Cloning vector

A cloning vector is a DNA molecule in which foreign DNA can be inserted and which is further
capable of replicating within host cell to produce multiple copies of itself and the foreign DNA.
Vector containing foreign DNA is termed recombinant DNA.

Features of a Cloning Vector

All commonly used cloning vectors in molecular biology have key features necessary for their
function. For reason of ease and convenience, cloning is often performed using E. coli. These
features include -

i. Origin of replication

The replication origin (ORI) is a specific DNA sequence of 50 – 100 base pairs that permits a
cloning a vector to replicate within host cell.

ii. Cloning site

All cloning vectors have features that allow a gene to be conveniently inserted into the vector or
removed from it. This may be a multiple cloning site (MCS) or polylinker, which contains many
unique restriction sites.

iii. Selectable marker

A selectable marker is carried by the vector to allow the selection of host cells which bear
recombinant DNA (called transformants) from those which do not bear recombinant DNA
(non-transformants).

Types of Cloning Vectors

A large number of cloning vectors are available and choosing the vector may depend on a number
of factors, such as the size of the insert, copy number and cloning method.

Below is a table of several kinds of vectors commonly used for genomic libraries and the insert size
that each generally holds.

[1]
 Vector type Maximum Insert size
Plasmids 15 kb
Phagemids 15 kb
Phage lambda (λ) 25 kb
Cosmids 45 kb
P1 artificial chromosomes (PACs) 300 kb
Bacterial artificial chromosomes (BACs) 300 kb
Yeast artificial chromosomes (YACs) 2000 kb

Plasmid

Plasmids are autonomously replicating circular extra-chromosomal DNA. They are the standard
cloning vectors and the ones most commonly used. Most general plasmids may be used to clone
DNA insert of up to 15 kb in size. One of the earliest commonly used cloning vector is
the pBR322 plasmid. Other cloning vectors include the pUC series of plasmids, for example pUC19.

pBR322 is a plasmid and was one of the first widely used E. coli cloning vectors. The p stands for
"plasmid" and BR for "Bolivar" and "Rodriguez." “322” distinguishes this plasmid from others
developed in the same laboratory (there are also plasmids called pBR325, pBR327, pBR328, etc).

pBR322 is 4363bp in size and has two antibiotic resistance genes - the ampR gene and the tetR
gene. Either ampicillin or tetracycline resistance can be used as a selectable marker for cells
containing the plasmid. Each marker gene includes unique restriction sites that can be used in
cloning experiments. It has a reasonably high copy number (30-45 molecules per E. coli cell).

Figure: Schematic representation of the pBR322 vector

[2]
Phagemid

Phagemid combines components of phage such as M13 with parts of plasmids. 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. However, infection of a bacterial host
containing a phagemid with a 'helper' phage provides the necessary viral components to enable
single stranded DNA replication and packaging of the phagemid DNA into phage particles.

Bluescript is a phagemid vector with a multiple cloning site (MCS) that contains many unique
restriction enzyme cleavage sites, two distinct origins of replication and a good selectable marker -
a gene that makes the host bacterium resistant to ampicillin.

Figure: Schematic representation of pBluescript II

Cosmid

A cosmid is a type of hybrid plasmid that carries a cos site from the lambda phage. This sequence
allows the cosmid to be packaged into bacteriophage λ particles. These particles-containing a
linearized cosmid are introduced into the host cell by transduction. Once inside the host, the
cosmids circularize with the aid of the host's DNA ligase and then function as plasmids.

It also needs a selectable marker, such as the ampicillin resistance gene, and a plasmid origin of
replication, as cosmids lack all the lambda genes and so do not produce plaques. Cosmids are
capable of carrying inserts up to 45kb in size. pJB8 is typical cosmid.

[3]
 Figure: Schematic representation of pJB8

Compare between Cosmid, bacteriophages & plasmid as cloning vectors

Features Cosmid Bacteriophages Plasmid

Host Bacteria Bacteria Bacteria
Insert Size Up to 45 kb up to 25 kb up to 15 kb

Entry into cells Infection Infection Transformation

Efficiency Very efficient Very efficient Less efficient

Outcome Multiply Multiply and Kill Multiply

Appearance of infected cells Colonies Plaques Colonies

[4]