CHAPTER 11

BIOTECHNOLOGY: PRINCIPLES AND

PROCESSES

What is Biotechnology?

The techniques of using live organisms or enzymes from organisms to

produce products and processes useful to humans is called Biotechnology.
Many processes like in vitro fertilization leading to ‘test-tube’ baby,
synthesizing gene and using it, developing a DNA vaccine or correcting a
defective gene are also parts of Biotechnology.

The European Federation of Biotechnology (EFB) has given a definition of

biotechnology that comprises both traditional and modern molecular
biotechnology. The definition is as follows: - “The integration of natural
science and organisms, cells, parts thereof, and molecular analogues for
products and services”.

Principles of Biotechnology

Modern biotechnology is based on two main principles-

• Genetic Engineering – Genetic Engineering is defined as the direct

manipulation of genome (DNA and RNA) of an organism. It involves the
transfer of new genes to improve the function or trait into host organisms
and thus changes the phenotype of the host organism.
• Maintenance of sterile condition in chemical engineering process to
enable growth of only desired microbes for manufacture of
biotechnological products like antibiotics, vaccine, enzymes etc.

Gene Cloning
• Traditional hybridization used in plants and animal breeding leads to
inclusion and multiplication of undesirable genes along with the desired
traits. The technique of genetic engineering which include creation of
recombinant DNA, use of gene cloning and gene transfer allow us to isolate
and introduce only one or a set of desirable genes without introducing
undesirable genes into the target organism.

• In a chromosome there is a specific DNA sequence called the origin of

replication, which is responsible for initiating replication. Therefore, for the
multiplication of any alien piece of DNA in an organism, it needs to be a
part of a chromosome which has a specific sequence known as ‘origin of
replication’. Thus, an alien DNA is linked with the origin of replication, so
that, this alien piece of DNA can replicate and multiply itself in the host
organism.

This is known as Cloning or making multiple identical copies of any

template DNA.

Recombinant DNA

 The construction of the first recombinant DNA emerged from the

possibility of linking gene encoding antibiotic resistance with a
native plasmid of Salmonella typhimurium.
 Stanley Cohen and Herbert Boyer in 1972 isolated the antibiotic
resistance gene by cutting out a piece of DNA from
a plasmid (autonomously replicating circular extra-chromosomal
DNA) of Salmonella typhimurium. The cutting of DNA at specific
locations became possible with the discovery of the so-called
‘molecular scissors’– restriction enzymes.
 The cut piece of DNA was then linked with the plasmid DNA. These
plasmid DNA act as vectors to transfer the piece of DNA attached to
it. A plasmid can be used as vector to deliver an alien piece of DNA
into the host organism.
  The linking of antibiotic resistance gene with the plasmid vector
become possible with the enzyme ligase, which acts on cut DNA
molecules and joins their ends. This makes a new combination of
autonomously replicating DNA created in vitro and known
as recombinant DNA.
 When this DNA is transferred into E.coli, it could replicate using the
new host DNA polymerase enzyme and make multiple copies. The
ability to multiply copies of antibiotic resistance gene in E.coli was
called cloning of antibiotic resistance gene in E.coli.

“Recombinant DNA technology” or also called “Genetic Engineering”

It deals about, the production of new combinations of genetic material

(artificially) in the laboratory. These “recombinant DNA” (rDNA) molecules
are then introduced into host cells, where they can be propagated and
multiplied.

Steps of Recombinant DNA Technology:

I. Identification of DNA with desirable genes.

II. Introduction of the identified DNA into the host.

III. Maintenance of introduced DNA in the host and transfer of the DNA to
its progeny.

Tools of Recombinant DNA Technology

• Restriction Enzymes

• Polymerase enzymes

• Ligases

• Vectors

• Host organisms
Restriction Enzymes (Molecular Scissors)

Restriction enzymes belong to a larger class of enzymes called Nucleases.

There are of two kinds; Exonucleases and Endonucleases. Exonucleases
remove nucleotides from the ends of the DNA whereas, endonucleases
make cuts at specific position within the DNA.
Example, the first restriction endonuclease – Hind II, always cut DNA
molecules at a particular point by recognizing a specific sequence of six
base pairs. This specific base sequence is known as the Recognition
Sequence for Hind II.

• Each restriction endonuclease recognizes a specific palindromic

nucleotide sequence in the DNA. Palindromes are group of letters that form
the same words when read both forward and backward for example
“MALYALAM”

The palindrome in DNA is a sequence of base pairs that reads same on two
stands when orientation of reading is kept the same.

• Restriction enzymes cut the strand of DNA a little away from the centre of
the palindrome site between the same two bases on the opposite strands
having sticky strand. The stickiness of the strands facilities the action of the
enzyme DNA ligase.

• Restriction endonucleases are used in genetic engineering to form

recombinant molecules of DNA which are composed of DNA from different
sources or genome.

• When cut with the same restriction enzyme the resultant DNA fragments
have the same kind of Sticky-ends and can be joined together using DNA
ligases.

Separation and isolation of DNA fragments/ Gel Electrophoresis

The fragment of DNA obtained by cutting DNA using restriction enzyme is
separated by technique called gel electrophoresis. Negatively charged DNA
fragments can be separated by forcing them to move towards the anode
under an electric field through medium. DNA fragments separate according
to their size through sieving effect provided by agarose gel.
• The separated DNA fragment can be visualized after staining the DNA
with ethidium bromide followed by exposure to UV light. Separated bands
of DNA are separated from agarose gel and extracted from gel,
called elution. The DNA fragment purified this way is used for
recombination.

Cloning Vector

Plasmids and Bacteriophages is commonly used vector for cloning. They

have ability to replicate within bacterial cells independent of the control of
chromosomal DNA. Bacteriophages because of their high number per cell,
have very high copy numbers of their genome within the bacterial cells.

Following features are required to facilitate cloning into a vector-

a. Origin of replication (ori) – the sequence from where replication starts

and any piece of DNA when linked to this sequence can be made to
replicate within the host cells. This sequence is responsible for controlling
the copy number of the linked DNA.
b. Selectable marker-help in the identifying and eliminating non
transformants and selectively permitting the growth of the
transformants. Transformation is a procedure through which a piece of
DNA is introduced in a host bacterium. Generally, the genes encoding
resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or
kanamycin, etc., are considered useful selectable markers for E. coli.
c. Cloning sites– to link the foreign DNA, the vector need to have single
recognition sites for the commonly used restriction enzymes as presence of
more than one recognition sites within the vector will generate several
fragments, which will complicate the gene cloning. The ligation of foreign
DNA is carried out at a restriction site present in one of the two antibiotic
resistance genes.

Insertional inactivation

The most efficient method of screening for the presence of recombinant

plasmids is based on the principle that the cloned DNA fragment disrupts
the coding sequence of a gene. This is termed as Insertional Inactivation.
For example, the powerful method of screening for the presence of
recombinant plasmids is referred to as Blue-White selection. This method
is based upon the insertional inactivation of the lac Z gene present on the
vector. The lac Z gene encodes the enzyme beta-galactosidase, which can
cleave a chromogenic substrate into a blue coloured product. If this lac Z
gene is inactivated by insertion of a target DNA fragment into it, the
development of the blue colour will be prevented and it gives white
coloured colonies. By this way, we can differentiate recombinant (white
colour) and non-recombinant (blue colour) colonies.

Vectors for cloning genes in plants and animals

Agrobacterium tumefactions (pathogen of dicot plant) 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. Retroviruses in animals have the ability to transform normal
cells into cancerous cells. The tumor inducing (Ti) plasmid of Agrobacterium
tumefaciens has been modified into cloning vector having no more
pathogenic to plant. Similarly retrovirus have been modified into cloning
vector for animals.

Competent host (For Transformation with Recombinant DNA)

1) Simple chemical treatment with divalent calcium ions increases the

efficiency of host cells (through cell wall pores) to take up the rDNA
plasmids.
2) rDNA can also be transformed into host cell by incubating both on
ice, followed by placing them briefly at 42oC (Heat Shock), and then putting
them back on ice. This enables the bacteria to take up the recombinant
DNA.
3) In Microinjection method, rDNA is directly injected into the nucleus
of cells by using a glass micropipette.
4) Biolistics / Gene gun method, it has been developed to introduce
rDNA into mainly plant cells by using a Gene / Particle gun. In this method,
microscopic particles of gold / tungsten are coated with the DNA of interest
and bombarded onto cells.
5) The last method uses “Disarmed Pathogen” Vectors (Agrobacterium
tumefaciens), which when allowed to infect the cell, transfer the
recombinant DNA into the host.

Processes of Recombinant DNA Technology

Recombinant DNA technology involves several steps in specific sequence-

a. Isolation of DNA

b. Fragmentation of DNA by restriction endonucleases

c. Isolation of a desired DNA fragment

d. Ligation of the DNA fragment into vector

e. Transforming the recombinant DNA into the host

f. Culturing the host cells in a medium at large scale

g. Extraction of the desired product.

•Isolation of Genetic material:

Genetic material is isolated from other macromolecules by using enzymes

such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus). DNA
that separate out can be removed by spooling. The RNA can be removed
by treatment with ribonuclease whereas proteins can be removed by
treatment with protease.

• Cutting of DNA at specific location and separation of fragments:

It is performed by using restriction enzyme and Agarose gel electrophoresis

to check the progression of a restriction enzyme digestion. After cutting
source DNA as well as vector DNA, the cut fragments are separated and
extracted by electrophoresis.

• Amplification of Gene of Interest using PCR (Polymerase Chain

Reaction):

This is done to get multiple copies of the DNA or gene of interest in vitro
by using set of primers and enzyme DNA polymerase. This repeated
amplification is done by the use of a thermostable DNA polymerase
(isolated from a bacterium, Thermus aquaticus), which remain active during
the high temperature induced denaturation of double stranded DNA.

• Insertion of Recombinant DNA into the Host Cell/Organism:

This includes making the recipient cells competent to receive, take up DNA
present in its surrounding etc. The recombinant DNA bearing gene for
resistance to an antibiotic is transferred into E.coli cells, the host cell
become transformed into ampicillin-resistance cells.

• Obtaining the foreign gene product:

The foreign DNA multiplies in plant or animal cell to produce desirable

protein. Expression of foreign genes in host cells involve, optimized
condition to obtain recombinant protein. The recombinant cell is multiplied
in a continuous culture system in which used medium is drained out from
one side while fresh medium is added from the other to maintain the cells
in their physiological active phase. A bioreactor provides the optimal
conditions for achieving the desired product by providing optimum growth
conditions (temperature, pH, substrate, salts, vitamins, oxygen).

• Downstream Processing:

It involves processes that make the product obtain ready for marketing.
This process includes separation and purification called as downstream
processing. Suitable preservatives are added to it and send for clinical trial
in case of drugs before releasing to market for public use.

NOTE: PRACTICE ALL THE DIAGRAMS GIVEN IN N.C.E.R.T. TEXTBOOK.