Ever since the days of Rene Descartes, the French philosopher,
mathematician and biologist of seventeenth century, all human
knowledge especially natural sciences were directed to develop
technologies which add to the creature comforts of human
lives, as also value to human life. The whole approach to
understanding natural phenomena became anthropocentric.
Physics and chemistry gave rise to engineering, technologies
and industries which all worked for human comfort and welfare.
The major utility of the biological world is as a source of food.
Biotechnology, the twentieth century off-shoot of modern
biology, changed our daily life as its products brought
qualitative improvement in health and food production. The
basic principles underlying biotechnological processes and some
applications are highlighted and discussed in this unit.
Chapter 11
Biotechnology : Principles and
Chapter 12
Biotechnology and Its
Herbert Boyer was born in 1936 and brought up in a corner of western
Pennsylvania where railroads and mines were the destiny of most young
men. He completed graduate work at the University of Pittsburgh, in
1963, followed by three years of post-graduate studies at Yale.
In 1966, Boyer took over assistant professorship at the University of
California at San Francisco. By 1969, he performed studies on a couple
of restriction enzymes of the E. coli bacterium with especially useful
properties. Boyer observed that these enzymes have the capability of
cutting DNA strands in a particular fashion, which left what has became
known as ‘sticky ends’ on the strands. These clipped ends made pasting
together pieces of DNA a precise exercise.
This discovery, in turn, led to a rich and rewarding conversation in
Hawaii with a Stanford scientist named Stanley Cohen. Cohen had
been studying small ringlets of DNA called plasmids and which float
about freely in the cytoplasm of certain bacterial cells and replicate
independently from the coding strand of DNA. Cohen had developed
a method of removing these plasmids from the cell and then reinserting
them in other cells. Combining this process with that of DNA splicing
enabled Boyer and Cohen to recombine segments of DNA in desired
configurations and insert the DNA in bacterial cells, which could then
act as manufacturing plants for specific proteins. This breakthrough was
the basis upon which the discipline of biotechnology was founded.
(1936 )
Biotechnology deals with techniques of using live
organisms or enzymes from organisms to produce products
and processes useful to humans. In this sense, making
curd, bread or wine, which are all microbe-mediated
processes, could also be thought as a form of
biotechnology. However, it is used in a restricted sense
today, to refer to such of those processes which use
genetically modified organisms to achieve the same on a
larger scale. Further, many other processes/techniques are
also included under biotechnology. For example, in vitro
fertilisation leading to a ‘test-tube’ baby, synthesising a
gene and using it, developing a DNA vaccine or correcting
a defective gene, are all part of biotechnology.
The European Federation of Biotechnology (EFB) has
given a definition of biotechnology that encompasses both
traditional view and modern molecular biotechnology.
The definition given by EFB is as follows:
‘The integration of natural science and organisms,
cells, parts thereof, and molecular analogues for products
and services’.
Among many, the two core techniques that enabled birth
of modern biotechnology are :
(i) Genetic engineering : Techniques to alter the
chemistry of genetic material (DNA and RNA),
11.1 Principles of Biotechnology
11.2 Tools of Recombinant DNA
11.3 Processes of Recombinant
DNA Technology
to introduce these into host organisms and thus change the
phenotype of the host organism.
(ii) Bioprocess engineering: Maintenance of sterile (microbial
contamination-free) ambience in chemical engineering processes
to enable growth of only the desired microbe/eukaryotic cell in
large quantities for the manufacture of biotechnological products
like antibiotics, vaccines, enzymes, etc.
Let us now understand the conceptual development of the principles
of genetic engineering.
You probably appreciate the advantages of sexual reproduction over
asexual reproduction. The former provides opportunities for variations
and formulation of unique combinations of genetic setup, some of which
may be beneficial to the organism as well as the population. Asexual
reproduction preserves the genetic information, while sexual reproduction
permits variation. Traditional hybridisation procedures used in plant and
animal breeding, very often lead to inclusion and multiplication of
undesirable genes along with the desired genes. The techniques of genetic
engineering which include creation of recombinant DNA, use of
gene cloning and gene transfer, overcome this limitation and allows us
to isolate and introduce only one or a set of desirable genes without
introducing undesirable genes into the target organism.
Do you know the likely fate of a piece of DNA, which is somehow
transferred into an alien organism? Most likely, this piece of DNA would
not be able to multiply itself in the progeny cells of the organism. But,
when it gets integrated into the genome of the recipient, it may multiply
and be inherited along with the host DNA. This is because the alien piece
of DNA has become part of a chromosome, which has the ability to
replicate. 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(s) 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 can also be called as cloning or
making multiple identical copies of any template DNA.
Let us now focus on the first instance of the construction of an artificial
recombinant DNA molecule. The construction of the first recombinant
DNA emerged from the possibility of linking a gene encoding antibiotic
resistance with a native plasmid (autonomously replicating circular
extra-chromosomal DNA) of Salmonella typhimurium. Stanley Cohen and
Herbert Boyer accomplished this in 1972 by isolating the antibiotic
resistance gene by cutting out a piece of DNA from a plasmid which was
responsible for conferring antibiotic resistance. 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. You probably know that mosquito
acts as an insect vector to transfer the malarial parasite into human body.
In the same way, 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 became possible with the enzyme DNA ligase,
which acts on cut DNA molecules and joins their ends. This makes a new
combination of circular autonomously replicating DNA created in vitro
and is known as recombinant DNA. When this DNA is transferred into
Escherichia coli, a bacterium closely related to Salmonella, it could
replicate using the new host’s 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.
You can hence infer that there are three basic steps in genetically
modifying an organism —
(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.
Now we know from the foregoing discussion that genetic engineering or
recombinant DNA technology can be accomplished only if we have the
key tools, i.e., restriction enzymes, polymerase enzymes, ligases, vectors
and the host organism. Let us try to understand some of these in detail.
11.2.1 Restriction Enzymes
In the year 1963, the two enzymes responsible for restricting the growth
of bacteriophage in Escherichia coli
were isolated. One of these added
methyl groups to DNA, while the other cut DNA. The later was called
restriction endonuclease.
The first restriction endonuclease–Hind II, whose functioning
depended on a specific DNA nucleotide sequence was isolated and
characterised five years later. It was found that Hind II always cut DNA
molecules at a particular point by recognising a specific sequence of
six base pairs. This specific base sequence is known as the
recognition sequence for Hind II. Besides Hind II, today we know more
than 900 restriction enzymes that have been isolated from over 230 strains
of bacteria each of which recognise different recognition sequences.
The convention for naming these enzymes is the first letter of the name
comes from the genus and the second two letters come from the species of
the prokaryotic cell from which they were isolated, e.g., EcoRI comes from
Escherichia coli RY 13. In EcoRI, the letter ‘R’ is derived from the name of