lecture 2 biotech
TRANSCRIPT
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Growth and Development of Biotechnology: Past, Present and Future
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What is Biotechnology?
• Biotechnology : techniques applied to organisms or parts thereof to produce, identify or design substances, or to modify organisms for specific applications.
• Cell fusion techniques, hybridomas, recombinant DNA technology, cloning through whole genome transfer, protein engineering, structure based molecular design, genomics, proteomics, bio informatics including biochips, cell therapy including cell, tissue or organ transplant, xeno - transplants or creation of artificial cells are modern biotechnology.
• Conventional biotechnology includes fermentation or conversion of substrates into desired products by biological processes; use of microbes or enzymes; sera, vaccines and diagnostics; reproduction, artificial insemination and embryo transfer technology for animal breeding; methods for fish spawning induction; plant cell or tissue culture; plant breeding; bio-fertilisers; bio-pesticides; plant growth stimulants; extraction and isolation of active principles from plants or animals or parts thereof etc.
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Conventional Biotechnology Existed Since Ages!
* 4000 BC : Egyptians used yeast for bread & wine making.* 3000 BC : Peruvians cultivated potatoes.
* 2000 BC : Egyptians, Sumerians and Chinese developed techniques of fermentation, brewing and cheese making. * 500 BC : Chinese used moldy soybean curds to treat boils. * Indian ancient literature (> 4000 BC) refers to Sura & Madira which are fermented beverages!
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Beginning of Understanding Science of Biotechnology
* 1590 Janssen invented the microscope. * 1802 : The terms biology first appeared in literature. * 1830 : Proteins were discovered.
* 1865 : Mendel discovered the existence of heredity.
* 1860 : Louis Pasteur’s experiments abolished the theory of spontaneous generation of living organisms.
* 1897 : Buchner’s discovered the biochemical basis of life process.* 1919 : A Hungarian agricultural engineer coined the term Biotechnology.
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Domain of Biotechnologists (Schematic)Basic Biotechnologists:
Use molecular and biological keys and tools
Cellular tools / keys Genomics & Proteomics
Molecular tools/keys
Libraries of small Molecules : natural, combinatorial & computational
Libraries of large molecules: proteins, carbohydrates, nucleotides etc.
Study of molecular interactions: large-large, large-small molecules etc
Molecular evolutions: large and small molecules
Structural
Functional
Pharmacological
Informatics: Internet, sequence data, data searching, predictive methods etc.
Engineering Biotechnologists provide inputs for easing production
Products to Market
Feed back loop
Biotic Diversities & Interactions
Evolutionary Relationships
Metabolic Studies
Biological Models
Creating relation-ships among these disciplines
Main applications emerged in Healthcare Products, Agriculture, Environment Management & Others
THE HERSHEY-CHASE BLENDER EXPERIMENT, 1952
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No labeled sulphur detected in cells
LalLabeled sulphur detectedin supernatant
Labeled Phosphorus detected in cells
No labeled phosphorus Detected in supernatant
ROSALIND FRANKLIN’S X-RAY DIFFRACTION PHOTO OF STRUCTURE B OF DNA
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Sodium deoxyribose nucleate from calf thymus, Structure B, Photo 51, taken by Rosalind E. Franklin and R.G. Gosling. Linus Pauling's holographic annotations are to the right of the photo., May 2, 1952.
DNA DOUBLE HELICAL STRUCTURE
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Restriction Enzymes
SANGER’S DIDEOXY CHAIN TERMINATION METHOD FOR DNA SEQUENCING
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THE GENETIC CODE
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THE BASIC CONSTITUENTS OF LAC OPERON
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THE CENTRAL DOGMA OF LIFE
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FROM GENE TO PROTEIN
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PRINCIPLE OF PCR
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GENE CLONING (RECOMBINANT DNA TECHNOLOGY)
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Expression of Recombinant Proteins
Recombinant proteins are expressed as:
1) Intracellular Substances
•Insoluble protein•Soluble protein
CytoplasmicPeriplasmic
2) Extracellular Substances
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Recombinant Protein Purification : ISSUES
• Folding for activity• Purity
Primary Techniques for purification
• Ion exchange chromatography• Hydrophobic interaction chromatography• Gel filtration Chromatography• Affinity Chromatography
•Precipitation - pH and salt addition
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Intracellular Insoluble Proteins: as Inclusion Bodies
Dense aggregates of mainly the desired protein
Concentration basedUnable to fold correctly in the environment
Simple purification steps using centrifugation are used
Cell lysis: Enzyme treatment Sonication French press Dynomill Freeze-thaw
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1 2 3 4 5 6 7 8 9 10
14.3
68.0 43.0
29.0
20.0
1. Mol. Wt marker
2. Total cell no.1
3. Inclusion Bodies no.1
4. Total cell no. 2
5. Inclusion Bodies no.2
6. Total cell no.3
7. Inclusion Bodes no.3
8. Total cell no.4
9. Inclusion Bodies no.4
Expression and Purity of Intracellularly Expressed and Isolated Insoluble Proteins
Product obtained - insoluble and non-native
In-vitro refolding is essential
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Intracellular Soluble Protein
Expressed protein is soluble
In-vitro refolding may not be required as protein may attain native structure
Protein is extracted by simple lysis methods
Protein has large number of cellular protein contaminants, compared with partially purified inclusion bodies
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Purification of a Soluble Cytoplasmic Protein: rp24 of HIV-1
Expression of the protein in E. coli
Lysis of the cells
Centrifugation
Supernatant
Ion exchange Chromatography
Gel filtration Chromatography
Ready for use in a formulation
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Important Globally Approved Recombinant Therapeutics
ProductProduct Therapeutic IndicationTherapeutic IndicationFactor VIII Factor VIII Hemophilia Type AHemophilia Type A
Factor IXFactor IX Hemophilia Type BHemophilia Type B
Tissue Plasminogen activatorTissue Plasminogen activator Acute myocardial infarctionAcute myocardial infarction
InsulinInsulin DiabetesDiabetes
Human growth hormoneHuman growth hormone hGH deficiency in childrenhGH deficiency in children
Thyrotropin-a Thyrotropin-a Thyroid CancerThyroid Cancer
ErythropoietinErythropoietin Treatment of anemiaTreatment of anemia
GM-CSF / G-CSFGM-CSF / G-CSF Chemotherapy induced neutropeniaChemotherapy induced neutropenia
PDGFPDGF Diabetic Neuropathic ulcersDiabetic Neuropathic ulcers
Interferon alpha 2aInterferon alpha 2a Hairy cell leukemiaHairy cell leukemia
Interferon alpha 2bInterferon alpha 2b Hairy cell leukemia / Hepatitis B & CHairy cell leukemia / Hepatitis B & C
Interleukin – IL-2Interleukin – IL-2 Renal cell carcinomaRenal cell carcinoma
Hepatitis B surface antigenHepatitis B surface antigen Vaccine against Hepatitis B Vaccine against Hepatitis B
Alpha-GlucocerebrosidaseAlpha-Glucocerebrosidase Treatment of Gaucher’s diseaseTreatment of Gaucher’s disease
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Important Globally Approved Genetically Modified Plants
ProductProduct Genetically Altered Traits Genetically Altered Traits
TomatoTomato Delayed ripening: Gene sequence for polygalaturonase Delayed ripening: Gene sequence for polygalaturonase production in tomato rearranged and reversed to minimise its production in tomato rearranged and reversed to minimise its expression by Antisense technology.expression by Antisense technology.
CottonCotton Bt gene incorporated plants (ballworm & budworm resistant): Bt gene incorporated plants (ballworm & budworm resistant): CRY 1A c gene from Bt Kurstaki.CRY 1A c gene from Bt Kurstaki.
SoybeanSoybean Reisitant to glyphosate for control of weeds: Reisitant to glyphosate for control of weeds: Enolpyruvylashikimate-3-phosphate synthase gene from Enolpyruvylashikimate-3-phosphate synthase gene from Agrobacterium sp.CP4Agrobacterium sp.CP4
PotatoPotato Bt gene incorporated (Colorado potato beetle resistant) : Cry Bt gene incorporated (Colorado potato beetle resistant) : Cry III (A) gene from Bt. Tenebrionis.III (A) gene from Bt. Tenebrionis.
Maize/CornMaize/Corn Bt gene incorporated (resistant to comborer) : Cry 1A b gene Bt gene incorporated (resistant to comborer) : Cry 1A b gene from Bt. Kurstakifrom Bt. Kurstaki
Rapeseed / CanolaRapeseed / Canola Altered oil composition (high lauric acid content): 12:0 acyl Altered oil composition (high lauric acid content): 12:0 acyl carrier protein thioesterase gene from Umbellularia carrier protein thioesterase gene from Umbellularia californica.californica.Resistant to glufosinate for Male sterility propertiesResistant to glufosinate for Male sterility properties
SquashSquash Resistant to viruses: Coat protein genes of watermelon Resistant to viruses: Coat protein genes of watermelon mosaic virus 2 and Zucchini yellow mosaic virus.mosaic virus 2 and Zucchini yellow mosaic virus.
PapayaPapaya Resistant to Papaya ring spot virus: Coat protein gene of p Resistant to Papaya ring spot virus: Coat protein gene of p type of PRSV HA-5-1 from Hawai.type of PRSV HA-5-1 from Hawai.
ChicoryChicory Male starility resistant to glufosinate and fertility restores Male starility resistant to glufosinate and fertility restores genes from bacteria.genes from bacteria.
MICROARRAY TECHNOLOGY
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DOLLY- THE CLONING GLORY
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Global Segment Estimated consumption in billion US$
2001 2005 2010
USA & Canada 28 34 50
Europe and Japan 7.5 10 15
Rest of the World 2.5 5 12
Total 38 49 77
Overlap of the Segments of the rest of the world
India 0.1 0.3 1.0
China 0.6 1.2 3.0
South Korea 0.3 0.5 1.3
Latin American countries and the rest of the world
1.5 3.0 6.7
Subtotal 2.5 5.0 12.0
Estimated Global Consumption of Modern Biotech Products
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Strategies for Technology Development In Developing Country Industries
Alliances through Alliances through Research Research GrantsGrants
Alliances through Alliances through Institutions & Companies Institutions & Companies :In-Country & Abroad:In-Country & Abroad
Sponsored Sponsored ResearchResearch
Technology Technology Sourcing &Sourcing & AcquisitionsAcquisitions
A A Developing Country Developing Country
CompanyCompany
TechnologiesTechnologies
In-House R&DIn-House R&D
Production InfrastructureProduction Infrastructure
Products and ServicesProducts and ServicesCommercializationCommercialization
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Capacity in handling sterile fermentation processes
Skills in handling microbes and animal cells
Skills in plant cell/tissue culture
Competence in chemical synthesis, proficiency in immunology and hands-on experience in microbiology
Capacity in downstream processing and isolation methods
Skills in cloning
Skills in extraction and isolation of plant and animal products
Competence in plant and animal breeding
Infrastructure and skills in fabricating bio-reactors and processing equipment of diverse kinds
Indian Strength in Biotechnology Development
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INDIA’S ENTRY WILL REDUCE COSTS
• Health care products : Hepatitis B Vaccine, Interferon Alpha, Erythropoietin and Insulin doses cost less.
• Agriculture : Genetically modified planting materials will be available at cheaper prices.
• Prices of Industrial products like Enzymes will also come down.
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Modern Biotechnology has made tremendous progress during Modern Biotechnology has made tremendous progress during the last 5 decades.the last 5 decades.
The number of Biotech products are increasing at a galloping The number of Biotech products are increasing at a galloping speed in Health care area, Agriculture and Industrial speed in Health care area, Agriculture and Industrial applications.applications.
Industries in Developed Countries shall hold Major Industries in Developed Countries shall hold Major Technologies.Technologies.
Developing countries incl. India shall also make progress. Developing countries incl. India shall also make progress. All Biotech Industries shall need strong support from All Biotech Industries shall need strong support from
Governments. Governments. Indian development would contribute to providing Biotech Indian development would contribute to providing Biotech
products at cheaper prices though total contribution in products at cheaper prices though total contribution in global context would be small.global context would be small.
Skilled Biotechnologists would have plenty of opportunitiesSkilled Biotechnologists would have plenty of opportunities
Concluding Remarks