Ch-9 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 use of a gene.
• Preparation of DNA vaccine.
• Correcting a defective gene.

PRINCIPLES OF BIOTECHNOLOGY
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.
• Bioprocess engineering: Maintenance of sterile ambience in chemical engineering processes for
growing desired microbe/eukaryotic cells for the manufacture of antibiotics, vaccines, enzymes etc.

Basic steps in genetically modifying an organism

• Identification of DNA with desirable genes: Traditional hybridisation leads to the inclusion and multiplication of
undesirable genes along with desired genes. In genetic engineering, only desirable genes are introduced.
• 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.
• 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.
• The process of joining and inserting a foreign piece of DNA into a host organism to produce new genetic
combinations is called recombinant DNA technology.
• First recombinant DNA (rDNA) was produced by Stanley Cohen & Herbert Boyer (1972).
• They isolated an antibiotic resistance gene (a piece of DNA) from a plasmid of Salmonella typhimurium. It was
linked with a plasmid vector and transferred into E. coli. As a result, the gene was expressed & multiplied in E. coli.

TOOLS OF RECOMBINANT DNA TECHNOLOGY

1. Restriction Enzymes (‘molecular scissors’)
• The enzymes that cut DNA at specific sites into fragments.
• They belong to a class of enzymes called nucleases.
• In 1963, two enzymes responsible for restricting the 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:

➢ The first letter indicates the genus. The second two letters indicate species of prokaryotic cells 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).
Types of Restriction Enzymes:
• Exonucleases: They remove nucleotides from the ends of the DNA.
• Endonucleases:
➢ They cut at specific positions within the DNA. E.g. EcoRI.
➢ They bind to specific recognition sequences of the DNA and cut the two strands at specific points.
➢ 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 sequence in the DNA. It is
a sequence of base pairs that read the same on the two strands in the 5' → 3' direction and in the 3' →
5' direction. E.g. Palindromic nucleotide sequence for EcoRI is

5' —— GAATTC —— 3'

3' —— CTTAAG —— 5'

Steps in the formation of recombinant DNA by EcoRI

• 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 the 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.
 2. Cloning Vector
• It is a DNA molecule that can carry a foreign DNA segment and replicate inside the host cells.
o 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
bacterial cells.
• 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 replication starts.

• A piece of DNA linked to ori can replicate within the host cells. This also controls the copy number of
linked DNA. So, to get many copies of the target DNA, it should be cloned in a vector whose origin
supports a high copy number.

b. Selectable marker (marker gene)

• It is a gene that helps to select the transformants and eliminate the non-transformants.
• If a piece of DNA is introduced in a host bacterium, it is called transformation. Such bacterium is
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.

c. Cloning sites

• These are the recognition sites for restriction enzymes.

• To link the alien DNA, the vector needs a single or very few recognition sites.
• More than one recognition sites generate several fragments. It complicates gene cloning.
• Ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic-
resistance genes.
. In vector pBR322, foreign DNA is ligated at the Bam H I site of the tetracycline resistance gene. As a
ult, recombinant plasmid is formed. If ligation does not occur, it is called a 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.

➢ When a foreign DNA is inserted within a gene of bacteria, that gene is inactivated. It is
called insertional inactivation. Here, the recombinant plasmids lose tetracycline resistance due to the
insertion of foreign DNA.
➢ When the plasmids are introduced into E. coli cells, 3 types of cells are obtained:

▪ Non-transformants: They have no plasmid. So, they are not resistant to either tetracycline or
ampicillin.
▪ Transformants with non-recombinant plasmid: They are resistant to both tetracycline &
ampicillin.
▪ Transformants with recombinant plasmid: They are resistant only to ampicillin.

• Recombinant plasmids can be selected from non-recombinant ones by plating transformants

on ampicillin medium. Then the transformants are transferred on a tetracycline medium.
• The recombinants grow in ampicillin medium but not in tetracycline medium. But, non-
recombinants grow on the medium containing both antibiotics.
• Thus, one antibiotic resistance gene helps to select the transformants. The inactivated antibiotic
resistance gene helps to select recombinants.
• But this type of selection of recombinants is a difficult procedure because it needs simultaneous plating
on 2 plates having different antibiotics. So, alternative selectable markers have developed based on
their ability to produce colour in the presence of a chromogenic substrate.
• In this, recombinant DNA is inserted into the coding sequence (gene) of an enzyme, b-
galactosidase. So, the gene is inactivated (insertional inactivation). Such colonies do not produce any
colour. These are identified as recombinant colonies.
• If the plasmid in bacteria has no insert, it gives blue-coloured colonies in the 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 tumefaciens (a pathogen of many dicot plants) can deliver a piece of DNA (T-DNA)
to transform normal plant cells into a tumour. These tumour cells produce the chemicals required by
the pathogen. The tumour-inducing (Ti) plasmid of A. tumefaciens is modified into a cloning vector
which is not pathogenic but can use 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)

• Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. So bacterial cells are
made ‘competent’ to take up alien DNA or plasmid as follows:
➢ Treat bacterial cells with a specific concentration of a divalent cation (e.g. calcium) → DNA enters
the bacterium through pores in cell wall → Incubate the cells with recombinant DNA on ice → Place
them briefly at 420C (heat shock) → Put them back on ice → Bacteria take up recombinant DNA.
 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: They infect the cell and transfer the recombinant DNA into the host.
E.g. A. tumefaciens.

PROCESSES OF RECOMBINANT DNA TECHNOLOGY

1. Isolation of the Genetic Material (DNA)

• Treat the bacterial cells/plant or animal tissue with

enzymes like lysozyme (bacteria), cellulase (plants), chitinase (fungus) etc. The cell is broken
releasing DNA & other macromolecules (RNA, proteins, polysaccharides & 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. As a result, DNA digests. These DNA
fragments are separated by a technique called gel electrophoresis.

• Agarose gel electrophoresis is employed to check the progression of restriction enzyme digestion.
DNA is negatively charged. So, it moves towards the anode. DNA fragments are separated according
to their size through the sieving effect of the agarose gel (a polymer extracted from seaweeds). The
smaller fragment moves farther.
• The process is repeated with the vector DNA also.
• 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. It is called elution. The cut-out gene of interest and
cut vector are mixed 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 oligonucleotides that are complementary to the regions of DNA.

Steps of PCR

• Denaturation: It is the heating of target DNA (gene of interest) at high temperature (940 C) to separate
the strands. Each strands act as a template for DNA synthesis.
• Annealing: It is the joining of the two primers (at 520 C) at the 3’ end of the DNA templates.
• Extension: It is the addition of nucleotides to the primer using a thermostable DNA polymerase called Taq
polymerase. It is isolated from a bacterium, Thermus aquaticus. It remains active in high temperatures during the
denaturation of double-stranded DNA.
• Through continuous replication, the DNA segment is amplified up to 1 billion copies.
• The amplified fragment can be used to ligate with a vector for further cloning.

4. Insertion of Recombinant DNA into the Host Cell

• Using any method, the ligated DNA is introduced into the recipient (host) cell/organism. They take up
DNA from its surroundings.
• 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 a foreign gene is expressed in a heterologous host, it is called a recombinant
protein.
• The cells with foreign genes can be grown in the 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 the 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.

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 (pH, temperature, substrate, salts, vitamins,
oxygen) to get the desired product.
• The most commonly used bioreactors are of stirring type (stirred-tank bioreactor).
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 control system
• A temperature control system
• pH control system
• 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.