•Lipid membrane enveloped
•Host range determined by envelope proteins Retrovirus

ssRNA Genome

Reverse Transcription
into dsDNA

Random integration
into host genome

Host DNA

Host Cell
 The Retroviral Genome

LTR  LTR
gag pol env

Long Terminal Repeat (LTR): Necessary for integration into host genome

 (Psi): packaging signal

gag: Packages viral genome into viral particles

pol: viral polymerase necessary for viral replication

env: viral envelope proteins, necessary for entry into host cells, dictate host range
 Design of Replication Incompetent Lentiviral
Vectors (3rd Generation)

The viral vector is “gutted” as much as possible to create room for the
insert gene and to divide the viral genome into cis- and trans- acting
regions

Promoter and Insert Gene

LTR∆ LTR∆
gag pol env Modified LTR
To impede the
Virus from
Performing more
Than one round
Of reverse
Transcription
Transfer Packaging Envelope
Vector Vector Vector
“self inactivating”

Regulatory Signals Structural &

Packaging Genes Viral envelope
(LTR and ),
(may already be protein alters host
promoter and
present in packaging range of the viral
Insert Gene
line) vector
Principles of Retroviral Vector Design
It is possible to make replication-competent retroviral vectors
by adding sequences to existing viruses, but a more common
design involves the replacement of retroviral sequences to
create replication-defective vectors. In addition, the amount of
foreign DNA that can be accommodated in replication-
competent vectors is much smaller than can be
accommodated in replication-defective vectors. Expression of
retroviral proteins in most of the naturally occurring
oncogenic retroviruses is driven by a single promoter in the
5′long terminal repeat (LTR), and the expression of multiple
viral coding regions is achieved by alternative splicing.
However, vector design is not limited to the use of the single
retroviral promoter with alternative splicing. Other strategies
include the use of multiple promoters, insertion of genes in
the reverse orientation, and the use of internal ribosome
entry sites (IRESs).
Efficient gene transduction and integration depend on the inclusion in
the retroviral vector of a number of cis-acting viral elements. These
include
 (1) a promoter and polyadenylation signal in the viral genome;
 (2) a viral packaging signal (ψ or E) to direct incorporation of vector
RNA into virions;
(3) signals required for reverse transcription, including a transfer RNA-
binding site (PBS) and polypurine tract (PPT) for initiation of first- and
second-strand DNA synthesis, and a repeated (R) region at both ends of
the viral RNA required for transfer of DNA synthesis between templates;
 (4) short, partially inverted repeats located at the termini of the viral
LTRs required for integration.
An important general consideration in the design of retroviral vectors is
the effect of viral replication on vector structure. After one round of viral
replication, the U3 regions in both LTRs are derived from the U3 region
originally present in the 3′LTR in the plasmid form of the vector, and both
U5 regions are derived from the U5 region originally present in the 5′LTR
in the plasmid.
 Ordinarily, R sequences should arise primarily from the 5′plasmid LTR,
but they may also include 3′plasmid LTR sequences.
 Packaging Recombinant Lentiviral Particles

The three plasmids

containing the viral
genome components
are transfected into the
packaging line to create
the infectious viral
particles.
Multiple plasmids are
used so multiple
recombination events
would be required to
reconstitute a
replication competent
virus.

www.sigma.com/RNAI
 Viral Psuedotyping: A Double Edged Sword
Tropism: The ability of a virus to infect a particular type of
host cell
Psuedotyping: Altering the viral envelope protein to alter
tropism, thus allowing the virus to infect cells it originally
could not
Tropism Host Range Viral Envelope Receptor for
Protein Viral Envelope
Ecotropic Mouse / Rat Gap70 mCAT-1

Amphotropic Mammals 4070A Ram-1

/ Dualtropic / 10A1 / GALV

Pantropic All Animals VSV-G Phosphotidyl

serine
Phosphotidyl
inositol
GM3 ganglioside

Special care should be used when working

with pantropic or amphotropic viruses which
can infect humans!
 Replication Deficient Viral Vectors: Genetically Engineered So The
Viral Infection Cannot Spread

•The viral DNA does not contain the viral genes needed to make more
viruses.

Viral DNA Virus Target Cell Infected With Viral

Gene of Interest DNA Containing The Gene of
Interest

Cell’s DNA
Target Cell

No New Viral Particles are Created

Infection dose not spread
 Rescue of Replication Deficient Viruses
by superinfection with Wild Viruses

Wild
Viral DNA Virus Virus
Gene of Interest

Cell’s DNA
Target Cell

Complementation:
The genome from the wild virus provides the missing proteins
needed for the viral vector to replicate. The superinfected cell
functions similarly to a packaging line.
 Rescue of Replication Deficient Viruses
by superinfection with Wild Viruses

Wild
Viral DNA Virus Virus
Gene of Interest

Cell’s DNA
Target Cell

Recombination:
The genome from the wild virus randomly recombines with the
viral vector, providing sufficient genetic material for the viral
vector to replicate. The resulting rescued virus may possess pieces
of the original insert gene. The viral genome is impossible to
predict due to random recombination. The virus may exhibit
altered virulence.
 Risks Associated with Retroviruses: Insertional Mutagenesis

Viral DNA Virus

Gene of Interest

Target Cell
Host Cell DNA

Oncogene

Proto-Oncogene
Random integration of viral genome may
disrupt endogenous host genes. Of special concern
Is disruption of proto-oncogenes, which can lead
to increased cancer risk.
 Risks Associated with Retroviral Vectors: Viral
Transduction

Viral DNA Virus Target Cell Infected With Viral

Gene of Interest DNA Containing The Gene of
Interest

Cell’s DNA
Target Cell

Individuals infected with the viral vector may

express the insert gene at the site of infection.