Contents:

1. Project Report on Introduction to Recombinant DNA Technology

2. Project Report on the Purpose of Recombinant DNA Technology
3. Project Report on the Basic Steps of Recombinant DNA Technology
4. Project Report on Enzymes Involved in Recombinant DNA Technology
5. Project Report on Vectors Used in Recombinant DNA Technology

Project Report

1. Introduction to Recombinant DNA Technology:

Genetic engineering involves manipulation of the genetic material
towards a desired and in a directed and predetermined way. This
method aims at isolating DNA fragments and recombining them; i.e.,
two DNA molecules are isolated and cut into fragments by one or more
specialized enzymes and then the fragments are joined together in a
desired combination and restored to a cell for replication and
reproduction.

When the recipient organism is a microbe, then as the microbe

multiplies, it is possible to obtain millions of copies of a specific region
of DNA by allowing the cell to multiply.

Project Report # 2. Purpose of Recombinant DNA Techno-

logy:
The interest in genetic engineering principally is due to its
varied applications (Fig. 18.1):
 i. Production of varieties of plants having particular desirable
characteristics (e.g., resistance or tolerance to disease,
drought, development of CMS line, etc.).

ii. Improvement in the production of biochemicals and commercially

important organic chemicals.

iii. Correction of genetic disorder in higher organisms.

iv. The sequencing of gene, the prerequisite for mapping the genome
as well as utilizing the gene for horizontal transfer involved in raising
transgenic organism.
Recombinant DNA technology involves several steps in
specific sequence such as:
(1) Isolation of genetic material

(2) (2) Cutting of DNA at specific locations

(3) (3) Recombinant DNA formation

(4) Cloning of DNA

1. Isolation of Genetic Material (DNA):

In majority of organisms deoxynobonucleic acid or DNA is the
genetic material. It is present in chromosomes within the cell.
To isolate DNA, the cell at first is to be broken open by
treating cell with enzyme (lysozyme for bacteria, cellulose for
plant cell. Chitinase for fungus) so that DNA with other
macromolecules are released. To get pure DNA the other
necessary macro molecules such as RNA, protein,
polysaccharides are removed by treating with appropriate
enzymes. This is often called foreign DNA. It is then
incorporated into bacterial plasmid.
2. Cutting of DNA:
To cut DNA at specific location restriction endonuclease enzyme is
used. These enzymes are called as molecular scissor or molecular
scalpel and found in bacteria. These enzymes can cut the, DNA at
any known point. The enzymes can cut DNA of the plasmid as well
as foreign DNA at specific sites. These sites or points are mostly 8
palindromic i.e., the sequences which read the same both backward
and forward e.g., ‘Madam’.

3. Formation of Recombinant DNA:

As stated earlier a desired piece of DNA or a gene is first isolated.
This is generally called as foreign DNA. It is then incorporated into
bacterial plasmid (plasmids are rings of DNA other than main ring
shaped DNA of a bacterium which can replicate independent of
main DNA). For this DNA of the plasmid is cut open by
endonuclease enzyme leaving the sticky ends. The foreign DNA is
also cut out (by the same restriction endonuclease) and allowed to
 join the sticky ends of plasmid DNA. Such a plasmid DNA is now
known as recombinant DNA.

4. Cloning of DNA:
The recombinant plasmid DNA obtained above is allowed to
multiply to form a clone of recombinant DNA. To achieve this
recombinant plasmid DNA is introduced into a rapidly dividing
bacterium. Each time the bacterium divides and replicates its DNA,
it also copies the introduced recombinant plasmid and also the
foreign DNA. This method of introducing plasmid DNA into a
bacterium (usually E. coli.) is known as transformation. In this
process bacterial cell takes up pieces of naked DNA from the
surrounding medium.

These bacteria with recombinant plasmid DNA are grown in

nutrient medium where these double in number in every 20-30
minutes, producing millions of cells. In this way millions of copies of
recombinant plasmid DNA are formed.

To recover the foreign DNA from recombinant plasmid DNA, the

bacterial cells are broken. The foreign DNA is cut out of the
recombinant plasmid DNA by appropriate restriction enzyme and
separated by gel electro-phoresis.

Important Steps in Recombinant DNA technology:

i. Isolation of the gene (target DNA) to be cloned.

ii. Insertion of the gene into another piece of DNA called a vector
which allows it to be taken up by the recipient cell and replicated.

iii. Transfer of the recombinant vectors into cells of recipient

organisms, either by transformation or by infection using viruses.

iv. Selection of those cells which contain the desired recombinant

vectors.
v. Growth of the transformed organism.

vi. Expression of the gene to obtain the desired product.

Project Report # 4. Enzymes Involved in Recombinant DNA
Technology:
Genetic engineering became possible with the discovery of mainly two
types of enzymes: the cutting enzymes called restriction endonucleases
and the joining enzymes called ligases.

Restriction endonucleases or restriction enzymes, as they are called

popularly, recognize unique base sequence motifs in a DNA strand and
cleave the backbone of the molecule at a place within or, at some
distance from the recognition site. Whereas ligase is the enzyme that
joins a 5′ end of a DNA with a 3′ end of the same or of another strand.

(i) Restriction Endonuclease:

Ordinary nucleases are endonucleases or exonucleases. The former
cleaves the DNA backbone between two nucleotides, i.e., it cleaves the
double stranded DNA at any point except the ends, but it involves only
one strand of the duplex. The latter remove or digest one nucleotide at
a time starting from 5′ or 3′ end of a DNA strand.

The restriction endonucleases cleave only at specific regions in a

particular DNA, so that discrete and defined fragments are obtained at
the end of total digestion. The name ‘restriction’ endonuclease
originated from an observation of a system of restriction of the growth
of the phage lambda in particular strains of the E. coli host cell.
Most restriction enzymes recognize only one short base sequence in a
DNA molecule and make two single strand breaks, one in each strand,
generating 3′-OH and 5′-P groups at each position. The sequences
recognized by restriction enzymes are often palindromes, i.e., inverted
repetition sequences which are symmetrical.

Restriction enzymes can cut DNA in two ways to generate blunt ends
(cut precisely at opposite sites, e.g., Hpal) and staggered ends (cut at
asymmetrical position, e.g., Eco R!) with short single stranded
overhangs at each end. A large number of restriction enzymes have
been identified and classified into three categories (type 1, 11, III) on
the basis of their site of cleavage.
Restriction enzymes have three important features:
a. Restriction enzymes make breaks in palindromic sequences.

b. The breaks are usually not directly opposite to one another.

c. The enzymes generate DNA fragments with complementary ends.

The commonly employed restriction enzymes are listed in Table 18.1.

(ii) DNA Ligase:

Ends of DNA strands may be joined by the enzyme polynucleotide
ligase, called ‘glue’ of the recombinant DNA molecule. The enzyme
catalyses the formation of a phosphodiester bond between the 3′-OH
and 5′-P terminals of two nucleotides.
The enzyme is thus able to join unrelated DNA, repair nicks in single
strand of DNA and join the sugar phosphate backbones of the newly
repaired and resident region of a DNA strand.
The enzyme which is extensively used for covalently joining restriction
fragments is the ligase from E. coli and that encoded by T4 phage. As
the’ main source of DNA ligase is T4 phage, hence, the enzyme is
known as T4 DNA ligase.

The ligation reaction is controlled by several factors, such as pH,

temperature, concentration and kinds of sticky ends, etc. As ligase
uses the, ends of DNA molecules as substrates rather than the entire
DNA, the kinetics of joining depend on the number of ends
(concentration) available for joining.

Cloning Vector:
By cloning, one can produce unlimited amounts of any particular
fragment of DNA. In principle, the DNA isolated and cut pieces are
introduced into a suitable host cell, usually a bacterium such as
Escherichia coli, where it is replicated, as the cell grows and divides.

However, replication will only occur if the DNA contains a sequence

which is recognized by the cell as an origin of replication. Since such
sequences are infrequent, this will rarely be so, and therefore, the DNA
to be cloned, has to be attached to a carrier, or vector DNA which does
contain an origin of replication.

Criteria of an Ideal Vector:

Vectors are those DNA molecules that can carry a foreign DNA
fragment when inserted into it. A vector must possess certain
minimum qualifications to be an efficient agent for the transfer,
maintenance and amplification of the passenger DNA.

1. The vector should be small and easy to isolate.

2. They must have one or more origins of replication so that they will
stably maintain themselves within host cell.

3. Vector should have one or more unique restriction sites into which
the recombinant DNA can be inserted.
4. They should have a selectable marker (antibiotic resistance gene)
which allows recognition of transformants.

5. Vector DNA can be introduced into a cell.

6. The vector should not be toxic to host cell.

Types of Vector:
Based on the nature and sources, the vectors are grouped into
bacterial plasmids, bacteriophages, cosmids and phagemids (Fig.
22.3).
(a) Plasmid:
(b) Plasmids are the extra-chromosomal, self-replicating, and
double stranded closed and circular DNA molecules present in
the bacterial cell. A number of properties are specified by
plasmids such as antibiotic and heavy metal resistance, nitro-
gen fixation, pollutant degradation, bacteriocin and toxin
production, colicin factors, etc.
(c) Plasmids have following advantages as cloning
vehicle (Cohen et a. 1973):
(d) 1. It can be readily isolated from the cells.

(e) 2. It possesses a single restriction site for one or more

restriction enzymes.

(f) 3. Insertion of foreign DNA does not alter the replication

properties.

(g) 4. It can be reintroduced into cell.

(h) 5. Selective marker is present.

(i) 6. Transformants can be selected easily by using selective

medium

(j) (b) Bacteriophage:

(k) The bacteriophage has linear DNA molecule, a single break
will generate two fragments, foreign DNA can be inserted to
generate chimeric phage particle. But as the capacity of phage
head is limited, some segments of phage DNA, not having
essential genes, may be removed. This technique has been
followed in λ (Lambda) phage vectors to clone large foreign
particle.

(l) Plasmid can clone up to 20 to 25 kb long fragments of

eukaryotic genome. The examples of different Lambda phage
vectors are λ gt 10, λ gt 11, EMBL 3, etc. M-13 is a filamentous
bacteriophage of E. coli whose single stranded circular DNA
has been modified variously to give rise M-13 series of cloning
vectors.

(m) (c) Cosmid:

(n) Cosmids are plasmid particles, into which certain specific
DNA sequences, namely those for cos sites are inserted which
enable the DNA to get packed in X particle. Like plasmids, the
 cosmids perpetuate in bacteria without lytic development. The
cosmids have high efficiency to produce a complete genomic
library

Gebiticaly ;;;;;;;;

Genetically Modified Organisms in Agriculture

Genetically modified crops are the crops whose genes are modified using genetic engineering
techniques. These are also called transgenic crops. The main goal of using GMO’s is to increase the
yield of the crop and to produce disease-resistant crops.
An example of the genetically modified crop is Golden rice. It was genetically modified to generate
beta-carotene twenty times more than the previous varieties of rice. Golden rice is intended for Asia.
One more rice variety was also created to fight iron deficiency. A gene from the bean plant is taken
and inserted into the rice gene and the resultant rice helps to fight iron deficiency.

Genetically modified organisms in Medicine

GMO’s in medicine has been nothing short of a revolutionary idea. This helps to cure some disease
like diabetes, hypertension, and also helps in rectifying certain genetic disorders. Scientists are
conducting research on it since the 1980’s. Insulin is one of the outcomes of such research. Insulin
is generated by injecting a gene from the pig intestine into a bacterium. The bacterium with pig gene
grows and produces insulin. Many other products like Aspartame, Remicade, Avastin etc. are
the result of GMO application in medicine. Certain genetically engineered plants even produce
edible vaccines which are used to prevent diseases.

Role of GMO’s the management of the Environment

GMO’s offer many potential benefits related to environmental management. It helps in reducing
ecological damage. For instance, certain genetically engineered bacteria could produce
biodegradable plastics. A bacterium called Ralstonia eutropha convert glucose and some other acids
into a flexible polymer. This polymer is used in the making of plastic.

4.3. Environment
Genetic engineering has wide applications in solving the environmental issues. The release of
genetically engineered microbes, for example, Pseudomonas fluorescens strain designated
HK44, for bioremediation purposes in the field was first practiced by University of Tennessee
and Oak Ridge National Laboratory by working in collaboration [99, 100]. The engineered strain
contained naphthalene catabolic plasmid pUTK21 [101] and a transposon-based
bioluminescence-producing lux gene fused within a promoter that resulted in improved
naphthalene degradation and a coincident bioluminescent response [102]. HK44 serves as a
reporter for naphthalene bioavailability and biodegradation whereas its bioluminescence
signaling ability makes it able to be used as an online tool for in situ monitoring of
bioremediation processes [102]. The production of bioluminescent signal is detectable using
fiber optics and photon counting modules [101].
4.3.1. Phytoremediation and Plant Resistance Development
Genetic engineering has been widely used for the detection and absorption of contaminants in
drinking water and other samples. For example, AtPHR1 gene introduction into garden
plants Torenia, Petunia, and Verbena changed their ability for Pi absorption. The
AtPHR1 transgenic plants with enhanced Pi absorption ability can possibly facilitate effective
phytoremediation in polluted aquatic environments [103]. A fragment of the AtPHR1 gene was
inserted into binary vector pBinPLUS, which contains an enhanced cauliflower mosaic virus 35S
promoter. This plasmid was named pSPB1898 and was used for transformation [104] in Petunia
and Verbena using Agrobacterium tumefaciens [105]. AtPHR1 is effective in other plant species,
such as Torenia, Petunia, and Verbena [103] but posttranscriptional modification of the
endogenous AtPHR1 counterpart might be inhibited by overexpression of AtPHR1 [103].

6. Conclusions
Recombinant DNA technology is an important development in science that has made the human
life much easier. In recent years, it has advanced strategies for biomedical applications such as
cancer treatment, genetic diseases, diabetes, and several plants disorders especially viral and
fungal resistance. The role of recombinant DNA technology in making environment clean
(phytoremediation and microbial remediation) and enhanced resistace of plants to different
adverse acting factors (drought, pests, and salt) has been recognized widely. The improvements it
brought not only in humans but also in plants and microorganisms are very significant. The
challenges in improving the products at gene level sometimes face serious difficulties which are
needed to be dealt for the betterment of the recombinant DNA technology future. In
pharmaceuticals, especially, there are serious issues to produce good quality products as the
change brought into a gene is not accepted by the body. Moreover, in case of increasing product
it is not always positive because different factors may interfere to prevent it from being
successful. Considering health issues, the recombinant technology is helping in treating several
diseases which cannot be treated in normal conditions, although the immune responses hinder
achieving good results.