Genetic Engineering

The use of experimental techniques to produce DNA

molecules containing new genes or new combination
of genes is called Genetic Engineering.
Gene cloning
1 A fragment of DNA, containing the gene to be cloned, is inserted into a
circular DNA molecule called a vector, to produce a recombinant DNA
molecule.

2 The vector transports the gene into a host cell, which is usually a
bacterium, although other types of living cell can be used.

3 Within the host cell the vector multiplies, producing numerous identical
copies, not only of itself but also of the gene that it carries.

4 When the host cell divides, copies of the recombinant DNA molecule are
passed to the progeny and further vector replication takes place.

5 After a large number of cell divisions, a colony, or clone, of identical host

cells is produced. Each cell in the clone contains one or more copies of the
recombinant DNA molecule; the gene carried by the recombinant molecule is
now said to be cloned.
PCR is carried out in a single test tube
simply by mixing DNA with a set of
reagents and placing the tube in a
thermal cycler, a piece of equipment that
enables the mixture to be incubated at a
series of temperatures that are varied in
a preprogrammed manner.
1 The mixture is heated to 94°C, at which temperature the hydrogen bonds that hold
together the two strands of the double-stranded DNA molecule are broken, causing the
molecule to denature.
2 The mixture is cooled down to 50–60°C. The two strands of each molecule could join back
together at this temperature, but most do not because the mixture contains a large excess
of short DNA molecules, called oligonucleotides or primers, which anneal to the DNA
molecules at specific positions.
3 The temperature is raised to 74°C. This is a good working temperature for the Taq DNA
polymerase that is present in the mixture. All we need to understand at this stage is that the
Taq DNA polymerase attaches to one end of each primer and synthesizes new strands of
DNA, complementary to the template DNA molecules, during this step of the PCR. Now we
have four stands of DNA instead of the two that there were to start with.

The temperature is increased back to 94°C. The double-stranded DNA molecules, each of
which consists of one strand of the original molecule and one new strand of DNA, denature
into single strands. This begins a second cycle of denaturation–annealing–synthesis, at the
end of which there are eight DNA strands. By repeating the cycle 30 times the
double-stranded molecule that we began with is converted into over 130 million new
double-stranded molecules, each one a copy of the region of the starting molecule
delineated by the annealing sites of the two primers.
Why gene cloning and PCR is so important

The answer is largely because both techniques can provide a pure

sample of an individual gene, separated from all the other genes in the
cell.
Vectors for Gene Cloning: Plasmids and Bacteriophages
• Vectors transport the gene into host cell and is responsible for its replication.

Features to be able to act as a vector for gene cloning:

• Most importantly it must be able to replicate within the host cell, so that
numerous copies of the recombinant DNA molecule can be produced and passed
to the daughter cells.
• A cloning vector also needs to be relatively small, ideally less than 10 kb in size,
as large molecules tend to break down during purification, and are also more
difficult to manipulate.

• Two kinds of DNA molecule that satisfy these criteria can be found in bacterial
cells.
A) plasmids
B) bacteriophage chromosomes
The term plasmid was introduced by
Lederberg in 1952.

From genetic point of view, a plasmid is a replicon (unit of genetic material

capable of independent replication) that is stably inherited (maintained
without specific selection) in an extrachromosomal state.

From molecular point of view, plasmids are circular, ds deoxyribonucleic

acid molecule that replicate autonomously in a host cell.
 Characteristics of plasmids

• Extra-chromosomal DNA
• Plasmids are circular ds molecules of DNA that lead an independent
existence in the bacterial cell. Normally they are supercoiled in form which
makes them more stable during chemical isolation.
• Heritable (capable of passing into progeny cells).
• Most plasmids possess at least one DNA sequence that can act as an origin
of replication, so they are able to multiply within the cell independently of
the main bacterial chromosome.
• Small in size, which makes them easy to isolate and manipulate. Less than
10 kb size is desirable for a cloning vector. Plasmids range from about 1.0
kb for the smallest to over 250 kb for the largest plasmids, so only a few
are useful for cloning purposes.
• larger plasmids can be adapted for cloning under some
circumstances.
Multiple copy number:
• The copy number refers to the number of molecules of an individual plasmid that are
normally found in a single bacterial cell.
• The factors that control copy number are not well understood.
• Some plasmids, especially the larger ones, are stringent and have a low copy number
(just one to three per cell). Their replication is coupled to that of the host.
• Others, called relaxed plasmids, are present in multiple copies of 50 or more per cell.
They can carry out multiplication independent of cell division. Copy number can be
further increased to several thousands per cell if host protein synthesis is stopped (for
example, by treatment with chloramphenicol)
• Generally speaking, a useful cloning vector needs to be present in the cell in multiple
copies so that large quantities of the recombinant DNA molecule can be obtained.
Conjugation
Plasmids fall into two groups:
• conjugative
• non-conjugative

Conjugative plasmids are characterized by the ability to

promote sexual conjugation between bacterial cells a
process that can result in a conjugative plasmid spreading
from one cell to all the other cells in a bacterial culture.

Conjugation and plasmid transfer are controlled by a set of

transfer or tra genes, which are present on conjugative
plasmids but absent from the non-conjugative type.
 • Plasmids almost always carry one or more genes,
which encodes as drug resistant marker.
• For example, the ability to survive in normally toxic
concentrations of antibiotics such as
chloramphenicol or ampicillin is often due to the
presence in the bacterium of a plasmid carrying
antibiotic resistance genes.

• In the laboratory, antibiotic resistance is often used as a selectable marker to

ensure that bacteria in a culture contain a particular plasmid.
• Almost all plasmid contain a closely arranged series of synthetic cloning site
termed as polycloning site that consists of sequence recognized by restriction
endonuclease.
• Sometimes the ancillary sequence into the plasmid vector makes it useful for
variety of cloning purposes. For example expression of large amount of foreign
proteins.
• The smaller plasmids make use of the
host cell’s own DNA replicative enzymes
in order to make copies of themselves
(Non integrative).
• Some of the larger ones carry genes that
code for special enzymes that are
specific for plasmid replication.
• A few types of plasmid are also able to
replicate by inserting themselves into
the bacterial chromosome. These
integrative plasmids or episomes may be
stably maintained in this form through
numerous cell divisions, but always at
some stage exist as independent
elements.
 Compatibility
• Several different kinds of plasmid may be found in a single cell, including more
than one different conjugative plasmid at any one time.

• In fact, cells of E. coli have been known to contain up to seven different

plasmids at once.

• To be able to coexist in the same cell, different plasmids must be compatible.

• If two plasmids are incompatible, then one or the other will be rapidly lost from
the cell.

• Different types of plasmid can therefore be assigned to different incompatibility

groups on the basis of whether or not they can coexist, and plasmids from a
single incompatibility group are often related to each other in various ways.

• The basis of incompatibility is not well understood, but events during plasmid
replication are thought to underlie the phenomenon.
Structural configuration of plasmids
• Plasmids may exist in several structural configuration.
• A linear double stranded form
• An open circle form (OC) when one of the two DNA strands is broken
down
• Covalently closed circular (CCC) form when both strands of plasmid
DNA are covalently bonded.
• Supercoiled (SC) form when the DNA helix is twisted around itself. This
is the found when plasmid is isolated extracellularly.
 Bacteriophage

• 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 for replication of
the phage, surrounded by a
protective coat or capsid made up
of protein molecules.
 The phage infection cycle:
1 The phage particle attaches to the
outside of the bacterium and injects its
DNA chromosome into the cell.
2 The phage DNA molecule is replicated,
usually by specific phage enzymes
coded by genes in the phage
chromosome.
3 Other phage genes direct synthesis of
the protein components of the capsid,
and new phage particles are assembled
and released from the bacterium.
With some phage types the entire infection cycle is completed very quickly, possibly in
less than 20 minutes.
This type of rapid infection is called a lytic cycle, as release of the new phage particles is
associated with lysis of the bacterial cell.
The characteristic feature of a lytic infection cycle is that phage DNA replication is
immediately followed by synthesis of capsid proteins, and the phage DNA molecule is
never maintained in a stable condition in the host cell.
 Lysogenic phages
• lysogenic infection is characterized by retention of
the phage DNA molecule in the host bacterium,
possibly for many thousands of cell divisions.
• With many lysogenic phages the phage DNA is
inserted into the bacterial genome, in a manner
similar to episomal insertion.
• The integrated form of the phage DNA (called the
prophage) is quiescent, and a bacterium (referred
to as a lysogen) that carries a prophage is usually
physiologically indistinguishable from an
uninfected cell.
• However, the prophage is eventually released from
the host genome and the phage reverts to the lytic
mode and lyses the cell.
A limited number of lysogenic phages follow a
rather different infection cycle.
When M13 or a related phage infects E. coli, new
phage particles are continuously assembled and
released from the cell.
The M13 DNA is not integrated into the bacterial
genome and does not become quiescent.
With these phages, cell lysis never occurs, and
the infected bacterium can continue to grow and
divide, albeit at a slower rate than uninfected
cells.
 Prominent Fields of Genetic Engineering
1. Medical Biotechnology (Gene therapy, biopharmaceuticals, personalized medicine, etc.)
2. Agriculture Biotechnology (GMO, Animal biotechnology, Sustainable agriculture, etc.)
3. Industrial Biotechnology (Biofuel production, bioremediation, bioproducts, etc.)
4. Environmental Biotechnology (Conservation biotechnology, synthetic biology, etc.)
5. Research and Development (Functional genomics, proteomics, metabolomics, etc.)
6. Forensic Biotechnology (DNA profiling)
7. Synthetic Biology (Genetic circuits, metabolic engineering, etc.)
8. Veterinary Biotechnology (Animal health, genetic improvement, etc.)
9. Plant Biotechnology (Plant health, genetic improvement, etc.)
10. Food Biotechnology (Nutraceuticals, food safety, etc.)
11. Space Biotechnology (Astrobiology, terraforming, etc.)