Biotechnology

Term biotechnology was coined by Karl Ereky, a Hungarian Engineer in 1919
steps involved in recombinant DNA technology are
1. Isolation of a DNA fragment containing a gene of interest that needs to be

cloned.
2. Generation of recombinant DNA (rDNA) molecule by insertion of the DNA
fragment into a carrier molecule called a vector that can self-replicate within the
host cell.
3. multiplying the rDNA molecule.
4. The entire process thus generates a large amount of rDNA
PCR: Polymerase Chain Reaction is a common laboratory technique used to make

copies (millions) of a particular region of DNA.
The basic tools are

1. enzymes,
2. vectors
3. host organisms.
enzymes required for genetic engineering are

1. restriction enzymes,
2. DNA ligase
3. alkaline phosphatase
restriction enzymes
1. restriction endonuclease is an enzyme that cleaves DNA into fragments at or
near specific recognition
2. restriction enzymes are called as molecular scissors.
3. These act as foundation of recombinant DNA technology
restriction enzymes 2 types:

1. Exonucleases are enzymes which remove nucleotides - from the end of a DNA
molecule.
2. Endonucleases are enzymes which remove the internal bonds within a DNA
molecule
DNA ligase enzyme joins the sugar and phosphate molecules

Alkaline Phosphatase:
It is a DNA modifying enzyme and adds or removes specific phosphate group
Vector
Vector is a small DNA molecule capable of self-replication and is used as a carrier
and transporter of DNA fragment which is inserted into it for cloning experiments.
Following are the features that are required to facilitate cloning into a vector.
1. Origin of replication (ori)
2. Selectable marker
3. Cloning sites:
Types of vector
1. Plasmids
Plasmids are extra chromosomal, self replicating ds circular DNA molecules
2. pBR 322 Plasmid

B and R respectively the names of scientist Boliver and Rodriguez who
developed this plasmid. The number 322 is the number of plasmid developed
from their laboratory
3. Ti Plasmid
Ti plasmid is found in Agrobacterium tumefaciens, a bacterium
responsible for inducing tumours in several dicot plants. It is also known as the
natural genetic engineer of plants.
E.coli is the most widely used organism as its genetic make-up has been
extensively studied
In Agarose GEL Electrophoresis method,

The bands of DNA in the gel are stained with the dye Ethidium Bromide and DNA
can be detected as visible fluorescence illuminated in UV light will give orange
fluorescence, which can be photographed.
Nucleic Acid Hybridization - Blotting Techniques

Blotting techniques are widely used analytical tools for the specific identification of
desired DNA or RNA fragments from larger number of molecules
Types of Blotting Techniques
Southern Blotting: The transfer of DNA from agarose gels to nitrocellulose
membrane.
Northern Blotting: The transfer of RNA to nitrocellulose membrane.
Western Blotting: Electrophoretic transfer of Proteins to nitrocellulose membrane.
genome
1. The whole complement of gene that determine all characteristic of an organism is
called genome.
2. The genome may be nuclear genome, mitochondrial genome or plastid genome.
CRISPR-Cas9, which is short form of Clustered Regularly Interspaced Short

Palindromic Repeats and CRISPR associated protein 9.
Rice, was among the first plants to be used to demonstrate the feasibility of CRISPR
RNAi:
RNA interference is a biological process in which RNA molecules inhibit gene
expression or translation.
Transgenic Plants / Genetically Modified Crops (Gm Crops)
Herbicide Tolerant – Glyphosate
Herbicide Tolerant - Basta

1. Trade name ‘Basta’ refers to a non-selective herbicide containing the chemical
compound phosphinothricin.
2. Basta herbicide tolerant gene PPT (L-phosphinothricin) was isolated from
Medicago sativa plant.
3. The PPT gene was introduced into tobacco and transgenic tobacco produced
Insect resistance - Bt Crops: i. Bt Cotton
Strains of the bacterium Bacillus thuringiensis produce over 200 different Bt toxins,
each harmful to different insects.
The advantages of Bt cotton are:
• Yield of cotton is increased due to effective control of bollworms.
• Reduction in insecticide use in the cultivation of Bt cotton
• Potential reduction in the cost of cultivation.
Disadvantages Bt cotton has some limitations:

• Cost of Bt cotton seed is high.
• Effectiveness up to 120 days after that efficiency is reduced
• Ineffective against sucking pests like jassids, aphids and whitefly.
• Affects pollinating insects and thus yield.
Bt Brinjal
Bt brinjal is another transgenic plant created by inserting a crystal protein gene
(Cry1Ac) from the soil bacterium Bacillus thuringiensis
Dhara Mustard Hybrid (DMH)
1. DMH -11 is transgenic mustard developed by Delhi University under

Government sponsored project
2. genetically modified variety of Herbicide Tolerant (HT) mustard.
3. genes from soil bacterium that makes mustard, a self-pollinating plant.
4. DMH -11 contains three genes viz. Bar gene, Barnase and Barstar
5. bar gene had made plant resistant to herbicide named Basta.
FlavrSavr Tomato
1. the ripening process of the tomato is slowed down and thus prevent it from
softening and to increase the shelf life.
2. The tomato was made more resistant to rotting by Agrobacterium mediated
gene transfer
Golden rice - Biofortification
1. rice developed by Ingo Potrykus and his group

2. aim is to produce a fortified food to be grown and consumed in areas with a
shortage of dietary Vitamin-A,
3.
addition of three beta-carotene biosynthesis genes namely
1. ‘psy’ (phytoene synthase) from daffodil plant Narcissus pseudonarcissus
2. ‘crt-1’ gene from the soil bacterium Erwinia auredorora
3. ‘lyc’ (lycopene cyclase) gene from wild-type rice endosperm.
Golden rice can control childhood blindness - Xerophthalmia.
Risks
• Affect liver, kidney function and cancer
• Hormonal imbalance and physical disorder
• Anaphylactic shock (sudden hypersensitive reaction) and allergies.
Adverse effect in immune system because of bacterial protein.
• Loss of viability of seeds seen in terminator seed technology of GM crops
Polyhydroxybutyrate (PHB)
1. Polyhydroxyalkanoates (PHAs) and polyhydroxybutyrate (PHB) are group of
degradable biopolymers which have several medical applications such as
drug delivery, scaffold and heart valves.
2. PHAs are biological macromolecules and thermoplastics which are
biodegradable and biocompatible.
Polylactic acid or polylactide (PLA) is a biodegradable and bioactive

thermoplastic derived from renewable resources, such as corn starch, cassava root,
chips or starch or sugarcane.
Green Fluorescent Protein (GFP)

GFP refers to the protein first isolated from the jellyfish Aequorea victoria.
It has been used in modified forms to make biosensors.
Biopharming, “molecular farming or pharming”:

use of transgenic plants genetically engineered to produce pharmaceutical
substances for use of human beings.
Bioremediation
1. use of microorganisms or plants to manage environmental pollution.
2. used to treat wastes including wastewater, industrial waste and solid waste.
3. Bioremediation process is applied to the removal of oil, petrochemical
residues, pesticides or heavy metals from soil or ground water.
4. bioremediation is less expensive and more sustainable
examples of bioremediation technologies
Phytoremediation - use of plants to bring about remediation of environmental

pollutants.
Mycoremediation - use of fungi to bring about remediation of environmental
pollutants.
Bioventing - a process that increases the oxygen or air flow to accelerate the
degradation of environmental pollutants.
Bioleaching - a use of microorganisms in solution to recover metal pollutants from
contaminated sites.
Bioaugmentation - an addition of selected microbes to speed up degradation
process.
Composting - a process by which the solid waste is composted by the use of
microbes into manure which acts as a nutrient for plant growth.
Rhizofiltration - uptake of metals or degradation of organic compounds by
rhizosphere microorganisms.
Rhizostimulation - stimulation of plant growth by the rhizosphere by providing better
growth condition or reduction in toxic materials.
Biofuel: Algal Biofuel

1. algal fuels are an alternative to commonly known biofuel sources obtained from
corn and sugarcane.
2. Botryococcus braunii is normally used to produce algal biofuel.
Biological hydrogen production by algae
1. In normal photosynthesis the alga, Chlamydomonas reinhardtii releases

oxygen.
2. When it is deprived of sulfur, it switches to the production of hydrogen during
photosynthesis
Bioprospecting
Bioprospecting is the process of discovery and commercialization of new products
obtained from biological resources.
Applications of Biotechnology
1. transgenic varieties of plants e.g. transgenic cotton (Bt-cotton), rice, tomato,

tobacco, cauliflower, potato and banana.
2. pesticide resistant, stress resistant and disease resistant varieties of

agricultural crops
3. synthesis of human insulin and blood protein in E.coli
4. synthesis of vaccines, enzymes, antibiotics, dairy products and beverages
5. Biochip based biological computer is one of the successes of biotechnology
6. Genetic engineering involves genetic manipulation, tissue culture involves

aseptic cultivation of totipotent plant cell into plant clones
7. Single cell protein from Spirulina is utilized in food industries
8. Production of secondary metabolites, biofertilizers, biopesticides and enzymes.
9. Biomass energy, biofuel, Bioremediation, phytoremediation for environmental

biotechnology

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Term biotechnology was coined by Karl Ereky, a Hungarian Engineer in 1919

steps involved in recombinant DNA technology are

1. Isolation of a DNA fragment containing a gene of interest that needs to be

PCR: Polymerase Chain Reaction is a common laboratory technique used to make

The basic tools are

enzymes required for genetic engineering are

restriction enzymes 2 types:

DNA ligase enzyme joins the sugar and phosphate molecules

2. pBR 322 Plasmid

In Agarose GEL Electrophoresis method,

Nucleic Acid Hybridization - Blotting Techniques

CRISPR-Cas9, which is short form of Clustered Regularly Interspaced Short

Transgenic Plants / Genetically Modified Crops (Gm Crops)

Herbicide Tolerant – Glyphosate

Herbicide Tolerant - Basta

Insect resistance - Bt Crops: i. Bt Cotton

Disadvantages Bt cotton has some limitations:

Dhara Mustard Hybrid (DMH)

1. DMH -11 is transgenic mustard developed by Delhi University under

Golden rice - Biofortification

1. rice developed by Ingo Potrykus and his group

Polylactic acid or polylactide (PLA) is a biodegradable and bioactive

Green Fluorescent Protein (GFP)

Biopharming, “molecular farming or pharming”:

Phytoremediation - use of plants to bring about remediation of environmental

Biofuel: Algal Biofuel

2. Botryococcus braunii is normally used to produce algal biofuel.

Biological hydrogen production by algae

1. In normal photosynthesis the alga, Chlamydomonas reinhardtii releases

1. transgenic varieties of plants e.g. transgenic cotton (Bt-cotton), rice, tomato,

2. pesticide resistant, stress resistant and disease resistant varieties of

3. synthesis of human insulin and blood protein in E.coli

4. synthesis of vaccines, enzymes, antibiotics, dairy products and beverages

5. Biochip based biological computer is one of the successes of biotechnology

6. Genetic engineering involves genetic manipulation, tissue culture involves

7. Single cell protein from Spirulina is utilized in food industries

8. Production of secondary metabolites, biofertilizers, biopesticides and enzymes.

9. Biomass energy, biofuel, Bioremediation, phytoremediation for environmental

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