VECTOR BIOLOGY:

CLONING AND
EXPRESSION VECTORS
GROUP 3
 OUTLINE
• Introduction
• Definition of terms
• Essential features of cloning and expression vectors
• Types of cloning vectors
• Plasmids
• Virus-based vectors (λ phage, M13 phage)
• Hybrid vectors (Cosmids, phagemids)
• Artificial chromosomes
• Cloning vectors in animals
• Cloning vectors in higher plants
• Expression vectors
 Introduction
• Many recombinant DNA methods require numerous copies
of a specific DNA fragment.
• gene cloning provides a means of generating identical
copies (clones) of an original piece of DNA.
• It involves separating a specific gene or DNA segment from
a larger chromosome, attaching it to a small molecule of
carrier DNA (vector), and then replicating this modified
DNA thousands or millions of times through both an
increase in cell number and the creation of multiple copies
of the cloned DNA in each cell.
• The result is selective amplification of a particular gene or
DNA segment.
 Definition of terms
• A clone is a population of identical cells, generally those
containing identical recombinant DNA molecules.
• A vector (in molecular biology) is a DNA molecule capable of
replication in a host organism, which serves as a carrier for a
foreign DNA molecule
• A cloning vector is a stable, self-replicating DNA molecule to
which a foreign DNA fragment can be attached for
introduction into a cell in order to generate multiple identical
copies of the foreign DNA.
• An expression vector is a cloning vector designed so that a
foreign gene inserted into the vector will be expressed in the
host organism.
• Host – cell in which the recombinant DNA is introduced, for
which the vector has replication functions- bacteria, yeast,
 Essential features of Cloning vectors
• An effective cloning vector has three important characteristics:

• An origin of replication, which ensures that the vector is

replicated within the cell;

• Selectable markers, which enable any cells containing the

vector to be selected or identified.

• One or more unique restriction sites into which a DNA

fragment can be inserted.
 Major types of cloning vectors
• There are four major types of cloning vectors
applied for cloning in bacteria and yeast host
cells:
1. Plasmids
2. Virus-based vectors (Bacteriophages )
3. Hybrid vectors (Cosmids, phagemids)
4. Artificial chromosomes (Yeast and
Bacterial artificial chromosomes).
 PLASMIDS
• extra-chromosomal elements of DNA, which are
relatively small, covalently closed circular molecules
carrying genes for antibiotic resistance, conjugation,
virulence or the metabolism of “unusual” substrates.
• Plasmids lead an independent existence in the
bacterial cell.
• pBR322 one of the most notable plasmids named
after its developers Bolivar and Rodriguez in the
early 1970s.
 Features
• At least one DNA sequence that can act as an origin of replication.
• Possess at least one unique restriction site.
• antibiotic resistance is often used as a selectable marker to ensure
that bacteria in a culture contain plasmid.
• Range in size from about 1.0kb for the smallest to over 250kb for
the largest plasmids
• May exhibit stringent or relaxed regulation of replication
• Insert capacity: approx 10-20kb.
• A few types of plasmid are also able to replicate by inserting
themselves into the bacterial chromosome. These integrative
plasmids - episomes
 Classification of plasmids
• Fertility or ‘F’ plasmids: carry genes (tra genes) that possess the ability to
promote sexual conjugation between bacterial cells.

• Resistance or ‘R’ plasmids: carry genes conferring on the host bacterium

resistance to one or more antibacterial agents

• Col plasmids: these contains genes that code for colicins i. e. proteins that kill
other bacteria. An example is ColE1 of Escherichia coli.

• Degradative plasmids: these allow the host bacterium to metabolize unusual

molecules e.g. toluene and salicylic acid.

• Virulence plasmids: these confer pathogenicity on the host bacterium.

 
Cloning with pBR322
 pUC plasmids
• Allows for identification of the foreign DNA containing cells
in a single screening step.
• Have the advantage of a High copy number, A multiple
cloning site or polylinker.
• pUC8, for example, is a Lac-selection plasmid. This plasmid
carries the ampicillin resistance gene and a gene called lac
Z’, which codes for part of the enzyme beta-galactosidase.
• Cloning with pUC8 involves insertional inactivation of the
lac Z’ gene, with recombinants identified because of their
inability to synthesize the amino terminal portion of β-
galactosidase called the α peptide.
 Cloning with pUC8
• Cells that harbor a normal pUC8 plasmid are ampicillin resistant
and able to synthesize beta-galactosidase; recombinants are also
ampicillin resistant but are unable to make beta-galactosidase.
• Screening for beta-galactosidase presence or absence is done
using a lactose analogue called X-gal (5- bromo-4-chloro-3 indoyl-
β-D-galactopyranoside) which is broken down by beta-
galactosidase to a product that is colored deep blue.
• If X-gal is added to the agar, along with ampicillin, then non-
recombinant colonies, the cells of which synthesize beta-
galactosidase will be colored blue, whereas recombinants with a
disrupted lac Z ’gene and unable to synthesize beta-galactosidase
will be white.
 Bacteriophage λ
• a typical example of a head-and-tail phage
• λ DNA molecule is 49 kb in size.
• λ is a linear, double-stranded DNA phage and consists of two
self-complementary 12bp single-stranded tail at both ends,
called cohesive termini (COS SITES).
• Being complementary, they can base-pair with one another
to form a circular, completely double-stranded molecule, a
necessary prerequisite for insertion into the bacterial
genome.
• It selectively infects bacteria and replicates by a lytic or non-
lytic (lysogenic) pathway.
• Two basic types of λ vectors:
• 1. insertional vectors
• 2. replacement vectors
 M13 phage vectors
• M13 is an example of a filamentous phage. It is also a male-specific
lysogenic phage
• Its DNA molecule is much smaller than the λ genome ((about 6407
nucleotides in length).
• It is circular and is unusual in that it consists entirely of single-stranded
DNA.
• Because M13 switches between a single-stranded form and a double-
stranded replicative form it is of utmost importance in in vitro
mutagenesis experiments.
• normal M13 genome is 6.4kb 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 inter-genic sequence into which
new DNA could be inserted.
• Inserts larger than a few fragments tend to make the vector unstable.
 Cosmid vectors
• hybrids between a phage DNA molecule and a
bacterial plasmid.
• two cos sites from λ phage that are separated
by 37–51 kbp of intervening sequence.
• Cosmids can be isolated as dsDNA for in vitro
manipulation.
 Phagemids
• Phagemids are plasmids that contain the f1
phage origin of replication for the production
of single stranded DNA.
• They are small plasmids and have the ability to
accept larger inserts than M13-based vectors
• Phagemids can only produce ssDNA in the
presence of a wild-type M13 or f1 helper
phage.
• In the absence of a helper phage, dsDNA can
be isolated as a normal plasmid
phagemids
Artificial Chromosoomes
 ARTIFICIAL CHROMOSOMES
• Bacterial artificial chromosomes (BACs),
• mammalian artificial chromosomes(MACs)
• viral PI artificial chromosomes (PACs)
• Yeast artificial chromosomes (YACs).
 EXPRESSION VECTORS
• Especially useful in the production of large amounts
of a low-abundance protein e.g. many protein
hormones and other signaling or regulatory proteins
are normally expressed at very low concentrations,
precluding their isolation and purification in large
quantities by standard biochemical techniques.

• insulin, growth hormone, factor VIII (a blood clotting

factor), granulocyte colony-stimulating factor (G-CSF)
and other human proteins with therapeutic uses.
• Expression vectors, in bacteria, in addition to the usual origin of
replication, restriction sites, and selectable markers, contains
sequences required for transcription and translation in bacterial
cells. These additional sequences may include:
1. A bacterial promoter, such as the lac promoter.

2. A DNA sequence that, when transcribed into RNA, produces a

prokaryotic ribosome binding site.
3. Prokaryotic transcription initiation and termination sequences.
 4. Sequences that control transcription initiation, such as regulator
genes and operators
The general layout of expression vectors