11.

BIOTECHNOLOGY: PRINCIPLES & PROCESSES

Biotechnology deals with techniques of using live organisms Exonucleases
or their enzymes for products and processes useful to humans. They remove nucleotides from the ends of the DNA.
The European Federation of Biotechnology (EFB) Endonucleases
defines Biotechnology as the integration of natural science - They cut at specific positions within the DNA. (For
and organisms, cells, parts thereof, and molecular figures see TB page: 196).
analogues for products and services. - Each restriction endonuclease can bind to specific
Biotechnology deals with: recognition sequence of the DNA and cut each of the two
- Microbe-mediated processes (making curd, bread, wine etc). strands at specific points in their sugar-phosphate
- In vitro fertilisation (test-tube baby programme) backbones. Each restriction endonuclease recognizes a
- Synthesis and using of a gene specific palindromic nucleotide sequences in the DNA.
- Preparation of a DNA vaccine - The palindrome in DNA is a sequence of base pairs that
- Correcting a defective gene read the same on the two strands in 5' 3' direction and
PRINCIPLES OF BIOTECHNOLOGY in 3' 5' direction. E.g.
The two core techniques of modern biotechnology are: 5' GAATTC 3'
a. Genetic engineering: The technique in which the genetic 3' CTTAAG 5'
material (DNA & RNA) is chemically altered and - Restriction enzymes cut the strand a little away from the
introduced into host organisms to change the phenotype. centre of the palindrome sites, but between the same two
b. Maintenance of sterile ambience: It is necessary in bases on the opposite strands. This leaves single stranded
chemical engineering processes for growing only the overhanging stretches at the ends. They are called sticky
desired microbe/ eukaryotic cell in large quantities for ends. They form H-bonds with their complementary cut
the manufacture of antibiotics, vaccines, enzymes, etc. counterparts. This stickiness facilitates action of the
- Traditional hybridisation techniques lead to inclusion and enzyme DNA ligase. (For figure see TB page: 197).
multiplication of undesirable genes along with desired - When cut by the same restriction enzyme, the resultant
genes. Genetic engineering helps to isolate and introduce DNA fragments have the same kind of sticky-ends and
only desirable genes into the target organism. these are joined together by DNA ligases.
- A piece of DNA is not able to multiply itself in the Separation and isolation of DNA fragments:
progeny cells of the organism. But, when it gets - DNA fragments formed by restriction endonucleases can
integrated into the recipient genome, it multiplies and be separated by a technique called gel electrophoresis.
inherits along with the host DNA. (For figure see TB page: 198).
- First recombinant DNA was emerged from the possibility - DNA fragments are negatively charged. So they can be
of linking a gene of antibiotic resistance with a native separated by moving them towards the anode under an
plasmid of Salmonella typhimurium. Stanley Cohen & electric field through a medium/matrix such as agarose
Herbert Boyer (1972) isolated the antibiotic resistance (a natural polymer extracted from sea weeds).
gene by cutting out a piece of DNA from a plasmid. - The DNA fragments separate (resolve) according to their
3 basic steps in genetically modifying an organism: size through sieving effect provided by the agarose gel.
a) Identification of DNA with desirable genes The smaller sized fragment move farther.
b) Introduction of the identified DNA into the host - The separated DNA fragments can be visualized after
c) Maintenance of introduced DNA in the host and staining the DNA with ethidium bromide followed by
transfer of the DNA to its progeny. exposure to UV radiation. Bright orange coloured DNA
bands can be seen.
TOOLS OF RECOMBINANT DNA TECHNOLOGY - The separated DNA bands are cut out from agarose gel
1. Restriction Enzymes (molecular scissors) and extracted from gel piece. This step is called elution.
- In 1963, two enzymes responsible for restricting the These purified DNA fragments are used in constructing
growth of bacteriophage in E. coli were isolated. One of recombinant DNA by joining them with cloning vectors.
these added methyl groups to DNA. The other 2. Cloning Vectors
(restriction endonuclease) cut DNA. - They are the DNA molecules that can carry a foreign
- The first restriction endonuclease is Hind II. It always DNA segment and replicate inside the host cells.
cuts DNA molecules at a particular point by recognizing E.g. Plasmids (circular extra-chromosomal DNA of
a specific sequence of six base pairs. This is known as bacteria) and bacteriophages.
the recognition sequence for Hind II. - Bacteriophages (high number per cell) have very high
- Today more than 900 restriction enzymes have been copy numbers of their genome within the bacterial cells.
isolated from over 230 strains of bacteria. Some plasmids have only 1-2 copies per cell. Others may
- Naming of the restriction enzymes: First letter indicates have 15-100 copies per cell.
genus and the second two letters indicate species of the - When the cloning vectors are multiplied in the host the
prokaryotic cell from which they were isolated. linked piece of DNA is also multiplied to the numbers
E.g. EcoRI comes from E. coli RY 13 (R = the strain. equal to the copy number of the vectors.
Roman numbers = the order in which the enzymes were
Features of cloning vector:
isolated from that strain of bacteria).
- Restriction enzymes belong to a class of enzymes called a. Origin of replication (ori): This is a sequence from
nucleases. They include exonucleases & endonucleases. where replication starts. A piece of DNA linked to ori

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 can replicate within the host cells. This also controls the The tumor inducing (Ti) plasmid of A. tumifaciens is
copy number of the linked DNA. So, for getting many modified into a cloning vector which is not pathogenic
copies of the target DNA it should be cloned in a vector to the plants but is able to use the mechanisms to
whose origin support high copy number. deliver genes of interest into plants.
b. Selectable marker (marker gene): It helps to select the Retroviruses in animals can transform normal cells into
transformants and eliminate the non-transformants. cancerous cells. So they are used to deliver desirable
Transformation is a procedure in which a piece of DNA genes into animal cells.
is introduced in a host bacterium. 3. Competent Host (For Transformation with
Selectable markers of E. coli include the genes encoding
Recombinant DNA)
resistance to antibiotics like ampicillin, chloramphenicol,
tetracycline or kanamycin, etc. The normal E. coli cells - DNA is a hydrophilic molecule. So it cannot pass
do not carry resistance against any of these antibiotics. through cell membranes.
c. Cloning sites: In order to link the alien DNA, the vector - To avoid this problem, bacterial cells are treated with a
needs very few recognition sites for restriction enzymes. specific concentration of a divalent cation (e.g. calcium).
Presence of more than one recognition sites generates So DNA enters the bacterium through pores in cell wall.
several fragments, which complicates the gene cloning. - Such cells are incubated with recombinant DNA on ice.
The ligation of alien DNA is carried out at a restriction Then they are placed briefly at 420C (heat shock) and put
site present in one of the two antibiotic resistance genes. them back on ice. This enables the bacteria to take up the
E.g. ligation of a foreign DNA at the Bam H I site of recombinant DNA.
tetracycline resistance gene in the vector pBR322. 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 is suitable for plants.
Disarmed pathogen vectors: which when infect the
cell, transfer the recombinant DNA into the host.
PROCESSES OF RECOMBINANT DNA TECHNOLOGY
1. Isolation of the Genetic Material (DNA)
- To get pure DNA (free from other macro-molecules), the
- The recombinant plasmids lose tetracycline resistance bacterial cells/plant or animal tissue are treated with
due to insertion of foreign DNA. But they can be selected enzymes such as lysozyme (bacteria), cellulase (plant
out from non-recombinant ones by plating the cells), chitinase (fungus) etc. The cell is broken to
transformants on ampicillin containing medium. release DNA along with other macromolecules (RNA,
- Then these transformants are transferred on tetracycline proteins, polysaccharides and lipids).
medium. The recombinants grow in ampicillin medium - Genes (DNA) are interwined with proteins such as
but not on tetracycline medium. But, non-recombinants histones. RNA is removed by treating with ribonuclease.
will grow on the medium containing both the antibiotics. Proteins are removed by treatment with protease. Other
- In this case, one antibiotic resistance gene helps to select molecules are removed by appropriate treatments.
the transformants, whereas the other antibiotic resistance - When chilled ethanol is added purified DNA precipitates
gene gets inactivated due to insertion of alien DNA, and out as a collection of fine threads in the suspension.
helps in selection of recombinants. 2. Cutting of DNA at Specific Locations
- Selection of recombinants due to inactivation of antibiotics - Restriction enzyme digestions are performed by
requires simultaneous plating on 2 plates having different incubating purified DNA with the restriction enzyme, at
antibiotics. Therefore, alternative selectable markers have the optimal conditions.
developed to differentiate recombinants from non- - Agarose gel electrophoresis is employed to check the
recombinants on the basis of their ability to produce colour progression of a restriction enzyme digestion. As DNA is
in the presence of a chromogenic substrate. negatively charged, it moves towards the anode. The
- A recombinant DNA is inserted within the coding process is repeated with the vector DNA also. (For figure
sequence of an enzyme, -galactosidase. So the enzyme see TB page: 198).
is inactivated. It is called insertional inactivation. Such - After cutting the source DNA and the vector DNA, the
colonies do not produce any colour. These are identified cut out gene (DNA segment) of interest from the source
as recombinant colonies. DNA and the cut vector are mixed and ligase is added.
- If the plasmid in bacteria have no an insert it gives blue This creates recombinant DNA.
coloured colonies in presence of chromogenic substrate. 3. Amplification of Gene of Interest using PCR
d. Vectors for cloning genes in plants and animals: - Polymerase Chain Reaction (PCR) is the synthesis of
Genetic tools of some pathogens can be transformed into multiple copies of the gene of interest in vitro using 2
useful vectors for delivering genes to plants & animals. E.g. sets of primers & the enzyme DNA polymerase.
Agrobacterioum tumifaciens (a pathogen of many dicot Primers are small chemically synthesized oligonucleotides
plants) can deliver a piece of DNA (T-DNA) to that are complementary to the regions of DNA.
transform normal plant cells into a tumor. These tumor - The enzyme extends the primers using the nucleotides
cells produce the chemicals required by the pathogen. and the genomic DNA (template). Through continuous

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 DNA replication, the DNA segment is amplified up to 1 produces a larger biomass leading to higher yields of
billion copies. (For figures see TB page: 202). desired protein.
- For amplification a thermostable DNA polymerase Bioreactors
(isolated from a bacterium, Thermus aquaticus) is used. - To produce large quantities of products, the bioreactors
It remains active in high temperature during the are used where large volumes (100-1000 litres) of culture
denaturation of double stranded DNA. can be processed.
- The amplified fragment can be used to ligate with a - Bioreactors are the vessels in which raw materials are
vector for further cloning. biologically converted into specific products, enzymes
4. Insertion of Recombinant DNA into the Host etc., using microbial plant, animal or human cells.
Cell/Organism - A bioreactor provides the optimal growth conditions
- There are several methods of introducing the ligated (temperature, pH, substrate, salts, vitamins, oxygen) for
DNA into recipient cells. Recipient cells take up DNA achieving the desired product.
present in its surrounding. - The most commonly used bioreacters are of stirring type
- If a recombinant DNA bearing ampicillin resistant gene (stirred-tank reactor) (For figures see TB page: 204).
(a selectable marker gene) is transferred into E. coli cells, It is usually cylindrical or with a curved base to facilitate
the host cells become ampicillin-resistant cells. the mixing of the reactor contents. The stirrer facilitates
- If the transformed cells are spread on agar plates even mixing and oxygen availability. Alternatively air
containing ampicillin, only transformants will grow, can be bubbled through the reactor. The bioreactor has
untransformed recipient cells will die. An agitator system
An oxygen delivery system
5. Obtaining the Foreign Gene Product A foam control system
- The ultimate aim of recombinant DNA technology is to A temperature control system
produce a desirable protein. The foreign gene gets pH control system
expressed under appropriate conditions. Sampling ports (for periodic withdrawal of the culture).
- If a protein encoding gene is expressed in a heterologous
host, it is called a recombinant protein. 6. Downstream Processing
- The cells with foreign genes may be grown on a small - It is a series of processes such as separation and
scale in the laboratory. The cultures may be used to purification of products after the biosynthetic stage.
extract the desired protein and purify it by using different - The product is formulated with suitable preservatives.
separation techniques. Such formulation undergoes thorough clinical trials as in
- The cells can also be multiplied in a continuous culture case of drugs. Strict quality control testing for each
system. Here, the used medium is drained out from one product is also required.
side while fresh medium is added from the other. It - The downstream processing and quality control testing
maintains the cells more physiologically active and so vary from product to product.

Prepared by:
K.C. MUHAMMED ALI
mailtokcm@gmail.com
bankofbiology.blogspot.in