Cloning vector

Vectors are self- replicating DNA molecules . that serves
as the carrier for the transfer or insertion of foreign gene(s)
into another cell, where it can be replicated and/or
expressed.
Vector is a latin word derived from ‘vehere’ means carrier
Examples
Plasmid
Phage
Hybrid vector
Artificial Chromosomes.

Cloning vector
A cloning vector is a genome
that can accept the target
DNA and increase the number
of copies through its own
autonomous replication.
These vectors are only useful
for storing a genetic
sequence.
Expression Vector
A vector that is used for
expressing a gene contained
within the cloned DNA. For a
gene to give rise to a protein
product, an expression vector
must be used that contains the
necessary elements for a host
cell to transcribe and translate
the gene.

Major types of cloning Vectors
Plasmid. Circular extrachromosomal DNA that autonomously replicates inside
the bacterial cell. Plasmids generally have a high copy number, such
as pUC19 which has a copy number of 500-700 copies per cell.
Phage. Linear DNA molecules derived of bacteriophage lambda. Can be
replaced with foreign DNA without disrupting its life cycle.
Cosmids. Another circular extrachromosomal DNA molecule that combines
features of plasmids and phage.
Bacterial Artificial Chromosomes. Based on bacterial mini-F plasmids.
Yeast Artificial Chromosomes. This is an artificial chromosome that contains
telomeres (disposable buffers at the ends of chromosomes which are cut off
during cell division) with origins of replication, a yeast centromere (part of a
chromosome that links sister chromatids or a dyad), and a selectable marker for
identification in yeast cells.

Plasmids
• Plasmid vectors are used to clone DNA ranging in size from
several base pairs to several thousands of base pairs (100bp -
10kb).
• The term plasmid was first introduced by the American molecular
biologist Joshua Lederberg in 1952.

Some plasmids may integrate into the bacterial chromosome. They are
called episome or integrative plasmids. At this stage they replicate along
with the bacterial chromosome.
The plasmids in this way are classified into 2 types
Stringent & Relaxed plasmid
• Some plasmids, especially the larger ones, are stringent and have a low
copy number of perhaps just one or two per cell. stringent plasmids
encode protein factors that are necessary for their own replication,
• Relaxed plasmids, are present in multiple copies of 50 or more per cell.
relaxed plasmids replicate using host derived proteins

• One way of grouping plasmids is by their ability to transfer to
other bacteria. Conjugative plasmids contain tra genes, which
perform the complex process of conjugation, the transfer of
plasmids to another bacterium.
• Non-conjugative plasmids are incapable of initiating
conjugation, hence they can be transferred only with the
assistance of conjugative plasmids.

Another way to classify plasmids is by function. There are
five main classes:
•Fertility F-plasmids: which contain tra genes. They are
capable of conjugation and result in the expression of sex
pilli.
•Resistance plasmids: which contain genes that provide
resistance against antibiotics or poisons. They were
historically known as R-factors.
•Col plasmids: which contain genes that code for
bacteriocins, proteins that can kill other bacteria.
•Degradative plasmids: which contains genes for the
digestion of unusual substances, e.g. toluene and salicylic
acid.
•Virulence plasmids: which turn the bacterium into a
pathogen.

 First vetors to develop was pBR322
 “P” indicates plasmid
 “BR” identifies the laboratory in which vector
was originally constructed.
 (BR stands for Bolivar and Rodriguez, the two
researchers who develop pBR322)
 “322” distinguishes the plasmid from others
developed in the same laboratory.

Cloning Vectors based on E. coli Plasmids
pBR322
• The plasmid pBR322 was the ﬁrst widely used plasmid vector. It is a
small plasmid (4363 bp). pBR322 contains the following
components.
• Origin of replication: carries the Col EI replication origin and rop
gene to ensure reasonably high plasmid copy number (15–20 copies
per cell)
• Antibiotic resistance genes. as selectable markers. The ampicillin
resistance gene (termed bla or, more commonly, AMPr), and the
tetracycline resistance gene (termed tet or TETr)
• Cloning sites. The plasmid carries a number of unique restriction
enzyme recognition sites. Some of these are located in one or other of
the antibiotic resistance genes. For example, sites for PstI, PvuI and
SacI are found within AMPR, and sites for BamHI and HindIII are
located within TETR

• The antibiotic resistance genes in pBR322 allow for the direct selection of
recombinants in a process called insertional inactivation. For example, if we
want to clone a DNA fragment into the BamHI site of pBR322
• The insert DNA will interrupt the gene responsible for tetracycline
resistance, but the gene for ampicillin resistance will not be altered.
• Transformed cells are ﬁrst grown on bacterial plates containing ampicillin to
kill all the cells that do not contain a plasmid.
• Those cells that grow on ampicillin are then replica plated onto medium
containing both ampicillin and tetracycline.
• Those cells that grow in the presence of the ampicillin, but die under
tetracycline selection, contain plasmids that have foreign DNA inserts
 In other words, the insertion of a foreign DNA fragment into an antibiotic
resistance gene inactivates the gene product and leads to antibiotic
sensitivity

pUC plasmids
Cloning Vectors based on E. coli Plasmids

A schematic representation of a typical
plasmid vector that can be used for blue-
white screening.

• Blue-white screening is a rapid and efficient technique for the identification of
recombinant bacteria. It relies on the activity of β-galactosidase, an enzyme occurring
in E. coli, which cleaves lactose into glucose and galactose.
• The presence of lactose in the surrounding environment triggers the lacZ operon in E.
coli. The operon activity results in the production of β-galactoisdase enzyme that
metabolizes the lactose. Most plasmid vectors carry a short segment of lacZ gene that
contains coding information for the first 146 amino acids of β-galactosisdase. The
host E. coli strains used are competent cells containing lacZΔM15 deletion mutation.
When the plasmid vector is taken up by such cells, due to α-complementation process,
a functional β-galatosidase enzyme is produced.
• The plasmid vectors used in cloning are manipulated in such a way that this α-
complementation process serves as a marker for recombination. A multiple cloning
site (MCS) is present within the lacZ sequence in the plasmid vector. This sequence
can be nicked by restriction enzymes to insert the foreign DNA. When a plasmid
vector containing foreign DNA is taken up by the host E. coli, the α-complementation
does not occur, therefore, a functional β-galactosidase enzyme is not produced. If the
foreign DNA is not inserted into the vector or if it is inserted at a location other than
MCS, the lacZ gene in the plasmid vector complements the lacZ deletion mutation in
the host E. coli producing a functional enzyme.

• For screening the clones containing recombinant DNA, a chromogenic substrate
known as X-gal is added to the agar plate. If β-galactosidase is produced, X-gal is
hydrolyzed to form 5-bromo-4-chloro-indoxyl, which spontaneously dimerizes to
produce an insoluble blue pigment called 5,5’-dibromo-4,4’-dichloro-indigo. The
colonies formed by non-recombinant cells, therefore appear blue in color while the
recombinant ones appear white. The desired recombinant colonies can be easily
picked and cultured.
• Isopropyl β-D-1-thiogalactopyranoside (IPTG) is used along with X-gal for blue-
white screening. IPTG is a non-metabolizable analog of galactose that induces the
expression of lacZ gene. It should be noted that IPTG is not a substrate for β-
galactosidase but only an inducer. For visual screening purposes, chromogenic
substrate like X-gal is required.

Advantages and Disadvantages of using Plasmids
• pBR322 was a breakthrough for molecular biology as the ﬁrst widely
used plasmid for molecular cloning, but the double screening procedure
required to identify recombinant clones was both time consuming and
error prone.
• The major cloning advantage of pUC plasmids over pBR322 is that
foreign DNA fragments can be cloned into a variety of restriction
enzyme sites and recombinants rapidly screened
• The efﬁciency at which the plasmid is transferred to a bacterial cell is
very low.
• The capacity of plasmids to carry large fragments of foreign DNA is
limited. Most plasmids become unstable if their overall size exceeds
about 15 kbp.

Bacteriophages
Bacteriophages, or phages as they are commonly known, are viruses
that speciﬁcally infect bacteria.
Like all viruses, phages are very simple in structure, consisting merely of a
DNA (or occasionally ribonucleic acid (RNA)) molecule carrying a number
of genes, including several genes for replication of the phage, and its
components protein coat and phage specific replicative enzymes. Alteration
in any of the gene can impair replicative ability of resultant molecule.

M13—a filamentous phage
The normal M13 genome is 6.4 kb in length, but most of this is taken up by ten
closely packed genes. Each essential for the replication of the phage.
There is only a single 507-nucleotide intergenic sequence into which new DNA
could be inserted without disrupting one of these genes, and this region includes
the replication origin which must itself remain intact. Clearly there is only limited
scope for modifying the M13 genome.

• The first step in construction of an M13 cloning vector was to
introduce the lacZ′ gene into the intergenic sequence. This gave
rise to M13mp1, which forms blue plaques on X-gal agar (Figure
a). M13mp1 does not possess any unique restriction sites in the
lacZ′ gene. It does, however, contain the hexanucleotide GGATTC
near the start of the gene. A single nucleotide change would make
this GAATTC, which is an EcoRI site. This alteration was carried
out using in vitro mutagenesis, resulting in M13mp2 (Figure b)

• The next step in the development of M13 vectors was to introduce additional
restriction sites into the lacZ′ gene. This was achieved by synthesizing in the
test tube a short oligonucleotide, called a polylinker, which consists of a
series of restriction sites and has EcoRI sticky ends (Figure a). This
polylinker was inserted into the EcoRI site of M13mp2, to give M13mp7
(Figure b), a more complex vector with four possible cloning sites (EcoRI,
BamHI, SalI, and PstI). The polylinker is designed so that it does not totally
disrupt the lacZ′ gene: a reading frame is maintained throughout the
polylinker, and a functional, though altered, b-galactosidase enzyme is still
produced.

Hybrid plasmid–M13 vectors
• Although M13 vectors are very useful for the production of single-stranded
versions of cloned genes, they do suffer from one disadvantage.
• There is a limit to the size of DNA fragment that can be cloned with an M13
vector, with 1500 bp generally being looked on as the maximum capacity,
though fragments up to 3 kb have occasionally been cloned.
• To get around this problem a number of hybrid vectors (“phagemids”) have
been developed by combining a part of the M13 genome with plasmid DNA.
• An example is provided by pEMBL8, which was made by transferring into pUC8
a 1300 bp fragment of the M13 genome. (Figure a)
• This piece of M13 DNA contains the signal sequence recognized by the enzymes
that convert the normal double-stranded M13 molecule into single-stranded DNA
before secretion of new phage particles.

• This signal sequence is still functional even though detached from the
rest of the M13 genome, so pEMBL8 molecules are also converted into
single-stranded DNA and secreted as defective phage particles (Figure
b).
• All that is necessary is that the E. coli cells used as hosts for a pEMBL8
cloning experiment are subsequently infected with normal M13 to act as
a helper phage, providing the necessary replicative enzymes and phage
coat proteins.
• pEMBL8, being derived from pUC8, has the polylinker cloning sites
within the lacZ′ gene, so recombinant plaques can be identified in the
standard way on agar containing X-gal.

Segments of the λ genome can be deleted without impairing viability
• The λ genetic map, showing the position of the main non-essential
region that can be deleted without affecting the ability of the phage to
follow the lytic infection cycle
• Removal of non-essential region decreases the size of the resulting λ
molecule by up to 15 kb. This means that as much as 18 kb of new
DNA can now be added.

Cosmid
Cosmids are hybrids between λ phage DNA molecule and a bacterial plasmid.
A cosmid is basically a plasmid that carries a cos site (Figure a). It also needs a
selectable marker, such as the ampicillin resistance gene, and a plasmid origin of
replication, as cosmids lack all the λ genes and so do not produce plaques. Instead
colonies are formed on selective media, just as with a plasmid vector.
Cosmids also possess a unique restriction enzyme recognition site into which
DNA fragments can be ligated. After the packaging reaction has occurred, the
newly formed λ particles are used to infect E. coli cells. The DNA is injected into
the bacterium like normal λ DNA and circularizes through complementation of the
cos ends.
Infected cells are plated onto a selective medium and antibiotic-resistant colonies
are grown.

• A bacterial artificial chromosome is an engineered large
DNA molecule used to clone DNA segment in a bacterial
cell.
• Based on well known natural F1 plasmid (allows
conjugation between two bacterial cells).
• MCS present for insertion of foreign DNA.
• 150-300 kb can be inserted.
• Copy no is 2 per cell.
• Used in analysis of large genome.
• First BACs vector is pBAC108L.
BACs

Advantages:
◦ Useful for cloning up to 200 kb, but can be handled like
regular bacterial plasmid vectors.
◦ Useful for sequencing large stretches of chromosomal DNA;
frequently used in genome sequencing projects.
◦ More stable
◦ Easier to work with than the YACs.
Disadvantage:
◦ Difficult to transform bacteria.

 Capable of carrying inserts of 200-2000 kbp in yeast
YACs Vector

Yeast artiﬁcial chromosome(YAC) vectors allow the
cloning, within yeast cells.
• A yeast centromere (CEN4): The yeast centromere is
specified by a 125 bp DNA segment.
• Yeast autonomously replicating sequence (ARS): Yeast
ARS elements are essentially origins of replication
• Yeast telomeres (TEL): Telomeres are the specific
sequences (5 - TGTGGGTGTGGTG-3) that are present
at the ends of chromosomes in multiple copies and are
necessary for replication and chromosome maintenance.

• YAC contains the telomere, centromere, and origin of
replication elements. If these elements are spliced into
DNA in the proper location and orientation, then a yeast
cell will replicate the artificial chromosome along with the
other, natural chromosomes. The target DNA is flanked by
the telomere regions that mark the ends of the
chromosome, and is interspersed with the centromere
region that is vital for replication. Finally, the start site for
the copying process is present. In essence, the yeast is
fooled into accepted genetic material that mimics a
chromosome.

Advantages:
• Very useful in mapping the human genome because
they could accommodate hundreds of thousands of
kilobases each.
• YACs containing a megabase or more are
known as “megaYACs.”
Disadvantages:
• They are inefficient (not many clones are obtained per
microgram of DNA)
• Hard to isolate from yeast cells unstable

 The TA cloning method takes advantage of the terminal
transferase activity of some DNA polymerases such as Taq
polymerase. This enzyme adds a single, 3'-A overhang to each
end of the PCR product. This makes it possible to clone this
PCR product directly into a linearized cloning vector with
single, 3'-T overhangs.

Ligase independent cloning (LIC) is a simple, fast and
relatively cheap method to produce expression constructs.
It makes use of the 3'--> 5'-activity of T4 DNA polymerase
to create very specific 10-15 base single overhangs in the
expression vector. PCR products with complementary
overhangs are created by building appropriate extensions
into the primers and treating them with T4 DNA
polymerase as well. The annealing of the insert and the
vector is performed in the absence of ligase by simple
mixing of the DNA fragments. This process is very
efficient because only the desired products can form.

Cloning vector

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