Samacheer Class 12 Bio Botany - Principles and Processes of Biotechnology

Today biotechnology is a billion-dollar business around the world, applies biotechnological tools for their product improvement.
* Pharmaceutical companies.
* Breweries.
* Agro Industries & others.
* Modern biotechnology – include all methods, rDNA technology, cell fusion technology, etc.,
* Major focus of Biotechnology (see the tabulation)

* The exact kind of cleavage produced by a v restriction enzyme is important in the • design of a gene cloning experiment.
* Some cleave both strands of DNA through the centre resulting in blunt or flush end known as symmetric cuts.
* Some restriction enzymes cut the strand of DNA, a little away from the centre of palindrome sites, between the same two bases on the opposite strands, protruding and recessed ends known as sticky or cohesive end, cuts known as asymmetric cut or
staggered cuts.
* It is necessary that the vector and the source DNA are cut with the same restriction enzyme, so that the resultant DNA fragments have the same sticky ends facilitating the action of DNA ligase to join them.

Director vector less Gene transfer is possible through several mediators
Chemical mediated gene transfer:
Certain chemicals like Poly Ethylene Glycol(PEG) and Dextran Sulphate.
These chemicals induce the uptake of DNA into plant protoplasts.

pBR332 – It is a reconstructed plasmid and most widely used as cloning vector.
* It contains 4361 base pairs.
* P denotes Plasmid.
* B&R – The names of Boliver and Rodriguez, the scientists developed this plasmid.
* 322 – The number of plasmid developed from their lab.
* It contains ampR & tet R – 2 different antibiotic resistant genes & the recognition sites for several restriction enzymes (Hindlll, ECoRI, Bam H-I, Sal I, Pvu II, Pst I, Cla I) ori & antibiotic resistance genes.
* Rop – Codes for the proteins involved in the replication of the plasmid.

Introduction: Most important applied interdisciplinary sciences of the 21 st century
It has promise for the benefits of Human Being.
Production of secondary metabolites – Biofertilizers, Biopesticides & Enzymes
Biomass Energy, Biofuel, Biorernediation phvtoremediation for environmental biotechnology.

Restriction enzymes are the enzymes of bacterial origin which cleaves DNA into fragments at or near specific recognition sites within DNA molecules. This principle is used in biotechnology to cut and insert the desired gene (gene of interest) thereby generating an rDNA with desirable characters.
a) Exonucleases – remove nucleotides one at a time from the end of DNA.
Eg: Bal 31, Exonuclease III
b) Endonucleases – break the internal phosphodiester bonds with in a DNA.
Eg: Hind II, EcoRI, Pvul, Bam HI, Taql.
Three classes of Restriction endonuclease
* Type 1, II & III – which differ slightly by their mode of action
* Type II – preferred in rDNA technology as they cut DNA with in a specific sequence consisting of 4 – 8 bp.
* Hind II – cut DNA at a point of specific sequence of 6 base pairs (recognition
sequence).
* From 200 strains 900 restriction enzymes isolated from over 230 strains of bacteria with different recognition sequences.
* Restriction endonucleases are named by a standard procedure.
* The first letter of the enzymes indicates the genus name, followed by the first two letters of the species, then comes the strain of the organism and finally a roman numeral indicating the order of discovery.
* For example ECORI is from Escherichia (E) coli (co) strain Ry 13 (R) and first endonuclease (I) to be discovered.
* It contains 2 different antibiotic resistance genes and recognition site for several restriction enzymes.
This sequence is referred to as a restriction site and is generally – palindromic which means that the sequence in both DNA strands at this site read same in 5′ – 3′ direction and in the 3′ – 5′ direction.

Yes, it is possible to transfer a suitable desired gene to a host plant using certain chemicals, microinjection method, electroporation or by biolistics.
a. Chemical mediated gene transfer:
Chemicals Poly Ethylene Glycol (PEG) & Dextran sulphate – induce DNA uptake into plant protoplasts.
b. Microinjection:
With a fine-tipped glass needle, DNA is directly injected into the nucleus.
The protoplasts are immobilized on solid support (agarose on a microscopic slide)
c. Electroporation method of gene transferJjjJU Protoplasts, cells or tissues subjected to a pulse of high voltage electric power to make transient pores in the plasma membrane, through which uptake of foreign DNA occurs.
d. Liposome – mediated methods of gene transfer
The gene or DNA is transferred in an encapsulated form from Liposome ( the artificial phospholipid vesicles) into the vacuole of plant cells.
e. Biolistics:
The DNA particle with gold or tungsten particle (1.3 gm) coating are bombarded into the target tissue by gene gun or microprojectile gun/shotgun The bombarded cells/tissues are cultured to regenerate plants from transformed cells.

Properties
Effect
Able to replicate automatically.
Multiple copies can be got along with insert in the host cell.
Small size, low molecular weight less than 10kbp
Entry into the host cell is easy.
Should contain ori
It can independently replicate within the host.
Contain suitable marker (Antibiotic resistance) etc.
It permit its detection in the transformed host cell.
Should have unique target sites for integration with DNA insert & should have ability to integrate with DNA insert.
So that it can be carried into the genome of the host cell.
Most of the cloning vectors have more than one restriction site (MCS) or polylinker.
Multiple cloning site (MCS) facilitates the use of restriction enzyme of choice.

Type. Transfer of DNA/RNA (From – To)
1. Southern Blotting
DNA from Agarose gels to Nitrocellulose membrane.
2. Northern Blotting.
RNA transferred to Nitrocellulose membrane.
3. Western Blotting.
Proteins transferred from protein to Nitrocellulose membrane.

Character
Effect
1. Weed control
Improves high yielding crops
2. Reduces spray of Herbicide
Economic effort – also ecofriendly & Nonhazardous
3. Use of low toxicity compounds
Does not harm soil because do not remain active in the soil.
4. Reduce competition between crop plant & weed
Healthy plant growth is assured

Advantages
Disadvantages
1. Yield – Increase due to effective control of bollworms
Cost of Bt cotton seeds are high.
2. Usage of insecticide is reduced
Remain effective only up to 120 days after that effectiveness is reduced
3. Cost of cultivation potentially reduced
Ineffective – against
sucking pests like 1. Jassids, 2.aphids, 3. Whitefly
Affects pollinating insects & thus yield.

Bioremediation:
It is defined as the use of microorganisms or plants to clean up the environmental pollution. It is an approach used to treat wastes including wastewater, industrial waste, and solid waste. The bioremediation process is applied to the removal of oil, petrochemical residues, pesticides, or heavy metals from soil or groundwater.
In many cases, bioremediation is less expensive and more sustainable than other physical and chemical methods of remediation. The bioremediation process is a cheaper and eco-friendly approach and can deal with lower concentrations of contaminants more effectively. The strategies for bioremediation in soil and water can be as follows:
* Use of indigenous microbial population as indicator species for the bioremediation process.
* Bioremediation with the addition of adapted or designed microbial inoculants.
* Use of plants for bioremediation – green technology.

Benefits
Risk (Believed to)
Yield:
High yield without pest.
Health Hazards: Liver, kidney function affected cause cancer.
Reduction in usage of chemical pesticides.
Hormonal Imbalance and Physical Disorder.
Reduction in soil pollution.
Anaphylactic shock (Sudden hypersensitive reaction) & Allergies.
Conservation of microbial population of soil.
Loss of viability of seeds as in terminator seed technology of GM crops.
Reduction in groundwater pollution.
Not favoured by agriculturists.
Reduction in air pollution (Chemical spray of pesticides – reduced)
12th Bio Botany Guide Principles and Processes of Biotechnology Additional Important Questions and Answers
I. Choose the correct answer

b. Mycoremediation – use of bacteria to bring about environmental remediation
II. Assertion and Reason
In each of the following questions, two statements are given. One is assertion (A) and the other one is reason(R) Mark the correct answer as
a) If both ‘A’ and ‘R’ are true and ‘R’ is the correct explanation of A
b) It both ‘A’ and ‘R’ are true but ‘R’ is not the correct explanation of A
c) It A is true but’R’is false
d) If both A&R are false

Plasmid DNA
* Extra chromosomal DNA
* Mostly circular double stranded (ds)
* Not associated with histones
* Show autonomous replication with in a suitable host
* Do not act as genetic factor
* Don’t have introns
Chromosomal DNA:
* Chromosomal DNA
* Associated with histone proteins
* They replicate with the genome
* Can be linear/circular ss or ds
* They act as genetic factor
* Have both introns & exons

• E.coli genetic makeup has been extensively studied
• It is easy to handle & grow in short time
• It can accept a range of vectors & also been studied for safety
• Under optimal growing conditions the cells divide every 20 minutes

• It is a method of transfecting cells by bombarding them with microprojectiles coated with DNA
• It is most useful for inserting genes(such as pesticide/ herbicide resistance genes) into plant cells
• The bombarded cells or tissues are cultured on selected medium to regenerate plants from the transformed cells

Yes – because the T1 plasmid of this bacterium is very large sized one known as(Tumour inducing) and a portion of it is referred as T-DNA (transfer DNA).Since upon infection of the cells at wound site the bacterium has the natural ability to transfer T- DNA region of its plasmid in to plant genome it is also known as Natural genetic engineer of plants

• In gene targeting experiments the nuclei has been targeted. This is known as gene knock out
• Two types of vectors are used for it. They are insertion vectors & the replacement vectors

• A process in biotechnology by which the nuitritive quantity of food material is increased by gene transfer technology.It is also known as Biofortification
• The nutritive protein, carbohydrate, Vitamins can be enriched by this process.
• Eg: Golden rice with vitamin A

• Weed control, improves higher crop yields
• Reduces usage of herbicides
• Reduces competition between crop & weed
• Use of low toxicity compounds ( not remain active in soil)
• Conservation of soil structure and soil microbes

BT.toxin is present in its inactive form called protoxin in bacillus thuringiensis
When the bacterium is ingested by the insect, the alkaline PH of the- alimentary canal of insect is activated.The toxin which binds to the epithelial cells of midgut forming pores -leading to swelling & lysis of the cells -leading to death of the insects

• Gene for BT.toxin is written as cry and the prote in as cry III At
• The first letter of protein symbol is always written capital form and written as cry III Ab.

It is an approach in which genetically engineered Micro organism (GEMS) or green plants etc., can be used to treat nonbiodegradable/toxic wastes suches
oil,petrochemical residues,pesticides or heavy metals in
i) Soil ii) Ground water iii) Marine environment and to make environment more sustainable.

• Only biodegradable contaminants can be degraded
• The process must be specific to the contaminated site
• Small scale tests to be conducted before carrying out on a pilot scale
• It is a costly affair also need more research in these areas.

• The use of Algae as a source of energy
• It is an alternative to i) Fossil fuels, ii) Fuel from corn, iii) Sugar cane
• It is also used for making bio-fuel or bio-iesal
• Land unsuitable for Agriculture can be utilised for (farming algae) algal culture. Eg.Botryococcus braunii

• The Technology is photo biological water splitting
• When thenormal condition of photosynthesis was altered, or when it is deprived of sulfur it switches to the production of Hydrogen and the electrons are transported to ferredoxins
• [Fe]-hydrogenase enzymes combine them into the production of Hydrogen gas, an alternative fuel for the next generation

• Electrophoresis is a separating technique used to separate different biomolecules with positive and negative charges.
• By applying electricity (DC) the molecules migrate according to the type of charges they have.
• The electrical charges on different molecules are variable.
• +ve charged cation will move towards -ve cathod.
• -ve charged anions will move towards +ve anode.

• After the introduction of r-DNA into a suitable host cell
• It is essential to identify those cells which have received the r-DNA molecule.
• This process is called screening

b) RNA probes are used in the identification of bacteria as pathogens
IV. Fill in the blanks Answer
1. The method that involved the growth of tissues & cells in a suitable new medium and away from the parent plant is known as…………………….
Tissue culture
2. The range of insects killed by Bt. Toxins are…………………….
Lepitopteron
3. The genes that code for Bt toxins are commercially called…………………….
Cry genes
4. The first company to produce insulin by rDNA technology is…………………….
Eli Lilly
5. The Indian scientist who was the innovator of ELISA in India is…………………….
Usha M.Joshi
6. PCR is usually used to detect the……………………. in a suspected …………………….patient.
HIV & AIDS
7. Are present in increased quantities in glutelin is…………………….
rice
8. Protein encoded by cry Ab control…………………….
Cotton borer
9. Use of microorganism in solution to recover toxic metal pollutants from contaminated sites is…………………….
Bioleaching
10. The endosperm of normal rice doesnot contain…………………….
Beta carotene
V. Two Marks

• Bio-pesticide: pesticide derived from plants bacteria, animals, etc.,
• Bio-fertilizer: all nutrient outputs of biological origin include plants, animals & microbes
• Bio venting: The process that increases oxygen to accelerate the degradation of environmental pollutants
• Bio leaching: Microbes in solution, used to recover poisonous metal pollutants in the soil
• Bioprospecting: The process of commercialization of new products of based on biological resources
• Bio – pharming: use of genetically engineered plants/microbes to produce molecular pharming: pharmaceutical products
• BioFuel: plant/microbes/algae used as an alternative fuel source
• Biofortification: Breeding crops to enrich the nutritional value either by conventional or gene by genetic engineering
• Bioremediation: use plants/microbes to clean up environmental pollutants
• Biopiracy: exploiting the traditional knowledge/invention of poor countries by MNC or developed countries without approval or proper compensation
• Bio patency: The legal exclusive right for the inventor and thereby excluding others from exploiting the knowledge/invention.
• Bio chip: microchip designed intended to function in a biological environment or inside the body of an organism

• They exist in many bacteria, where they function as a part of their defence mechanism called restriction-modification system
• It helps the bacteria to cut the genetic material of the virus that attack it and render them harmless. EgiE.coli

Cloning vector
It is used for cloning of DNA,insert inside the suitable host cell
Expression vector
It is used to express the DNA insert for producing specific protein inside the host

Origin replication (ori) is a sequence from
where replication starts and piece of DNA when linked to this sequence can be made to replicate within the host cells

The DNA sequence able to insert itself at a new location in the – Genome without having any sequence relationship with the target locus -,hence known as walking or jumping genes-or Transposons

• Ability to change the -genetic material for getting new products according to the requirement through r DNA technology
• Ownership of the newly developed technology and its social impact

It is a vessal or container, designed,
A. To provide an optimum environment, in which microorganism or their enzymes interact with a subtract to produce a product
B. It provide a controlled condition, aeration, agitation, temperature and PH.
C. It has 2 processes i) upstream ii) down stream

Upstream process:
* 1st part
* All the process-of preparation before the starting the process >
* Includes sterilization of the bioreactor, preparation & sterilization of culture medium and growth of the suitable inoculum
Downstream process:
* Follows upstream
* All the process after the fermentation process
* Includes distillation centrifuging, filteration & solvent extraction Mostly- involves the purification of the desired product

Ti plasmid:
* Ti plasmid is found in Agrobacterium tumefaciens a bacteria responsible for inducing tumours in several dicot plants.
* It plasmid carries transfer (tra) gene which help to transfer T-DNA from one bacterium to other bacterial or plant cell.
* It has one gene for oncogonecity, ori gene for origin for replication and inc gene for incompatibility.
* T-DNA of Ti-plasmid is stably integrated with plant DNA
* Agrobacterium plasmids have been used for introduction of genes of desirable traits in to plants.

• Protein supplement
• Cosmetic product for healthy hair & skin
• Poultry industry as excellent source of proteinacious food.
• In food industry – canbe carrier in production of aroma, tic compounds vitamin, emulsifying agent improve the nutritive value of baked products & ready to serve meals.
• In the processing of paper & leather as foam stabilizers.

• It is genetic form refer to the identify of the taxon based on its genetic makeup.
• It is an optical machine readable representation of data which describes about A the characters of any plants / objects.

• A group of technologies that has the ability to change an organism’s DNA.
• Genetic material can be added, removed or altered at particular locations in the genome – known as genome or gene editing.
• Eg. GRISPR – mediated gene replacement – Rice can be switched from sexual to an asexual mode.

• Affect Liver, Kidney functioning
• Carcinogenic (cause cancer)
• Hormonal imbalance & Physical disorder
• Anaphylactic shocks (sudden hypersensitive reaction) & Allergies
• Adverse effects on immune system – due to interference of bacterial protein
• Loss of viability of seeds, (shown in terminator seed technology of GM crops).

* Alwin et al. (1979) devised a special technique Northern Blot hybridization to
transfer RNA bands.
* Amino Benzyloxymethyl paper is the filter paper used, which can be prepared from what man 540 paper.
Western Blot
* It is electrophoretic transfer of protein to blotting papers.
* Nitrocellulose filter paper can be used.
* A particular protein is then identified by probing the blot with a radio-labelled antibody – binds on the specific protein to which the – antibody was prepared.

• The native genes in Tomato produce enzyme Polygalacturonase and this leads to ripening follow by senescence & fruits get spoit.
• When Anitsense RNA genes inserted into Tomato plant via Agrobacterium mediated gene transfer the gene interfere with the production of Polygalacturonase, there by delay ripening, softening and further spoiling (shelf life of fruits increased).
• Transgenic tomatos can be transported to long distance with out getting spoilt.

Both ELISA and Western Blotting are indirect tests – to measure he immune system’s response to an infections agent rather than looking for the components of the agent itself.
ELISA Test
Western Blot Test
It detects the antibodies which the
body starts to produce between 2-12 weeks after being infected,
It is a confirmative test. It is less likely to have false positive results – as it can effectively distinguish between the anti bodies of the particular disease from other antibodies
It is a qualitative, sensitive test – but not a confirmative test. Eg.: HIV – AIDS
It is a clear confirmative test.
Eg.: HIV – AIDS
VII. Five marks

1. Up-stream proces (Preparation)
It include,
* Sterilization of the fermenter
* Sterilization of the culture medium
* Growth of the suitable inoculum
2. Fermentation process
3. Down stream process (Purification)
It include,
* Distillation
* Centrifuging
* filtration &
* solvent extraction.
So; for fermentation process to occur the preparation process(upstream process) is essential. If no inoculum we can’t produce culture. Also without sterilization contami-nation occur leading to spoilage of the culture by the harmful microbes.

• Antibiotic Resistant Marker (ARM) is – a gene when introduced into bacterial cells – (Recombinant) produce – a protein that provide resistance to antibiotics.
• Recombinants (A) may grow well in a medium with antibiotics (such as ampicillin, chioramphenicol, teiracycline or kanamycinetc)
• Non recombinants (B) may not be able to grow in these media with these antibiotics.
• Thus Antibiotic resistant marker is a useful selectable marker in distinguishing the two.

Technique:
Pattern of colonies growing on a culture plate can be copied.
Procedure:
* A culture plate with growing bacterial colonies is taken (A) – infected.
* A sterile filter plate (B) – is pressed against culture plate (A) – infected.
* The filter (B) got infected and then it is pressed against a sterile culture plate (C)
* New plate (C) got infected with same relative positions as colonies in the original.
The study can be repeated on different conditions.
(i) with an Antibiotic
(ii) with a growth factor etc.,

• Agarose GEL Electrophoresis is a,medium used to separate DNA fragements of larger sizes (few 100S to 20,000 bp)
• Polycrylamide is a medium used to separate DNA fragments of smaller sizes.
• Agarose GEL provides – a three dimensional matrix & DNA molecules migrates through the – gel and DNA bands can be readily detected at highter sensitivity.
• Energy – The electric field provide energy
• Technique – DNA are negatively charged and migrate towards the positive pole (anode)
• (The marker DNA fragments of known size which allow accurate size determination of an unknown DNA molecule by interpolation)
• The bands of DNA can be stained by a dye Ethium bromide and can be detected as visible orange fluorescence under UV light and can also be photographed.

Definition:
RNAi (is a phenomena in which ds RNA molecules targetedly select m RNA – molecule and inactive or inhabit or neutralise its gene expression into protein (Translation)

1. Basta refers to nonselective herbicide with chemical compoumd Phosphinothricin – which inhibit, the enzyme glutamine synthetase involved in ammonia assimilation.
Steps:
2. Like wise PAT – similer FPT was extracted
to get Herbicide resistant transgenic plant:

Transgenic plants
Useful application of transgenic plants
1 Bt.cotton
Resist bollworms & gat flies
Increased yield
Reduction in insecticide use
2 Bt.Brinjal
Resist lepidopteron insects such as fruit and shoot borer- Leucinodes or bonalis
3 Golden rice
Bio fortified rice-modified to produce beta carotene (used by the body to make vitamin A)
4 Dhara mustard hybrid (DMH-I)
Transgenic mustard is resistant to herbicide Basta
Yield is increased
5 Flavrsavr tomato
Transgenic tomato, retaining color & flavor ripening is delayed & can be transported to long distance without getting spoiled.

Bt.cotton is a transgenic plant
Bacillus thuringiensis produces 200 different Bt. toxins
Most of the toxins are effective against moths, Butterflies, Beeltes, cotton bollworms & gatflies
Cry genes produce crytoxins, when dissolved in the alkaline PH of gut of insect the toxins become active, form pores on the epithelial cells, there by sufficient regulation of potassium ions are lost resulting in the death of the epithelial cells leading to death of the larves.

Nature:
* Green Fluorescent protein (GFF) – contain aminoacid residues of 26.9 KD a that exhibits bright green fluorescence when exposed to blue UV range (395 nm)
Properties:
* This protein is 1st isolated from a Jelly fish Aequorea victoria
* It has the ability to form internal chromophore without any co factor except molecular oxygen Uses:
* GFP is used as a reporter of expression
* It is used in modified forms to make biosensors

It has wide applications in various sectors
I. Agriculture – Transgenic plants
Bt.cotton, Bt.brinjal, Golden rice, Flavr Savr tomato, Cauliflower, Potato, and Banana – are
the outcome of Biotechnology Resistant varieties They are Resistant to pest, stress, disease, etc.,
II. Medicine:
* Insulin – is produced by r DNA technology is a breakthrough in medicine
* Vaccines, enzymes, antibiotics, dairy products & beverages are also products of biotechnology
III. Biochip:
Bio chip-based biological computer
IV. Genetic engineering:
It involves
* gene manipulation
* Tissue culture
* Single-cell protein (food industry) SCP
* secondary metabolites & etc.,
* biofertilizers – biopesticides etc.,
V. Environmental aspects Include
* Bio mass-energy
* Biofuel
* Bio & phytoremediation
* Environmental biotechnology etc.,