27.

Biotechnology – Principles and Process,

Biotechnology – Applications
Summary Sheet
• The science of biotechnology is mainly based on two core technologies:
o Genetic engineering: It refers to artificial synthesis, isolation, modification, combination, addition
and repair of the genetic material (DNA) by man to alter the phenotype of the host organism to suit
human needs. It includes formation of ‘recombinant DNA (rDNA)’, use of gene cloning and gene
transfer.
o Biochemical engineering: It involves processes that help in the growth of desired microbes,
eukaryotic cells in large quantities in a sterile medium (tissue culture technique) for the manufacture
and multiplication of biotechnological products (antibiotics, vaccines, enzymes, medicines,
hormones, etc.).
• Genetic engineering was started by Paul Berg (1972). He is often considered as the “Father of genetic
engineering”. Genetic engineering is alternately called recombinant DNA technology or gene cloning.
The first recombinant DNA was constructed by Stanley Cohen and Herbert Boyer in 1972. They cut
the piece of DNA from a plasmid carrying antibiotic-resistance gene in the bacterium Salmonella
typhimurium and linked it to the plasmid of Escherichia coli. The cutting of DNA at specific locations
became possible with the discovery of the so-called ‘molecular scissors’— restriction enzymes.
• The process of rDNA technology involves: isolation of the genetic material, cutting of DNA at specific
locations, amplification of gene of interest using PCR, preparation and insertion of rDNA into host cell
and obtaining desirable gene product.
Tools of recombinant DNA technology
Enzymes
• Cleaving enzymes are used to break DNA molecules. They are further of 3 kinds: (a) exonucleases
(b) endonucleases (c) restriction endonucleases.
• Exonucleases cut off nucleotides from 5’ or 3’ terminal ends of DNA molecule. Endonucleases cleave
DNA duplex at any point except the terminal ends. (Restriction endonucleases were isolated for the first
time by W. Arber in 1963). They act as “molecular scissors” or “chemical scalpels”. They recognize
the specific base sequence at palindrome sites in DNA duplex and cut its strands. The palindromes in
DNA are base pair sequences that reads same on two strands when orientation of reading is kept the
same, e.g.: sequence reads the same on two strands in 5’ to 3’ direction or 3’ to 5’ direction.
Restriction enzyme Source Recognition sequence and Site of cleavage
EcoR I Escherichia coli RY 13 5'− G −↓ A − A − T − T − C − 3'
3'− T − T − C − G − A − A − 5'
↑

• Restriction enzymes cut the strand of DNA at the palindrome sites, but between the same bases on
opposite strands. This leaves single-stranded unpaired bases at cut ends. These overhanging ends with
unpaired bases are called sticky ends or cohesive ends. These are named so because they form hydrogen
bonds with their complementary cut counter parts. The sticky ends facilitate the action of the enzyme
DNA ligase.

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• DNA ligase or joining enzymes helps in sealing gaps and linking DNA fragments by forming
phosphodiester bonds. The linking of antibiotic resistance gene with the plasmid vector became possible
with the enzyme DNA ligase. e.g, T4 ligases.
• Separation and isolation of DNA fragments: Gel Electrophoresis is a technique of separation of
charged molecules under the influence of an electrical field so that they migrate in the direction of
electrode bearing the opposite charge, through a medium/matrix. As DNA fragments are negatively
charged molecules, move towards the anode under an electric field through matrix. DNA fragments
separate according to the size through the pores of agarose gel. Smaller the fragment size, farther it
moves. The most commonly used matrix is agarose which is a polysaccharide extracted from sea weeds.
The separated DNA fragments can be seen only after staining the DNA with a compound known as
ethidium bromide (EtBr) followed by exposure to UV radiation as bright orange coloured bands. The
separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is
called as elution.

Cloning Vectors
• The DNA used as a carrier for transferring of a fragment of foreign DNA into a suitable host is called
vehicle DNA or cloning vector.
• The DNA which is transferred from one organism into another by joining it with the vehicle DNA, is
called passenger DNA.
• Vectors
(i) Plasmids are extra-chromosomal, self replicating, usually circular double stranded DNA molecules
found naturally in many bacteria and also in some yeasts. pBR322 vector was the first artificial
cloning vector constructed in 1977 by Boliver and Rodriguez.
(ii) Agrobacterium tumefaciens, a pathogen of several dicot plants is able to deliver a piece of DNA
known as ‘T-DNA’ to transform normal plant cells into a tumor and direct these tumor cells to
produce the chemicals required by the pathogen. The tumor inducing (Ti) plasmid of
Agrobacteriuin tumefaciens has been modified into a cloning vector which is no more pathogenic to
the plants but is still able to use the mechanisms to deliver genes of our interest into a variety of
plants.
(iii) Bacterial artificial chromosome (BAC) vectors are based on natural, extra-chromosomal plasmid
of E. coli. These vectors can accommodate upto 300-350 Kb of foreign DNA.
(iv) Yeast artificial chromosome (YAC) vectors are used to clone DNA fragments of more than 1Mb in
size. Therefore, they have been exploited extensively in mapping the large genomes.

Characteristics of a cloning vector: The desirable characteristics of a cloning vector are:

o Origin of replication (Ori) is a sequence from where replication starts. The replication occurs inside
the host cells. A prokaryotic DNA has a single origin of replication while eukaryotic DNA may have
more than one origin of replication.
o Selectable markers (antibiotic resistance gene) are required to identify and eliminate non-
transformants and selectively permit the growth of the transformants. Generally, the genes encoding
resistance to antibodies such as tetracycline, ampicillin, kanamycin or chloramphenicol, etc., are
useful as selectable markers for E. coli.

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o A valuable feature of the cloning vector is the presence of specific restriction sites (or cloning
sites), where the enzyme restriction endonucleases make a cut so that a foreign DNA segment may
be introduced (joined) to the vector. This property helps a vector to act as trusted cloning vehicle
during gene transfer in genetic engineering. The ligation of alien DNA is carried out at a restriction
site present in one of the antibiotic resistance genes.

Competent host
• Competent host is essential for transformation with recombinant DNA. Many kinds of host cells,
including E. coli, yeast, animal and plant cells, are available for genetic engineering. For the expression
of some eukaryotic proteins, eukaryotic cells may be the preferred hosts.
• DNA is a hydrophilic molecule, it cannot pass through membranes, so bacterial cells must be made
capable to take up DNA. This is done by treating them with a specific concentration of a divalent cation,
such as calcium ion which increases the efficiency with which DNA enters the bacterium through pores
in its cells wall. In eukaryotic cells, the term transformation is replaced by the term transfection.
• Electroporation: This method involves suspension of cells in a suitable ionic solution containing
linearized recombinant plasmid DNA. This mixture is then exposed to low voltage-long pulses or high
voltage-short pulses for the desired number of cycles. The electrical pulses are thought to induce
transient pores in the plasma-lemma through which the DNA molecules are incorporated. Treated cells
are then cultured to obtain colonies.
• Particle bombardment or Gene gun method: In this method, gold or tungsten particles coated with
DNA are shooted into the plant cells using a helium pressure particle gun device. This method is also
known as biolistic technique. Important crop plants like, wheat, rice and maize have now been
transformed by this method.
• Microinjection: In this method, the DNA solution is injected directly inside the cell using capillary
glass micropipette or micro syringe. This technique was originally used in animal cells.

Processes of Recombinant DNA Technology

1. Isolation of the genetic material (DNA)
• In order to get pure DNA molecules the following steps are required : Bacterial cells are treated with
lysozyme to dissolve the bacterial cell walls. Plants cells are treated with enzyme cellulase to dissolve
cellulose cell wall. The enzyme chitinase is used to dissolve fungal cell wall. The plasma membrane
degrading enzymes are used to degrade membranes. This results in the release of DNA along with
several other macromolecules and impurities such as RNA, proteins, polysaccharides and lipids. The
eukaryotic DNA molecules are interwined with proteins such as histones. These proteins can be removed
by treatment with enzyme protease. The enzyme protease converts proteins into aminoacids. Similarly,
RNA are removed by treating with the enzyme ribonuclease. Other macromolecules can also be
removed by specific treatments. Finally the pure DNA molecules are precipitates out by adding chilled
ethanol and collected in the suspension.
2. Cutting and ligation of DNA at specific locations
• The vector DNA (e.g., plasmid DNA) and alien (foreign) DNA carrying gene of interest are cut by the
same restriction endonuclease to produce complementary sticky ends. This process of cutting DNA by
restriction enzyme is called restriction enzyme digestion. With the help of DNA ligase enzyme, the

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complementary sticky ends of the two DNAs are joined to produce a recombinant DNA
(rDNA/chimaera).
3. Amplification of Gene of interest using PCR technique- This reaction produces multiple copies of the
gene of interest in vitro using two sets of primers (chemically synthesized oligonucleoties that are
complementary to region of DNA) and enzyme thermostable DNA polymerase. Following are steps of
Polymerase Chain Reaction (PCR):
• Denaturation: The target DNA is heated to a high temperature (usually 940C to 98°C), resulting in the
separation of the two strands. Each single strand of the target DNA then acts as a template for DNA
synthesis.
• Primer Annealing: The two oligonucleotide primers anneal (hybridize) to each of the single-stranded
template DNA, since the sequence of the primers is complementary to the template DNA. This step is
carried out at a lower temperature (usually 50°C to 60°C) depending on the length and sequence of the
primers.
• Extension (Polymerisation) of primers: Taq DNA polymerase (from a thermophilic bacterium
Thermus aquaticus). synthesizes the DNA region from the primer, using dNTPs (deoxynucleoside
triphosphates) and Mg2+. It means the primers are extended so
that the DNA segment that lies between them will get copied.
This occurs at an optimum temperature of 72°C.
4. Insertion of recombinant DNA into the host cell/organism
• This method is to introduce the ligated DNA into recipient cells.
Recipient cells are made competent to receive the rDNA present
in its surrounding. Different techniques are used to select the
transformant. For example,
• If recombinant DNA bearing gene for resistance to an antibiotic
(ampicillin) is transferred into E.coli cells, the host cells (E.coli)
become ampicillin-resistant cells. If we spread the transformed
cells on agar plates containing ampicillin, only transformants will
grow, untransformed recipient cells will die. Ampicillin
resistance gene in this case is selectable marker.
5. Obtaining the desirable gene product
• The cells having cloned genes of interest can be grown on a small
scale in the laboratory. The cultures may be used for extracting
and purifying the desired protein. After the cloning of the gene of
interest one has to maintain the optimum conditions to induce the
expression of the target gene and consider producing it on a large
scale. If any protein encoding gene is expressed in a heterologous
host it is known as a “recombinant protein”.

• Bioreactors are considered as vessels in which raw materials are

biologically converted into specific products by microbes, plant and animal cells or their enzymes. It
provides the optimal conditions for obtaining the desired product by providing optimum growth
conditions such as temperature, pH, substrate, vitamins, oxygen and salts. The most commonly used

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bioreactors are of stirring type. Stirring type bioreactors are (i) simple stirred-tank bioreactor and (ii)
sparged stirred-tank bioreactor.

• A stirred-tank bioreactor is usually cylindrical or with a curved base to facilitate the mixing of the
reactor content. The stirrer facilitates even mixing the oxygen availability throughout the bioreactor.
Alternatively air can be bubbled through the reactor. The bioreactor has an agitator system, an oxygen
delivery system and foam control system, a temperature control system, pH control system and sampling
ports so that small volumes of the culture can be withdrawn periodically. After completion of the
biosynthetic stage, the product has to be subjected through a series of processes before it is ready for
marketing as a finished product. The processes include separation and purification, which are
collectively referred to as downstream processing. The product has to be formulated with suitable
preservatives. Such formulation has to undergo through clinical trials as in case of drugs.

Biotechnology Applications
• The applications of biotechnology include therapeutics, diagnostics, genetically modified crops,
processed food, bioremediation, waste treatment and energy production.
• Research areas of biotechnology– (i) Providing the best catalyst in the form of improved organism;
generally a microbe or pure enzyme. (ii) Creating optimal conditions through engineering for a catalyst
to act, and (iii) Downstream processing technologies to purify the protein/organic compound.

Genetically Modified Organisms (GMOs)

• The organisms which contain functional foreign gene experimentally introduced into their genome by
genetic engineering from another species are called genetically modified organisms or transgenic
organisms or transgenics. Foreign gene that alters the host cell genetically, is termed transgene and
production of transgenic organism is known as transgenesis.
Transgenic plants
• The plants in which foreign gene has been introduced through genetic engineering are called transgenic
plants.
• The vector used to introduce new genes into plant cells is most often a plasmid from the soil bacterium
Agrobacterium tumefaciens. This is the Ti plasmid (tumour inducing plasmid), so called because in
nature, it induces tumours in broad leaf plants. For using Ti plasmid as a vector, researchers have
eliminated its tumour causing properties while keeping its ability to transfer DNA into plant cells. The
part of Ti plasmid transferred into plant cell DNA, is called the T-DNA.
• Insect resistance in transgenic plants: Soil bacterium Bacillus thuringiensis produces proteins that kill
certain insects like lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans
(flies, mosquitoes). The Bt toxin protein exists as inactive protoxin but once an insect ingests the
inactive toxin, it is converted into an active form of toxin due to the alkaline pH of the alimentary canal
which solubilises the crystals. The activated toxin binds to the surface of midgut epithelial cells and
creates pores that cause cell swelling and lysis and eventually cause death of the insect. The choice of
genes depends upon the crop and targeted pest, as most Bt toxins are insect-group specific. The toxin is
coded by a gene named cry. There are numerous genes, Two cry genes: cry I Ac and cry II Ab have
been incorporated in cotton. This genetic By modified crop is called Bt cotton as it contains Bt toxin

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genes against cotton bollworms. Similarly, cry I Ab has been introduced in Bt corn to protect the same
from corn borer.
• Many nematodes live in plants and animals including human beings. A nematode Meloidogyne incognita
infests the roots of tobacco plants and causes a great reduction in yield. A novel strategy is adopted to
prevent this infection that was based on the process of RNA interference (RNAi).
• RNA interference (RNAi) is the phenomenon of inhibiting activity of a gene by synthesis of RNA
molecules complementary to the mRNA. The normal (in vivo synthesised) mRNA of a gene is said to be
“sense” because it carries the codons that are “read” during translation. Generally, the complement to the
mRNA “sense” strand will not contain a sequence of codons that can be translated to produce a
functional protein; thus, this complementary strand is called “anti-sense RNA”. The anti-sense RNA
and mRNA molecules will anneal to form duplex RNA molecules (or double stranded RNA) and the
duplex RNA molecules cannot be translated. Thus, the presence of anti-sense RNA will block translation
of the mRNA of the affected gene.
• Different steps involved in making tobacco plant resistant to nematode are as following
o Double-stranded RNAs are processed into approximately 21-23 nucleotide RNAs. An RNase
enzyme, called Dicer, cuts the dsRNA molecules (from a virus, transposon, or through
transformation) into small interfering RNAs (siRNAs).
o Each siRNA complexes with ribonucleases (distinct from Dicer) to form an RNA-induced
silencing complex (RISC).
o The sRNA unwinds and RISC is activated.
o The activated RISC targets complementary mRNA molecules. The siRNA strands act as guides
where the RISCs cut the transcripts. This destroys the mRNA.
o When mRNA of the parasite is destroyed no proteins are synthesized. It results in the death of the
parasite (nematode) in the transgenic host. Thus, the transgenic plant gets itself protected from the
parasite.
• Some other agricultural applications are:
o Flavr Savr tomato was the transgenic variety that prevents over-ripening of fruit.
o Golden rice is a transgenic variety of rice (Oryza sativa) which contains good quantities of beta
carotene (precursor of vitamin A synthesis).

Biotechnological applications in medicine

• Insulin is made up of 51 amino acids arranged in two polypeptide chains, A-chain having 21 amino
acids and B-chain with 30 amino acids that are linked together by disulphide bridges. In mammals,
including humans, insulin is synthesised as a pro-hormone which contains an extra stretch called the
C peptide. This C peptide is not present in the mature insulin and is removed during maturation into
insulin. The main challenge for production of insulin using rDNA techniques was getting insulin
assembled into a mature form. In 1983, Eli Lilly an American company, prepared two DNA sequences
corresponding to A and B chains of human insulin and introduced them in plasmids of E.coli to produce
insulin chains. Chains A and B were produced separately, extracted and combined by creating disulphide
bonds to form human insulin (humulin).

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• Gene therapy is a collection of methods that allows genes to be inserted into a person’s cells and tissues
to treat a disease. Correction of a genetic defect involves delivery of a normal gene into the individual or
embryo to take over the function of and compensate for the non-functional gene. The first clinical gene
therapy was given in 1990 to a 4-year old girl with adenosine deaminase (ADA) deficiency. This
enzyme is very important for the immune system to function. SCID is caused due to defect in the gene
for the enzyme adenosine deaminase. In some children, ADA deficiency can be cured by bone marrow
transplantation. However in others it can be treated by enzyme replacement therapy, in which functional
ADA is given to the patient by injection. But in both approaches the patients are not completely cured.
However if the isolated gene from bone marrow cells producing ADA is introduced into cells at early
embryonic stages, it can be a permanent cure.

Molecular Diagnosis
• An effective treatment of a disease, early diagnosis and understanding its pathophysiology is very
important.
• Recombinant DNA technology, Polymerase Chain Reaction (PCR) and Enzyme Linked Immuno-sorbent
Assay (ELISA) are some of the techniques that serve the purpose of early diagnosis.
• Detection of pathogen - bacteria or virus (at a time when the symptoms of the disease are not yet visible)
can be detected by amplification of their nucleic acid by PCR.
• PCR is now routinely used to detect HIV in suspected AIDS patients.
• It is being used to detect mutations in genes in suspected cancer patients too.
• A single stranded DNA or RNA, tagged with a radioactive molecule (probe) is allowed to hybridise to its
complementary DNA in a clone of cells followed by detection using autoradiography.
• ELISA is based on the principle of antigen-antibody interaction. Infection by pathogen can be detected
by the presence of antigens (proteins, glycoproteins, etc.) or by detecting the antibodies synthesized
against the pathogen.

Transgenic animals
• Transgenic animals that produce useful biological products can be created by the introduction of the
portion of DNA (or genes) which codes for a particular product such as human protein (alfa-1-
antitrypsin) used to treat emphysema. Similar attempts are being made for treatment of
phenylketonuria (PKU) and cystic fibrosis. In 1997, the first transgenic cow, Rosie produced human
protein-enriched milk (2.4 grams per litre). The milk contained the human alpha-lactalbumin and was
nutritionally a more balanced product for human babies than natural cow milk.
• Transgenic mice are being developed for use in testing the safety of vaccine before they are used on
humans. Transgenic animals are made that carry genes which make them more sensitive to toxic
substances than non-transgenic animals. Toxicity testing in such animals will allow us to obtain results
in less time.

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Ethical issues related to biotechnology

• Some ethical standards are required to evaluate the morality of all human activities that might help or
harm living organisms Therefore the Indian Government has set up organisations such as GEAC
(Genetic Engineering Approval Committee), which will make decisions regarding the validity of GM
research and the safety of introducing GM research, the safety of introducing GM-organisms for public
services.
• A patent is the right granted by a government to an inventor to prevent others from commercial use of
his invention. When patents are granted for biological entities and for products derived from them, these
patents are called biopatents. Patents have been taken out on plants such as black pepper (Piper
nigrum), basmati rice (Oryza sativa), Indian mustard (Brassica campestris), pomegranate (Punica
granatum), turmeric and neem.
• Some organisations and multinational companies exploit and/or patent biological resources or
bioresources of other nations without proper authorization from the countries concerned, this is called
biopiracy. For example, a patent granted in U.S.A. covers the entire ‘basmati’ rice germplasm
indigenous to our country.
• Bioethics includes rules of conduct that may be used to regulate our activities in relation to the
biological world. The main bioethical concerns pertaining to biotechnology are briefly mentioned as
follows
o Introduction of a transgene from one species into another species violates the ‘Integrity of species’.
o Biotechnology may pose unforeseen risks to the environment including risk to biodiversity
o Transfer of human genes into animals (and vice-versa) dilutes the concept of ‘humanness’.
o When animals are used for production of pharmaceutical proteins, they are virtually reduced to the
status of a ‘factory’.
o Use of animals in biotechnology causes great suffering to them.
o It is disrespectful to living beings, and only exploits them for the benefit of human beings.
o Scientists cannot rule out the possibility of other biological damage.
o It can accidentally create new infectious agents.

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