🧬

Biotechnology
Introduction
Biotechnology deals with the techniques of using living organisms or
enzymes from organisms to produce products useful to humans.

The processes like in vitro fertilization leading to a ‘test-tube’ baby,

synthesizing a gene and using it, developing a DNA vaccine or correcting a
defective gene, are all parts of biotechnology.

Biotechnology can be defined as- ‘The integration of natural science and

organisms, cells, parts thereof, and molecular analogues for products and
services’.

Biotechnology 1
 Fig. test tube baby
Principles of biotechnology
The two core techniques that enabled birth of modern biotechnology are –

1. Genetic engineering
2. Maintenance of sterile conditions.

Genetic engineering is the technique of altering the chemistry of DNA and

RNA so that it can be introduced into the host organism to change the
phenotype of the host organism.

Sterile conditions should be maintained to enable growth of only the

desired microbe or eukaryotic cell in large quantities for the manufacture
antibiotics, vaccines, enzymes, etc.

Hybridization procedures 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 allows us to
isolate and introduce only one or a set of desirable genes without
introducing undesirable genes into the target organism.

Three basic steps in genetically modifying an organism —

Identification of DNA with desirable genes

Introduction of the identified DNA into the host

Biotechnology 2
 Maintenance of introduced DNA in the host and transfer of the DNA to its
progeny.

Fig. genetic engineering

Tools of recombinant DNA technology

Important tools of recombinant DNA technology are-

Restriction enzymes- Restriction enzymes are called as molecular scissors

because these enzymes cut DNA at specific sites.

Cloning vector- Plasmids and bacteriophages have the ability to replicate

within bacterial cells independent of the control of chromosomal DNA.

Competent host- The host should be competent enough to take up the

foreign DNA.

Bioreactors- Bioreactor is the cylindrical vessel in which biological

processes is carried out on a large scale.

Biotechnology 3
 Fig. recombinant DNA technology

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

Restriction enzymes are called as molecular scissors because these

enzymes cut DNA at specific sites.

The first restriction endonuclease is Hind II.

The restriction enzymes cut DNA at specific base sequence, and these
specific base sequence is known as the recognition sequence.

The convention for naming restriction enzymes –

The first letter of the name comes from the genus.

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.

Roman numbers following the names indicate the order in which the
enzymes were isolated from that strain of bacteria.

900 restriction enzymes that have been isolated from over 230 strains of
bacteria.

These are of two kinds

1. Exonucleases

2. Endonucleases

Exonucleases remove nucleotides from the ends of the DNA whereas,

endonucleases make cuts at specific positions within the DNA.

Each restriction endonuclease recognizes a specific palindromic nucleotide

sequences in the DNA.

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

Example- the following sequences reads the same on the two strands in 5' à 3'
direction, this is also true if read in the 3' à 5' direction.

5' —— GAATTC —— 3’
3' —— CTTAAG —— 5'

Biotechnology 4
 Restriction enzymes cut the strand of DNA a little away from the center of
the palindrome sites, but between the same two bases on the opposite
strands which leaves a single stranded portions at the ends and the
overhanging stretches called sticky ends on each strand.

When cut by the same restriction enzyme, the resultant DNA fragments
have the same kind of ‘sticky-ends’ and, these can be joined together using
DNA ligases.

Fig. restriction digestion

Cloning vectors
A cloning vector is a small piece of DNA, taken from any organism into
which a foreign DNA fragment can be inserted for cloning purposes.

Plasmids and bacteriophages have the ability to replicate within bacterial

cells independent of the control of chromosomal DNA.

If 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.

The following are the features that are required to facilitate cloning into a vector
are-

Origin of replication (ori)

Selectable marker

Cloning sites

Origin of replication (ori)- This is the sequence from where replication starts
and any piece of DNA when linked to this sequence can be made to replicate

Biotechnology 5
 within the host cells

Selectable marker-

It 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.

The genes encoding resistance to antibiotics such as ampicillin,

chloramphenicol, tetracycline or kanamycin, etc., are useful selectable
markers for E. coli as the normal E. coli cells do not carry resistance against
any of these antibiotics.

Antibiotic resistance genes help in selecting recombinants from non-

recombinants by a method called insertional inactivation where a
recombinant DNA is inserted within the coding sequence of an enzyme β-
galactosidase in the presence of a chromogenic substrate which results
into inactivation of the enzyme.

The presence of a chromogenic substrate gives blue colored colonies if the

plasmid in the bacteria does not have an insert.

Presence of insert results into insertional inactivation of the β-galactosidase

and the colonies do not produce any colour, these are identified as
recombinant colonies.

Cloning sites-

Cloning sites are the recognition sites of the restriction enzymes.

The ligation of alien DNA is carried out at a restriction site present in one of
the two antibiotic resistance genes.

For example- ligation of a foreign DNA at the Bam H I site of tetracycline

resistance gene in the vector pBR322.

Biotechnology 6
 Fig. pBR322

Vector for cloning genes in plants is Agrobacterioum tumifaciens, a

pathogen of several dicot plants which delivers 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 Agrobacterium tumifaciens has now

been modified into a cloning vector.

Fig. tumor formation by Agrobacterium tumifaciens

Vector for cloning genes in animals is retrovirus which transforms normal

cells into cancerous cells.

Retroviruses have been disarmed and used to deliver desirable genes into
animal cells.

Competent host
Since DNA is a hydrophilic molecule, it cannot pass through cell
membranes so the bacterial cells must first be made ‘competent’ to take up
DNA.

Several methods are followed to make the bacterial cells competent-

Treating them with a specific concentration of a divalent cation, such as

calcium, which increases the efficiency with which DNA enters the

Biotechnology 7
 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 42oC
(heat shock), and then putting them back on ice which enables the bacteria
to take up the recombinant DNA.

Recombinant DNA can be directly injected into the nucleus of an animal cell
by a method called micro-injection.

In biolistics or gene gun method, cells are bombarded with high velocity
micro-particles of gold or tungsten coated with DNA

Disarmed pathogen vectors can be allowed to infect the cell to transfer the
recombinant DNA into the host.

Fig. DNA is injected into the nucleus of the host cell by the process of micro-
injection.

Bioreactors
Bioreactor is the cylindrical vessel in which biological processes is carried
out on a large scale.

The recombinant cells can be multiplied in a continuous culture system

wherein the used medium is drained out from one side while fresh medium
is added from the other to maintain the cells.

Bioreactors vessels in which raw materials are biologically converted into

specific products, individual enzymes, etc., using microbial plant, animal or
human cells.

Biotechnology 8
 A bioreactor provides the optimal conditions for achieving the desired
product by providing optimum growth conditions such as temperature, pH,
substrate, salts, vitamins, oxygen.

Bioreactors are of two types-

1. Simple stirred tank bioreactor

2. Sparged stirred-tank bioreactor

A stirred-tank reactor is usually cylindrical or with a curved base to

facilitate the mixing of the reactor contents and the stirrer facilitates even
mixing and oxygen availability throughout the bioreactor.

In sparged stirred-tank bioreactor sterile air is sparged through the reactor.

The bioreactor has an agitator system, an oxygen delivery system and a

foam control system, a temperature control system, pH control system and
sampling ports so that small volumes of the culture can be withdrawn
periodically.

Fig. simple stirred-tank bioreactor and sparged stirred-tank bioreactor

Process of recombinant DNA technology

Biotechnology 9
 Isolation of the Genetic Material (DNA)-

The cells are broken and opened to release DNA along with other
macromolecules such as RNA, proteins, polysaccharides and also lipids
which can be achieved by treating the cells with enzymes such as
lysozyme (bacteria), cellulase (plant cells), chitinase (fungus).

The RNA can be removed by treatment with ribonuclease whereas proteins

can be removed by treatment with protease and purified DNA ultimately
precipitates out after the addition of chilled ethanol which can be seen as
collection of fine threads in the suspension.

Fig. DNA precipitate

Cutting of DNA at Specific Location-

Restriction enzyme digestions are performed by incubating purified DNA

molecules with the restriction enzyme, at the optimal conditions for that
specific enzyme which results in the fragments of DNA.

The fragments are separated by a technique known as gel electrophoresis.

Since DNA fragments are negatively charged molecules they can be

separated by forcing them to move towards the anode under an electric
field through agarose.

The DNA fragments separate according to their size through sieving effect
provided by the agarose gel.

The smaller the fragment size, the farther it moves and the separated DNA
fragments can be visualized only after staining the DNA with a compound
known as ethidium bromide followed by exposure to UV radiation.

Bright orange coloured bands of DNA can be observed in an 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 by the process known as

Biotechnology 10
 Fig . DNA bands
Amplification of Gene of Interest using PCR

PCR stands for Polymerase Chain Reaction.

Multiple copies of the gene of interest is synthesized in vitro using two sets
of primers and the enzyme DNA polymerase.

Primers are small chemically synthesized oligonucleotides that are

complementary to the regions of DNA.

PCR includes three major steps-

1. Denaturation
2. Annealing

3. Extension

Denaturation is the process of heating of target DNA at 94oC to seperate

the two strands of DNA.

Annealing is the process of pairing of primers with complimentary base

sequences of the two separated strands.

Extension is the process of adding complimentary deoxyribonucleotides

one by one to the 3/ OH ends of primers by the activity of DNA polymerase
and as a result new DNA strand is synthesized.

If the process of replication of DNA is repeated many times, the segment of

DNA can be amplified to approximately billion times by the use of a
thermostable DNA polymerase isolated from a bacterium, Thermus
aquaticus.

The amplified fragment can be used to ligate with a vector for further
cloning.

Biotechnology 11
 Fig. polymerase chain reaction
Insertion of Recombinant DNA into the Host Cell/Organism

Recipient cells after making them ‘competent’ to receive, take up DNA

present in its surrounding.

If a recombinant DNA bearing gene for resistance to an antibiotic is

transferred into E. coli cells, the host cells become transformed into
ampicillin-resistant cell.

Obtaining the Foreign Gene Product

The foreign gene when gets expressed under appropriate conditions,

produces desirable proteins.

If any protein encoding gene is expressed in a heterologous host, is called a

recombinant protein.

The cells harboring cloned genes of interest may be grown on a small scale
in the laboratory or on a large scale in a bioreactor.

Downstream Processing

Downstream processing is the separation and purification of the product.

The product has to be formulated with suitable preservatives and the

formulation has to undergo thorough clinical trials.

Biotechnology 12