CLONING VECTORS

-PRIYANKA TIWARI
 Phage vector or lambda phages
• Bacteriophage λ is a genetically complex but very extensively
studied virus of E. coli
• The DNA of phage λ, in the form in which it is isolated from the
phage particle, is a linear duplex molecule of about 48.5 kbp.
• At each end are short single-stranded 5′ projections of 12
nucleotides. which are complementary in sequence
• With help of this sequence the DNA adopts a circular structure
when it is injected into its host cell, i.e. λ DNA naturally has
cohesive termini, which associate to form the cos site.
• Functionally related genes of phage λ are clustered
together on the map, except for the two positive
regulatory genes N and Q.
• Genes on the left of the conventional linear map code for
head and tail proteins of the phage particle.
• Genes of the central region are concerned with
recombination and the process of lysogenization, in which
the circularized chromosome is inserted into its host
chromosome and stably replicated along with it as a
prophage.
• Much of this central region, including these genes, is not
essential for phage growth and can be deleted or replaced
without seriously impairing the infectious growth cycle
 ADVANTAGE PHAGE VECTOR
• Since phage λ can accommodate only about 5% more than its
normal complement of DNA, vector derivatives are
constructed with deletions to increase the space within the
genome.
• The shortest λ DNA molecules that produce plaques of nearly
normal size are 25% deleted.
• Apparently, if too much non-essential DNA is deleted from the
genome, it cannot be packaged into phage particles
efficiently.
• If the replaceable fragment of a replacement-type vector is
either removed by physical separation or effectively destroyed
by treatment with a second restriction endonuclease that cuts
it alone, then the deleted vector genome can give rise to
plaques only if a new DNA segment is inserted into it.
• This amounts to positive selection for recombinant phage
carrying foreign DNA.
 COSMID VECTOR
• Plasmids have been constructed which contain a fragment of DNA including
the cos site.
• These plasmids have been termed cosmids and can be used as gene-cloning
vectors in conjunction with the in vitro packaging system.
• Packaging the cosmid recombinants into phage coats imposes a desirable
selection upon their size.
• With a cosmid vector of 5 kb, the insertion of 32–47 kb of foreign DNA – much
more than a phage- vector can accommodate.
• After packaging in vitro, the particle is used to infect a suitable host.
• The recombinant cosmid DNA is injected and circularizes like phage DNA but
replicates as a normal plasmid without the expression of any phage functions.
• Transformed cells are selected on the basis of drug-resistance marker.
• Cosmids provide an efficient means of cloning
large pieces of foreign DNA.
• Because of their capacity for large fragments
of DNA, cosmids are particularly attractive
vectors for constructing libraries of eukaryotic
genome fragments.
• Partial digestion with a restriction
endonuclease provides suitably large
fragments.
 PHAGEMID
• A phagemid (plasmid + phage) is a plasmid that contains an f1 origin of
replication from an f1 phage.
• It can be used as a type of cloning vector in combination with filamentous phage
M13.
• A phagemid can be replicated as a plasmid, and also be packaged as single
stranded DNA in viral particles.
• Phagemids contain an origin of replication (ori) for double stranded replication,
as well as an f1 ori to enable single stranded replication and packaging into
phage particles.
• M13 is a filamentous coliphage with a single-stranded circular DNA genome.
• Upon infection of E. coli harbouring male-specific structure termed the F-pilus,
the DNA replicates initially as a double-stranded molecule but subsequently
produces single-stranded virions for infection of further bacterial cells (lytic
growth).
• M13 or phagemids infect E. coli harbouring They enter the cell by
adsorption to this structure
• Once inside the phage DNA is converted to a double-stranded replicative
form (RF DNA).
• Replication then proceeds rapidly until some 100 RF molecules are
produced within the E. coli cell.
• DNA synthesis then switches to the production of single strands
• DNA is assembled and packaged into the capsid at the bacterial periplasm.
• The bacteriophage DNA is then encapsulated by the major coat protein,
gene VIII protein, of which there are approximately 2800 copies with three
to six copies of the gene III protein at one end of the particle.
• The extrusion of the bacteriophage through the bacterial periplasm
results in a decreased growth rate of the E. coli cell rather than host cell
lysis.
• Phagemids are very similar to M13 and replicate in a
similar fashion.
• One of the first phagemid vectors, pEMBL, was
constructed by inserting a fragment of another phage
termed f1 containing a phage origin of replication and
elements for its morphogenesis into a pUC8 plasmid.
• Following superinfection with helper phage the f1 origin is
activated allowing single-stranded DNA to be produced.
• The phage is assembled into a phage coat extruded
through the periplasm and secreted into the culture
medium in a similar way to M13.
 SHUTTLE VECTOR
• A shuttle vector is a vector (usually a plasmid)
constructed so that it can propagate in two
different host species.
• Therefore, DNA inserted into a shuttle vector
can be tested or manipulated in two different
cell types.
• The main advantage of these vectors is they can
be manipulated in E. coli, then used in a system
which is more difficult or slower to use (e.g.
yeast).
• Shuttle vectors include plasmids that can
propagate in eukaryotes and prokaryotes (e.g.
both Saccharomyces cerevisiae and
Escherichia coli) or in different species of
bacteria (e.g. both E. coli and Rhodococcus
erythropolis).
• There are also adenovirus shuttle vectors,
which can propagate in E. coli and mammals.
• Adenoviruses (members of the family Adenoviridae) are
medium-sized (90–100 nm), nonenveloped (without an
outer lipid bilayer) viruses with an icosahedral
nucleocapsid containing a double-stranded DNA genome.
• Adenovirus biology has been extensively studied;
adenovirus genomes are relatively easy to manipulate, and
they are capable of carrying large amounts of exogenous
DNA.
• They are capable of infecting both dividing and non-
dividing cells.
• The adenovirus genome is linear, non-segmented double-
stranded (ds) DNA that is between 26 and 48 Kbp.
• First-generation vectors contain deletions in the E1 region, often
lacking E1A and E1B.
• while second-generation vectors lack even more nonessential regions,
such as E2 and E4 transcription units, to increase the capacity for
exogenous DNA.
• Helper-dependent adenovirus vectors, also referred to as gutted
vectors, contain only viral sequences that are essential for DNA
replication including the inverted terminal repeats that serve as DNA
replication origins and viral packaging sequences.
• These vectors have an even greater cloning capacity than the first- and
second-generation adenovirus vectors. Because they lack early and
late genes that are essential for the production of virus particles,
helper-dependent adenovirus vectors must be grown in the presence
of a complementing helper adenovirus that provides these missing
elements.
 EXPRESSION VECTOR OR TRANSCRIBABLE
VECTOR
• An expression vector, is usually a plasmid or virus designed for gene
expression in cells.
• The vector is used to introduce a specific gene into a target cell, and can
command the cell's mechanism for protein synthesis to produce the
protein encoded by the gene.
• The vector is engineered to contain regulatory sequences that act as
enhancer and promoter regions and lead to efficient transcription of the
gene carried on the expression vector.
• The goal of a well-designed expression vector is the efficient production
of protein, and this may be achieved by the production of significant
amount of stable messenger RNA, which can then be translated into
protein.
An expression vector has features that any vector
may have, such as:
• An origin of replication,
• A selectable marker
• A suitable site for the insertion of a gene like the
multiple cloning site.

The cloned gene may be transferred from a

specialized cloning vector to an expression vector,
although it is possible to clone directly into an
expression vector. The cloning process is normally
performed in Escherichia coli.
 ELEMENTS OF EXPRESSION
• An expression vector must have elements necessary for gene expression.
• These may include a promoter, the correct translation initiation sequence such as a
ribosomal binding site and start codon, a termination codon, and a transcription
termination sequence.
• There are differences in the machinery for protein synthesis between prokaryotes
and eukaryotes, therefore the expression vectors must have the elements for
expression that are appropriate for the chosen host.
• For example, prokaryotes expression vectors would have a Shine-Dalgarno
sequence at its translation initiation site for the binding of ribosomes.
• While eukaryotes expression vectors would contain the Kozak consensus sequence
.
• The promoter initiates the transcription and is therefore the point of control for
the expression of the cloned gene.
• The promoters used in expression vector are normally inducible, meaning that
protein synthesis is only initiated when required by the introduction of an inducer
such as IPTG.
 EXAMPLES OF EXPRESSION VECTORS IN
VARIOUS HOST
• Bacteria- pKK233-2, pKK233-2 and pET
• Yeast- There are three main classes of S.
cerevisiae expression vectors: episomal, or
plasmid, vectors (yeast episomal plasmids
[YEps]), integrating vectors (yeast integrating
plasmids [YIps]); and YACs.
• Insects- Baculovirus Expression Vectors.
• Mammals-SV40 vector
 Pkk233-2
• A number of convenient vector systems that incorporate both
transcriptional and translational signals for the expression of cloned
eukaryotic genes in E. coli have been developed.
• One such system is called expression vector pKK233-2.
• It includes elements like -an ampicillin resistance gene as a selectable
marker, the tac promoter, the lacZ ribosome-binding site, an ATG start
codon located 8 nucleotides downstream from the ribosome-binding
site, and the transcription terminators T1 and T2 from bacteriophage λ.
• The cloned gene is inserted into an NcoI, PstI, or HindIII site that lies
between the ribosome binding site and the transcription terminators
so that it is in the same reading frame as the ATG start codon.
• After induction and transcription,the mRNA of a cloned gene is
efficiently translated.
 Bacterial Artificial Chromosome (BAC)
• Bacterial artificial chromosomes (BACs) are designed for the
cloning of large DNA insert (typically 100 to 300 kb).
• BAC vectors contain a single copy F-plasmid origin of
replication (ori).
• The F (fertility) plasmid is relatively large and vectors derived
from it have a higher capacity than normal plasmid vectors.
• F-plasmid has F (fertility) factor which controls the replication
and maintain low copy number.
• Conjugation can take place between F+ bacteria (male) and F-
bacteria (female) to transfer F-plasmid via pilus.
 Common gene components of a bacterial
artificial chromosome
• oriS, repE – F responsible for plasmid replication and
regulation of copy number.
• parA and parB for maintaining low copy number and avoiding
two F plasmids in a single cell during cell division.
• Chloramphenicol resistance gene-A selectable marker for
antibiotic resistance .
• Few BACs also have blue/white selection having lacZ gene at
the cloning site.
• T7 and Sp6 phage based promoters examining transcription of
genes of interest.
• the λ cos N and P1 loxP sites are cleavage site
• BAC system (bacterial artificial chromosome) is based
on the single-copy sex factor F of E. coli.
• This vector includes the λ cos N and P1 loxP sites, two
cloning sites (HindIII and BamHI), and several G+C
restriction enzyme sites (e.g. SfiI, NotI, etc.) for
potential excision of the inserts.
• The cloning site is also flanked by T7 and SP6
promoters for generating RNA probes.
 Yeast artificial chromosome (YAC)
• Yeast artificial chromosomes (YACs) are linear molecules
composed of a centromere, telomere and a replication
origin termed an ARS element (autonomous replicating
sequence).
• The YAC is digested with restriction enzymes at the SUP4
site (a suppressor tRNA gene marker) and BamHI sites
separating the telomere sequences.
• This produces two arms and the foreign genomic DNA is
ligated to produce a functional YAC construct.
 YAC vectors have following elements:

1. coli origin of replication

2. Yeast origin of replication
3. Elements of eukaryotic yeast chromosome
(centromere and telomere region)
4. Selection markers for both the hosts (Bacterial
as well as Yeast)
• Yeast artificial chromosomes are created artificially joining
centromere (CEN), telomeres (TEL), and an origin of
replication as autonomous replicating sequence (ARS)
elements necessary for replication and conservation of YAC in
host yeast.
• A circular plasmid is used to create YAC by cleaving it in to two
linear fragments by appropriate restriction endonucleases.
• These two linear fragments ligated with desired foreign DNA
using DNA ligase enzyme forming single large linear DNA
molecule.
• TRP1 and URA3 genes are integrated in the YAC vector to
provide a selection system for identifying transformed yeast
cells that include YAC by complementing recessive alleles trp1
and ura3 in yeast host cell.
 ADVANTAGES OF YAC
• DNA fragments with repeat sequences are sometimes
difficult to clone in bacterial-based vectors but may
be successfully cloned in YAC systems.
• The main advantage of YAC-based vectors, however,
is the ability to clone very large fragments of DNA.
• Thus the stable maintenance and replication of
foreign DNA fragments of up to 2000 kb have been
carried out in YAC vectors.
• They are the main vector of choice in the various
genome mapping and sequencing projects.