Biotechnology

Principles and processes

1
Index
1.1 Principles of Biotechnolog

1.2 Tools of Recombinant DNA Technology

➢ Restriction Enzyme
➢ Cloning vector
➢ Competent Host

1.3 Processes of Recombinant DNA Technology

➢ Isolation of the Genetic Material (DNA)

➢ Cutting of DNA at Specific Location

➢ Amplification of Gene of Interest using PCR

➢ Insertion of Recombinant DNA into the Host Cell/Organism

➢ Obtaining the Foreign Gene Product

➢ Downstream Processing
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.

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'.
1.1 PRINCIPLES OF BIOTECHNOLOGY

Among many, the two core techniques that

enabled birth of modern biotechnology are:

Genetic engineering: Techniques to alter the

chemistry of genetic material (DNA and RNA)
to introduce these into host organisms and thus
change the phenotype of the host organism.
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.

The techniques of genetic engineering which

include creation of recombinant DNA. use of gene
cloning and gene transfer, overcome this limitation
and allows u to isolate and introduce only one or a
set of desirable genes withou introducing
undesirable genes into the target 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.

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.

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 enzyme . The cut plece of DNA then
linked with the plasmid DNA. These plasmid DNA
act as vectors to transfer the piece of DNA
attached to it. 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.
There are three basic steps in genetically
modifying an organism

● Identification of DNA with desirable gene

● Introduction of the identified DNA into the
host .
● Maintenance of introduced DNA in the host
and transfer of the DNA to its progeny.
 1.2 TOOLS OF RECOMBINANT DNA
TECHNOLOGY

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.

● Restriction enzyme

In the year 1963. the two enzymes responsible for

restricting the growth 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.

Naming these enzyme: 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
strain.

Restriction enzymes belong to a larger class of

enzymes called nucleases
These are of two kinds; exonucleases and
endonucleases .
Exonucleases remove nucleotides from the ends
of the DNA whereas, endonucleases make cuts at
specific positions within the DNA.

Each restriction endonuclease recognises a

specific palindromic nucleotide sequences in the
DNA.
Restriction enzymes cut the strand of DNA a little
away from the centre of the palindrome sites, but
between the same two bases on the opposite
strands. This leaves single stranded portions at
the ends. There are overhanging stretches called
sticky ends on each strand.
Restriction endonucleases are used in genetic
engineering to form recombinant molecules of
DNA, which are composed of DNA from different
sources/genomes.

Separation and isolation of DNA fragments.

The cutting of DNA by restriction endonucleases
results in the fragments of DNA. These fragments
can be separated by a technique known as
gel electrophoresis.
DNA fragments are negatively charged molecules
they can be separated by forcing them to move
towards the anode under an electric field through
a medium/matrix. Nowadays the most commonly
used matrix is agarose which is a natural polymer
extracted from sea weeds. The DNA fragments
separate (resolve) according to their size through
sieving effect provided by the agarose gel. Hence,
the smaller the fragment size, the farther it moves.

The separated DNA fragments can be visualised

only after staining the DNA with a compound
known as ethidium bromide followed by exposure
to UV radiation (you cannot see pure DNA
fragments in the visible light and without staining).
You can see bright orange coloured bands of DNA
in a ethidium bromide stained gel exposed to UV
light.
The separated bands of DNA are cut out from the
agarose gel and extracted from the gel piece. This
step is known as elution.The DNA fragments
purified in this way are used in constructing
recombinant DNA by joining them with cloning
vectors.
 ● Cloning Vectors
You know that plasmids and bacteriophages have
the 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. Some plasmids
may have only one or two copies per cell whereas
others may have 15-100 copies per cell. Their
numbers can go even higher If we are able to link
an alien piece of DNA with bacteriophage or
plasmid DNA, we can multiply its numbers equal
to the copy number of the plasmid or
bacteriophage. Vectors used at present, are
engineered in such a way that they help easy
linking of foreign DNA and selection of
recombinants from non-recombinants.

Origin of replication (ori) : This is a 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
also responsible for controlling the copy number
of the linked DNA. So, if one wants to recover
many copies of the target DNA it should be cloned
in a vector whose origin support high copy
number.

Selectable marker : Selectable marker: In addition

to 'ori', the vector requires a selectable marker,
which helps in 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 (you will study the
process in subsequent section). Normally, the
genes encoding resistance to antibiotics such as
ampicillin, chloramphenicol, tetracycline or
kanamycin, etc., are considered useful selectable
markers for E. coli. The normal E. coli cells do not
carry resistance against any of these antibiotics.

Cloning sites: In order to link the alien DNA, the

vector needs to have very few. preferably single,
recognition sites for the commonly used restriction
enzymes. Presence of more than one recognition
sites within the vector will generate several
fragments, which will complicate the gene cloning.
The ligation of alien DNA is carried out at a
restriction site present in one of the two antibiotic
resistance genes. For you can ligate a foreign
DNA
E. coli cloning vector pBR322 showing restriction
sites (Hind III. EcoR I, BamHI, Sal I, Pvu II. Pst I.
Cla I), ori and antibiotic resistance genes (amp
and tet). rop codes for the proteins involved in the
replication of the plasmid.

Vectors for cloning genes in plants and animals:

You may be surprised to know that we have learnt
the lesson of transferring genes into plants and
animals from bacteria and viruses which have
known this for ages-how to deliver genes to
transform eukaryotic cells and force them to do
what the bacteria or viruses want. For example.
Agrobacterium tumifaciens, 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.
Similarly, retroviruses in animals have the ability
to transform normal cells into cancerous cells. A
better understanding of the art of delivering genes
by pathogens in their eukaryotic hosts has
generated knowledge to transform these tools of
pathogens into useful vectors for delivering genes
of interest to humans. The tumor inducing (Ti)
plasmid of Agrobacterium tumifaciens has now
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.

● Competent Host (For Transformation with

Recombinant DNA)

Since DNA is a hydrophilic molecule, it cannot

pass through cell membranes, Why? In order to
force bacteria to take up the plasmid, the bacterial
cells must first be made 'competent' to take up
DNA. This is done by treating them with a specific
concentration of a divalent cation. such as
calcium, which increases the efficiency with which
DNA enters the bacterium through pores in its cell
wall. Recombinant DNA can then be forced into
such cells by incubating the cells with
recombinant DNA on ice, followed by placing
them briefly at 42°C (heat shock), and then
putting them back on ice. This enables the
bacteria to take up the recombinant DNA.

This is not the only way to introduce alien DNA

into host cells. In a method known as
micro-injection, recombinant DNA is directly
injected into the nucleus of an animal cell. In
another method, suitable for plants. cells are
bombarded with high velocity micro-particles of
gold or tungsten coated with DNA in a method
known as biolistics or gene gun. And the last
method uses 'disarmed pathogen' vectors, which
when allowed to infect the cell, transfer the
recombinant DNA into the host.

1.3 PROCESSES OF RECOMBINANT DNA

TECHNOLOGY

Recombinant DNA technology involves several

steps in specific sequence such as isolation of
DNA, fragmentation of DNA by restriction
endonucleases, isolation of a desired DNA
fragment, ligation of the DNA fragment into a
vector, transferring the recombinant DNA into the
host, culturing the host cells in a medium at large
scale and extraction of the desired product.

Isolation of the Genetic Material (DNA)

Recall that nucleic acid is the genetic material of

all organisms without exception. In majority of
organisms this is deoxyribonucleic acid or DNA. In
order to cut the DNA with restriction enzymes, it
needs to be in pure form, free from other
macro-molecules. Since the DNA is enclosed
within the membranes, we have to break the cell
open to release DNA along with other
macromolecules such as RNA. proteins.
polysaccharides and also lipids. This can be
achieved by treating the bacterial cells/plant or
animal tissue with enzymes such as lysozyme
(bacteria), cellulase (plant cells), chitinase
(fungus).
 DNA that separates out can be removed by
spooling
Cutting of DNA at Specific Locations

The joining of DNA involves several processes.

After having cut the source DNA as well as the
vector DNA with a specific restriction enzyme. the
cut out 'gene of interest' from the source DNA and
the cut vector with space are mixed and ligase is
added. This results in the preparation of
recombinant DNA.

Restriction enzyme digestions are performed by

incubating purified DNA molecules with the
restriction enzyme, at the optimal conditions for
that specific enzyme. Agarose gel electrophoresis
is employed to check the progression of a
restriction enzyme digestion. DNA is a negatively
charged molecule, hence it moves towards the
positive electrode (anode) (Figure 11.3). The
process is repeated with the vector DNA also.
Amplification of Gene of Interest using PCR
PCR stands for Polymerase Chain Reaction.
In this reaction, multip copies of the gene (or
DNA) of interest is synthesised in vitro using two
sets of primers (small chemically synthesised
oligonucleotides that are complementary to the
regions of DNA) and the enzyme DNA
polymerase The enzyme extends the primers
using the nucleotides provided in the reaction and
the genomic DNA as template. (If the process of
replication of DNA is repeated many times, the
segment of DNA can be amplified to
approximately billion times, i.e., 1 billion copies
are made. (Such repeated amplification is
achieved 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

There are several methods of introducing the

ligated DNA into recipient cells. Recipient cells
after making them competent to receive, take up
DNA present in its surrounding. So, if a
recombinant DNA bearing gege for resistance to
an antibiotic (e.g.. ampicillin) is transferred into E.
coll cells, the host cells become transformed into
ampicillin-resistant cells. If we spread the
transformed cells on agar plates containing
ampicillin, only transformants will grow,
untransformed recipient cells will die. Since, due
to ampicillin resistance gene, one is able to select
a transformed cell in the presence of ampicillin.
The ampicillin resistance gene in this case is
called a selectable marker
Obtaining the Foreign Gene Product
In recombinant technologies, the desired gene
selected which is followed by selecting a perfect
vector into which the desired gene has to be
integrated and then recombinant DNA is formed
by ligating the gene of interest with the vector.
Once this foreign DNA is inserted, the host is
multiplied and ultimately desirable protein is
produced. The rDNA has to be maintained in the
host and carried forward to the offspring. For the
production of the desired protein, the gene
encodes for it needs to be expressed. This
happens only under optimized conditions. Not
only the target protein has to be expressed but
has to be produced on a large scale.

The recombinant cells can be multiplied on a

large scale using a continuous culture system.
Here the cells are cultured in a large vessel and
the medium is refreshed on a regular interval to
maintain the optimum conditions. This helps to
culture a large mass of the desired protein. This
can be achieved by using a bioreactor.
Bioreactor
A bioreactor helps to produce a large volume of
culture. The bioreactor is a large vessel where the
different cells such as human or plant, or animal
cells can be cultured to obtain new biological
products. It provides optimum conditions like
temperature, pH, substrate, oxygen, etc required
for the culturing of cells producing desired
products. Simple stirred-tank bioreactor and
sparged stirred-tank bioreactor are the two types
of bioreactors used for this purpose.

Simple stirred-tank bioreactor

Downstream Processing
Downstream processing is a sequential step in
which the isolation, purification and preservation
of final products are done before it is marketed. In
this stage, the final product is formulated with
additives like preservatives, colours, etc., followed
by clinical trials.