CHAPTER

11
biotechnology:
principles & Processes
 • Biotechnology is the technique of using live organisms or their enzymes
for products & processes useful to humans. The European Federation of
Biotechnology (EFB) defines Biotechnology as ‘the integration of natural
science and organisms, cells, parts thereof, and molecular analogues for
products and services’.

Biotechnology deals with:

• Microbe-mediated processes(making curd, bread, wine etc).
• In vitro fertilization (test-tube baby programme).
• Synthesis and using of a gene.
• Preparation of DNA vaccine.
• Correcting a defective gene�

PRINCIPLES OF BIOTECHNOLOGY
1. Core techniques of modern biotechnology
• Genetic engineering: The technique in which genetic material (DNA & RNA)
is chemically altered and introduced into host organisms
to change the phenotype.
• Maintenance of sterile ambience: It is necessary is chemical engineering
processes for growing desired microbe/
eukaryotic cell for the manufacture of antibiotics, vaccines, enzymes etc.

2. Basic steps in genetically modifying an

organism
a) Identification of DNA with desirable genes: Traditional hybridisation
techniques lead to inclusion and
multiplication of undesirable genes along with desired genes. Genetic
engineering helps to isolate and introduce only desirable genes into the
target organism.
b) Introduction of the identified DNA into the host: A vector DNA such as plasmid
is used to deliver an alien piece
of DNA into the host organism.
c) Maintenance of introduced DNA in the host and transfer of the DNA to its
progeny: A piece of alien DNA has no the sequence called Origin of replication
(ori) needed for starting replication. So, it cannot multiply itself in the progeny
cells of the organism. Hence alien DNA is integrated into the recipient
genome (it has ori). It multiplies & inherits along with host DNA.

First recombinant DNA (rDNA) was produced by Stanley Cohen & Herbert
Boyer (1972). They isolated an antibiotic resistance gene by cutting out a
DNA piece from a plasmid. This gene was linked with a native plasmid of
Salmonella typhimurium

TOOLS OF RECOMBINANT DNA

TECHNOLOGY
1. Restriction Enzymes (‘molecular scissors’)
• These are the enzymes which cut DNAat specific sites into fragments.
• They belong to a class of enzymes called nucleases.
• In 1963, two enzymes responsible for restricting growth of bacteriophage
in E. coli were isolated. One enzyme added methyl groups to DNA. The
other (restriction endonuclease) cut DNA.
• More than 900 restriction enzymes have been isolated from over 230
strains of bacteria.

Naming of the restriction enzymes:

• First letter indicates genus and the second two letters indicate species
of the prokaryotic cell from which they were isolated.
E.g. EcoRI comes from E. coli RY 13 (R = the strain. Roman numbers = the
order in which the enzymes were isolated from that strain of bacteria).

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 Types of Restriction enzymes:
• Exonucleases: They remove nucleotides from the ends of the DNA.
• Endonucleases:
• They cut at specific positions within theDNA.
• They bind to specific recognition sequence of the DNA and cut the
two strands at specificpoints.
• The first restriction endonuclease is Hind II. It cuts DNA molecules by
recognizing a specific sequence of 6 base pairs. This is called the
recognition sequence for Hind II�

• Restriction endonuclease recognizes a specific palindromic nucleotide sequences

in the DNA. It is a sequence of base pairs that read the same on the two
strands in 5' – 3'
direction and in 3' – 5' direction. E.g.
5' —— GAATTC —— 3'
3' —— CTTAAG —— 5
• Restriction enzymes cut the strand a little away from the centre of the palindrome
sites, but between the same two bases on the opposite strands. This leaves
single stranded overhanging stretches at the ends. They are called sticky
ends. They form H-bonds with their complementary cut counterparts. This stickiness
facilitates action of the enzyme DNA ligase.
• When cut by the same restriction enzyme, the resultant DNA fragments have
the same kind of sticky-ends and these are joined together by DNA ligases.

Separation and isolation of DNA fragments:

• DNA fragments are separated by a technique called gel
• DNA fragments can be seen as bright orange coloured bands when they
are stained with ethidium bromide and exposed to UV radiation.
• DNA bands are cut out from agarose gel. This is called elution. These
purified DNA are used to construct recombinant DNA by joining
them with cloning vectors.

2. Cloning Vector
• It is a DNA molecule that can carry a foreign DNA segment and
replicate inside the hostcells. E.g. Plasmids, bacteriophages etc.
• Plasmids are autonomously replicating circular extra�chromosomal
DNA of bacteria. Some plasmids have only 1-2 copies per cell.
Others have 15-100 copies per cell.
• Bacteriophages (high number per cell) have very high copy numbers
of their genome within the bacterialcells.
• When the cloning vectors are multiplied in the host, the linked piece
of DNA is also multiplied to the numbers equal to the copy number
of the vectors.

Features required for cloning into a vector

a) Origin of replication (ori)

• This is a sequence where replicationstarts.
• A piece of DNA linked to ori can replicate within the host cells.
This also controls the copy number of linked DNA. So, for getting
many copies of the target DNA, it should be cloned in a vector whose
origin support high copy number.

b. Selectable marker (marker gene)

• It is a gene that helps to select the transformants and eliminate
the non-transformants.
• Transformation is a procedure through which a piece of DNA is
introduced in a host bacterium. Such bacterium is called
transformant. If transformation does not take place, it is
non-transformant.
• Selectable markers of E. coli include the genes encoding resistance
to antibiotics like ampicillin, chloramphenicol, tetracycline, kanamycin
etc. Normal E. coli cells have no resistance against these antibiotics.

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 c. Cloning sites
• To link the alien DNA, the vector needs a single or very few
recognition sites for restrictionenzymes.
• More than one recognition sites generate several fragments. It
complicates the gene cloning.
• Ligation of alien DNA is carried out at a restriction site present
in one of the two antibiotic resistance genes.
E.g. ligation of foreign DNA at Bam H I site of tetracycline resistance
gene in vector pBR322. As a result, recombinant plasmid is formed.
If ligation does not occur, it is called non-recombinant plasmid.
• Restriction sites: Hind III, EcoR I, BamH I, Sal I, Pvu II, Pst I, Cla I. ori
• Antibiotic resistance genes: ampR and tetR. Rop: codes for the
proteins involved in the replication of
plasmid.
• The recombinant plasmids lose tetracycline resistance due to insertion
of foreignDNA.
• When the plasmids are introduced into E. coli cells, 3 types of cells
are obtained:
o Non-transformants: They have no plasmid. So they are not resistant
to either tetracycline or ampicillin.
o Transformants with non-recombinant plasmid: They are resistant to
both tetracycline & ampicillin.
o Transformants with recombinant plasmid: They are resistant only toampicillin.
• Recombinant plasmids can be selected out from non�recombinant ones
by plating transformants on ampicillin medium. Then the transformants
are transferred on tetracycline medium.
• The recombinants grow in ampicillin medium but not on tetracycline
medium. But, non-recombinants grow on the medium containing both
the antibiotics.
• Thus, one antibiotic resistance gene helps to select the transformants.
The inactivated antibiotic resistance gene helps to selectrecombinants.
• Selection of recombinants due to inactivation of antibiotics requires
simultaneous plating on 2 plates having different antibiotics. Therefore,
alternative selectable markers have developed to differentiate
recombinants from non recombinants based on their ability to produce
colour in the presence of achromogenic substrate.
• In this, a recombinant DNA is inserted within the coding sequence of an
enzyme, þ -galactosidase. So, the enzyme is inactivated. It is called
insertional inactivation. Such colonies do not produce any colour. These
are identified as recombinant colonies.
• If the plasmid in bacteria have no an insert, it gives blue coloured colonies
in presence of chromogenic substrate.

d. Vectors for cloning genes in plants & animals

• Genetic tools of some pathogens can be transformed into useful vectors
for delivering genes to plants & animals.
E.g. Agrobacterium tumifaciens (a pathogen of many dicot plants) can
deliver a piece of DNA (T-DNA) to transform normal plant cells into a
tumor. These tumor cells produce the chemicals required by the pathogen.
The tumor inducing (Ti) plasmid of A. tumifaciens is modified into a
cloning vector which is not pathogenic to the plants but is able to use
the mechanisms to deliver genes of interest into plants.
Retroviruses in animals can transform normal cells into cancerous cells.
So, they are used to deliver desirable genes into animal cells.�

3. Competent Host (For Transformation with

Recombinant DNA)
• DNA is a hydrophilic molecule. So, it cannot pass through cell membranes.
• To avoid this problem, bacterial cells are treated with a specific concentration
of a divalent cation (e.g. calcium). So, DNA enters the bacterium through
pores in cell wall.
• Such cells are incubated with recombinant DNA on ice. Then they are placed
briefly at 420C (heat shock) and put them back on ice. This enables the bacteria
to take up recombinant DNA.

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 Other methods to introduce alien DNA into host cells
• Micro-injection : In this, recombinant DNA is directly injected into the
nucleus of an animal cell.
• Biolistics (gene gun) : In this, cells are bombarded with high velocity
micro-particles of gold or tungsten coated with DNA.
This method issuitable for plants.
• Disarmed pathogen’ vectors : They infect the cell and transfer the \
recombinant DNA into the host.

PROCESSES OF RECOMBINANT
DNA TECHNOLOGY
1. Isolation of the Genetic Material (DNA)
• The bacterial cells/plant or animal tissue are treated with enzymes like
lysozyme (bacteria), cellulase (plants), chitinase (fungus) etc. The cell
is broken releasing DNA & other macromolecules (RNA, proteins,
polysaccharides and lipids).
• RNA is removed by treating with ribonuclease. Proteins are removed by
treatment with protease. Other molecules are removed by appropriate
treatments.
• When chilled ethanol is added, purified DNA precipitates out as a collection
of fine threads in the suspension.

2. Cutting of DNA at Specific Locations

• Purified DNA is incubated with the restriction enzyme at optimal conditions.
As a result, DNA digests.
• Agarose gel electrophoresis is employed to check the progression of a restriction
enzyme digestion. DNA is negatively charged. So it moves towards the anode. The
DNA fragments separate according to their size through sieving effect of the
agarose gel (a polymer extracted from sea weeds). The smaller sized
fragment moves farther.
• The process isrepeated with the vector DNAalso.
• After cutting the source DNA and vector DNA, the cut-out gene of interest from
source DNA and cut vector aremixed and ligase is added. It creates recombinant DNA.

3. Amplification of Gene of Interest using PCR

• Polymerase Chain Reaction (PCR) is the synthesis of multiple copies of the gene of
interest in vitro using 2 sets of primers & the enzyme DNA polymerase.
• Primers are small chemically synthesized oligo nucleotides that are complementary
to the regions of DNA.
• The enzyme extends the primers using the nucleotides and genomic DNA (template).
Through continuous replication , the DNA segment is amplified up to 1 billion copies.
• For amplification, a thermostable DNA polymerase (isolated from a bacterium,
Thermus aquaticus) is used. It remains active in high temperature during the
denaturation of double stranded DNA.
• The amplified fragment can be used to ligate with a vector for further cloning.

4. Insertion of Recombinant DNA into HostCell

• Using any methods, the ligated DNA is introduced into recipient cells. They take up
DNA from its surrounding.
• If a recombinant DNA bearing ampicillin resistant gene is transferred into E. coli cells,
the host cells become ampicillin-resistant cells.
• If the transformed cells are spread on agar plates containing ampicillin, only
transformants will grow. Untransformed recipient cells will die.

5. Obtaining the Foreign Gene Product

• The aim of recombinant DNA technology is to produce a desirable protein.
• If a protein encoding foreign gene is expressed in a heterologous host, it is
called a recombinant protein.
• The cells with foreign genes can be grown in laboratory. The cultures are used to
extract the desired protein and purify it by using separation techniques.
• The cells can also be multiplied in a continuous culture system. Here, the used
medium is drained out from one side while fresh medium is added from the other. It
maintains the cells more physiologically active and so produces a larger biomass.
It yields more desired protein.

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 Bioreactors
• These are the vessels in which raw materials are biologically converted
to specific products, enzymes etc., using microbial plant, animal or
human cells.
• Bioreactors are used to produce large quantities of products. They can
process 100-1000 litres of culture.
• A bioreactor provides the optimal growth conditions (temperature, pH, substrate,
salts, vitamins, oxygen) for achieving the desired product.
• The most commonly used bioreactors are of stirring type (stirred-tank reactor).
It is usually cylindrical or with a curved base to facilitate the mixing of the
reactor contents. The stirrer facilitates even mixing and oxygen availability.
Alternatively, air can be bubbled through the reactor.

The bioreactor has

• An agitator system
• An oxygen delivery system
• A foam controlsystem
• A temperature controlsystem
• pH controlsystem
• Sampling ports (for periodic withdrawal of the culture).

6. Downstream Processing
• It is a series of processes such as separation and purification of products
after the biosynthetic stage.
• The product is formulated with suitable preservatives. Such formulation
undergoes thorough clinical trials and strict quality control testing.

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