Cloning Vectors

•A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher
organism, that can be stably maintained in an organism and into which a foreign DNA fragment
can be inserted where it can be replicated/ or expressed. Eg. plasmids, bacteriophages,
bacterial artificial chromosomes.

•An expression vector, otherwise known as an expression construct, 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, the protein that is encoded by the gene is produced by the cellular-transcription and
translation machinery ribosomal complexes. Examples of mammalian expression
vectors include the adenoviral vectors, the pSV and the pCMV series of plasmid vectors,
vaccinia and retroviral vectors, as well as baculovirus.

•A shuttle vector is a vector that can propagate in two different host species, hence, inserted
DNA can be tested or manipulated in two different cell types. The main advantage of these
vectors is that they can be manipulated in E. coli and then used in a system which is more
difficult or slower to use. Shuttle vectors can be used in both eukaryotes and prokaryotes.
Shuttle vectors are frequently used to quickly make multiple copies of the gene in E. coli
(amplification). They can also be used for in vitro experiments and modifications such as
mutagenesis and PCR. One of the most common types of shuttle vectors is the yeast shuttle
vector that contains components allowing for the replication and selection in both E. coli cells and
yeast cells.
• Most vectors are genetically engineered.
• The cloning vector is chosen according to
the size and type of DNA to be cloned.
• Scientists (Herbert Boyer, Keiichi Itakura
and Arthur Riggs) working in Boyer’s lab
(University of California) recognized a
general cloning vector with unique
restriction sites for cloning in foreign DNA
and the expression of antibiotic resistance
genes for selection of transformed
bacteria.
• In 1977, they described the first vector
designed for cloning purposes, pBR322 –
a plasmid.
• This vector was small, ~4 kb in size, and
had two antibiotic resistance genes for
selection.
Distinct Features of Cloning Vectors
1. Origin of Replication – The specific sequence of nucleotide in a DNA,
which acts, as the origin of the replication process is known as ORI.
- This makes autonomous replication in vector.
- ORI is a specific sequence of nucleotide from where replication starts.
- When foreign DNA is linked to the sequence along with vector replication,
foreign (desirable) DNA also starts replicating within host cell.

2. Cloning Site: Cloning site is a place where the vector DNA can be
digested and desired DNA can be inserted by the same restriction enzyme.
– It is a point of entry or analysis for genetic engineering work.
– Recently recombinant plasmids contain a multiple cloning site (MCS)
which have many (up to ~20) restriction sites. It must be not too big in
size.

3. Selectable Marker – Selectable marker is a gene that confers

resistance to particular antibiotics or selective agent that would normally
kill the host cell or prevent its growth.
– A cloning vector contains a selectable marker, which confer on the host
cell an ability to survive and proliferate in a selective growth medium
containing the particular antibiotics.
4. Reporter Gene or Marker Gene: Reporter genes are used
in cloning vectors to facilitate the screening of successful clones
by using features of these genes that allow successful clone to
be easily identified.
– Such feature present in cloning vectors is used in blue-white
selection.
- Blue colonies represent ampicillin resistance bacteria that
contain pVector and express functional alpha fragment from
and intact LacZ alpha coding sequence. White colonies
represent Ampicillin resistance bacteria that contain pInsert
and do not produce LacZ alpha fragment. Because the Pinsert
lins within the LacZ portion.
5. It should be short, small.
6. Compatible with host cell.
7. Incompatible with other vector.
8. Should become high in copy number.
9. It should able to express itself utilizing the host machinery.
10. It should be able to move under two system (Prokaryote
and Eukaryote system)
 Plasmid

• Plasmid is an autonomously replicating circular double stranded extrachromosomal DNA which is

physically separated from a chromosomal DNA and can replicate independently.
• They are most commonly found in bacteria, sometimes they are present in archaea and eukaryotic
organisms.
• The size of the plasmid varies from 1 to over 200 kb.
• Most general plasmids may be used to clone DNA insert of up to 10 kb in size.
• Many plasmids have high copy number and is useful as it produces greater yield of recombinant
plasmid for subsequent manipulation.
• However low copy number plasmids may be preferably used in certain circumstances, for example,
when the protein from the cloned gene is toxic to the cells.
• Example: pBR322, pUC18, F plasmid, Col Plasmid etc.

The nomenclature of the plasmid. Eg. pBR322 is as stated

below: P – Plasmid, BR - Stands for Bolivar and Rodriguez who
constructed this plasmid, 322 - Number given to distinguish
this plasmid from the other plasmids developed in the same
laboratory.
Eg. pBR322 Plasmid Vector

•pBR322 DNA is a commonly used plasmid cloning vector in E. coli .

•The molecule is a double-stranded circle 4,361* base pairs in length .
•pBR322 contains the genes for resistance to ampicillin and
tetracycline, and can be amplified with chloramphenicol.
•The molecular weight is 2.83 x 106 daltons.
•The circular sequence is numbered such that 0 is the middle of the unique
EcoRI site and the count increases through the TetR gene.
•The AmpR gene is penicillin beta-lactamase.
•Promoters P1 and P3 are for the beta-lactamase gene.
• P3 is the natural promoter, and P1 is artificially created by the ligation of
two different DNA fragments to create pBR322.
• It contains the origin of replication of pMB1, and the rop gene, which
encodes a restrictor of plasmid copy number.
•The plasmid has unique restriction sites for more than forty restriction
enzymes.
•There are two sites for restriction enzymes HindIII and ClaI within
the promoter of the TetR gene.
•There are six key restriction sites inside the AmpR gene.

SEE pSU101; pUC18/19, PGEM3Z

Why Plasmids are Good Cloning Vectors:
• Small size (easy to manipulate and isolate).
• Circular (more stable).
• Replication independent of host cell.
• Several copies may be present (facilitates replication).
• Frequently have antibiotic resistance (detection easy).

Disadvantages Using Plasmids:

• Cannot accept large fragments.
• Sizes range from 0 – 10kb.
• Standard methods of transformation are inefficient.
 Bacteriophage (Phage Lambda)
• The bacteriophages used for cloning are the phage λ and M13 phage.
• There is an upper limit on the amount of DNA that can be packed into a
phage (a maximum of 53 kb)..
• There is also a lower size limit for DNA that can be packed into a
phage, and vector DNA that is too small cannot be properly packaged
into the phage.
• Phage lambda is a bacteriophage or phage, i.e. bacterial virus, that
uses E. coli as host.
• Its structure is that of a typical phage: head, tail, tail fibres.
• Lambda viral genome: 48.5 kb DNA with a 12 base ssDNA "sticky end"
at both ends; these ends are complementary in sequence and can
hybridize to each other (this is the cos site: cohesive ends).
• Infection: lambda tail fibres adsorb to a cell surface receptor, the tail
contracts, and the DNA is injected.
• The DNA circularizes and lambda begins its life cycle in the E. coli host.
• There are two kinds of λ phage vectors
• - insertion vector and replacement vector.
• – Insertion vectors contain a unique cleavage site whereby foreign DNA
with size of 5–11 kb may be inserted.
• – In replacement vectors, the cleavage sites flank a region containing
genes not essential for the lytic cycle may be deleted and replaced by
the DNA insert in the cloning process.
 Phage Vectors Present Two Advantages Over Plasmid Vectors:

1. They are more efficient than plasmids for cloning of large DNA fragments; the largest
cloned insert in lambda phage is 24 kb, while for plasmid vector it is less than 10 kb.
2. It is easier to screen a large number of phage plaques than bacterial colonies for
identification of recombinant vectors.

Cosmid
• Cosmids are plasmids that incorporate a segment of
bacteriophage λ DNA that has the cohesive end site (cos)
which contains elements required for packaging DNA into
λ particles. • It is normally used to clone large DNA
fragments between 25 and 45 Kb. • They can replicate
as plasmids if they have a suitable origin of replication. •
They can also be packaged in phage capsids, which
allows the foreign genes to be transferred into cells by
transduction.
• Advantages : • High transformation efficiency. • The
cosmid vector can carry up to 45 kb whereas plasmid
and Lambda phage vectors are limited to 25 kb.