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Opportunities and Challenges
in modern Biotechnology
by
Prof. Dr. Rainer Fischer
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Kondratieff-Cycles: Key Innovations.
... initiate new industrial and social stages of development
linked world
1900 1950 2000
Steel,Railway,Transport
Internet,
Mobile
Communic.Cycles
EarlyIndustrialisation
1850 1900 1950 2000
Automobil,
Petrolchemistry
Microchip
Automation
Life-sciences
Solar technology
Steem-maschine,Clothing-industry
Innovation
LateIndustrialisation
Service-society
Knowledge-society
HealthAge
E-Technics,
Chemistry
Source: similar in Nefiodowin Capital 1/2 2000
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What is Biotechnology ?
bios = life
teuchos = tool
logos = study of or essence of
e.g. the study of tools from living things/organisms
classical definition Biotechnology is a set of tools that
utilize living things (and more recently, derivatives of living
things) to solve problems or to provide products
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What is Biotechnology ?
biotechnology is the application of various sciences (i.e.,
immunology, molecular biology, biochemistry, botany, animal
science, etc.) to develop products or to solve problems.
the office of Technology Assessment of the U.S. Congress defines
biotechnology as "any technique that uses living organisms or
their products to make or modify a product, to improve plants or
animals, or to develop microorganisms for specific uses."
the use of living things or parts of living things to create or modifydrugs and other substances
to modify food crops and other macroscopic organisms
to adapt microorganisms to agricultural, medical, or other purposes
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What is Biotechnology ?
all lines of work by which products are produced
from raw materials with the aid of living things
Karl Ereky 1917
rawmaterials livingsystem product
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What is Biotechnology ?
recombinant genetic engineering
.using biological process to develop products
G. Steven Burrill 1997
genetic
engineering
raw
materials
living
systemproduct
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What is Biotechnology ?
protein
bioproduction of drugs so complex
they can only be synthesized in a living system
DNA
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What is Biotechnology ?
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The Promise of Biotechnology
diagnosing disease
curing disease
nutritious food/feed
healthy food/feed
productive land
feeding the poor sustainable agriculture
healthy environment
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The Breakthrough Experiments in Genetics
Hershey and Chase 1952
T2 bacteriophage: 32P DNA not 35S protein encodes genetic inform.
Watson, Crick, Franklin and Wilkins (1953)
X-ray crystallography
1962 Nobel Prize awarded to three men
Chargaff DNA base ratios
structural model of DNA developed
Messelson and Stahl
14
N/15
N semi-conservative replication confirmed scientific foundation of modern biotechnology based on knowledge
of DNA, its replication, repair and use of enzymes to carry out in vitro splicin
DNA fragments DNA polymerase, DNA ligase, restriction endonucleases.
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Breaking the Genetic Code Finding the Central Dogma
an RNA Club organized by George Gamow (1954)
assembled to determine the role of RNA in protein synthesis
radioactive tagging experiments demonstrate intermediate
between DNA and protein = RNA
RNA moves from nucleus to cytoplasm site of protein synthesis
DNA RNA Protein
transcription translation
genetic code determined for all 20 amino acids by Marshal Nirenberg and
Heinrich Matthaei and Gobind Khorana Nobel Prize 1968
3 base sequence = codon
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Origins of Biotechnology
historical pharmaceutical biotechnology:
Alexander Fleming discovery of penicillin
from bread mold - 1928
Large scale broth tank production of
penicillin for WWII injuries Florey & Chain
modern pharmaceutical biotechnology:
interspecies genetic transplantation
hybridoma tumor cell and leukocyte fusions
heterologous protein production (microbes, animal and plant cells)
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Biotechnology Timelines
1750 B.C. Sumerians use yeast to brew beer
500 B.C. Chinese use mold as an antibiotic to treat boils
1863 Mendel discovers transmission of genetic traits
1906 first early study of genes; term genetics introduced
1919 term biotechnology first used by agriculturalist
1928 Penicillin discovered
1953 Watson and Crick discover
double-helix structure of DNA
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Biotechnology Timelines
1960 first synthetic antibiotic
1965 mouse-human cells successfully fused
1966 genetic code cracked
1973 recombinant DNA technology to cut and paste genes
1975 hybridoma technology (monoclonal antibodies)
1978 insulin gene cloned
1981 first transgenic animal
1983 first transgenic plant (tobacco)
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Biotechnology: last 20 Years
1983 first artificial chromosome
1985 genetically engineered plants field tested
1986 use of microbes to clean up oil spill
1988 first patent for genetically altered animal (transgenic mouse)
1995 first non-viral full gene sequence completed
1997 Dolly the cloned sheep unveiled
2002 mapping of human genome virtually complete
2005 human genome confirmed
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What is Genetic Engineering ?
genetic engineering is the basic tool set of biotechnology
genetic engineering involves:
isolating genes
modifying genes so they function better
preparing genes to be inserted
into a new species
developing transgenes
analysing transgenic organisms
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What is Genetic Engineering ?
Recombinant DNA Technology
Boyer and Cohen, 1973
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Biotechnology Applications
food processing (cheese, beer, dairy products)
production of new and improved crops/foods, industrial chemicals,
pharmaceuticals and livestock
diagnostics for detecting genetic diseases, forensic applications
gene therapy (e.g. ADA, CF)
vaccine development (recombinant vaccines)
environmental restoration & bioremediation
protection of endangered species & conservation biology
plant biotechnology (pathogen and stress resistance)
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Evolution of Biotechnology
Double helix ofDNA structure
DNAcloned
Monoclonalantibodiesproduced
Recombinantinsulin approved
Genetic codeelucidated
A T
C
T
A
T
C
T
G
G
A
G
T
G
T
G
A
A
C
C
PCRreported
Biologicalsapproved for
clinical use
Combinatorialchemistry
Pharmaco-genomics
Gene therapy
Genomics
Humangenomemapped
Human gene cloned
CG GC
T A A T
G C T A
1953 73 75 826165 77 86 1986 - 1999 2000 +
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Waves of Discovery Technologies
1975-1990 The Molecular Stone Age early Genentech, Amgen
1990-2000 Genomics
- Incyte - HGS- Millennium - Celera
1995-2008 Proteomics
- Large-Scale Biology
- Celera
- Oxford Glycosciences
1985-2050 Bioinformatics
2000-2050 Structure-based Design
2002-2040 (High Throughput) Imaging
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Products produced with Biotechnology based enzymes
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The Orgins of Biotechnology
fermentation
tools/sources
grains
yeast
vessels
products
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Industrial Biotechnology
the application of life sciences to conventional manufacturing
and synthesis processes: uses genetically engineered bacteria, yeasts,
plants
usually results in:
lower production costs
more profit - $
less pollution
resource conservation
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Industrial Biotechnology: range of activities
Biobased Products Manufacturing Nanotechnology
Bioenergy and Synthesis Biotech Interface
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Industrial Biotechnology based early stage products
different types of beer & wine
dairy products (joghurt, cheese)
vinegar glycerol
acetone
butanol
lactic & citric acid
antibiotics WWII (Bioreactor developed for large scale production, e.g.penicilin made by fermentation of penicillium)
today many different antibiotics are produced by microorganisms
cephalosporins, bacitracin, neomycin, tetracycline..)
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Fermented Foods and Beverages
long history of fermented foods since people began to settle (9000 BC):
often discovered by accident!
improved flavor and texture deliberate contamination with bacteria or fungi (molds)
examples:
bread
yogurt
sour cream
cheese (chymosin)
wine & beer
sauerkraut
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Beer
barley moistening and
germination; enzymatic
relase of carbohydrates
drying, crushing, and mashing:
further enzymatic release of
maltose, dextrins and proteins
addition of hops, and heat in brew
kettle; clarification
remove hops, add yeast to initiate
alcoholic fermentation
storage (lagering) and packaging
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Wine
grape pressing: must
sterilization & SO2 ; addition of
yeast starter culture
fermentation of must (sugar
content essential for product)
removal of excess yeast
malolactic fermentation
removal of excess yeast
aging & bottling
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Optimization of Yeasts
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Microbial based Products
amino acids to improve food & feed taste, quality or preservation
enzymes (cellulase, collagenase, diastase, glucose isomerase,
invertase, lipase, peroxidase, laccase, pectinase, protease)
vitamins
pigments (melanins)
chemical transformation: substrate + microbial enzyme product
examples: cholesterol steroids (cortisone, estrogen, progesterone) hydroxylation
reaction -OH group added to cholesterol ring
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The Perfect Enzyme (enhanced stability)
sampling nature & screening culture collections
improve available enzyme with rational design
random mutagenesis
natures catalysts are extremely well suited to support life;
they evolved to perform optimally in the context of aliving cell as part of a metabolic network
natural enzymes are usually not so well suited for biotechnology
applications, because of the distinct conditions and different demands
biocatalysis applications depend on methods to tailor nature's
catalysts or redesigning them anew
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The Perfect Enzyme: Exploring Natural Diversity
http://www.pmel.noaa.gov/vents/geology/video.html
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One Example
Diversa: has bioprospecting
agreement with USPS
samples hot pools and geysers in
Yellowstone National Park
finds microbes (extremophiles)
with unique genomes and then uses
gene shuffling to discover newenzymes for industrial applications
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Environmental Biotechnology
using life sciences to clean up pollution
bioremediation using:
microbes
enzymes
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How to make inexpensive sugars in large quantities
development of novel technologies for biobased energy and products
commercial viability
economic and ecological sustainability
sugars are the raw materials or
the crude oils that will
be used in biorefineries
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Renewable Sugar Resources
current:
conventional grain milling operations
near term:
microbial/enzymatic hydrolysis of
cellulosic biomass
(R&D -- enzymes being developed)
medium term:
genetically modified plants to produce more
sugars or starch, larger plants and biomass
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Cellulosic Biomass
plant matter made of tightly bonded sugars and lignin
cellulose is made of sugar building blocks:
but is a tough nut to crack
pre-treatment + enzyme (cellulase) treatment = technological
breakthrough
cellulose after
pre-treatment
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Cellulase: enzymatic conversion of cellulose to sugar
improved cellulase is required to make enzyme conversion of
cellulosic biomass economically viable
DOE/contract with Genencor and Novozymes for R&D
to greatly improve activity of cellulase
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Products that can be made from cellulose & sugars
ethanol (transportation fuel)
polymers PLA, PHA, PDO
fine chemicals
bulk chemicals
commodity chemicals
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The birth of an new Industry
University of California, San Francisco
Genentech Inc. South San Francisco
h i h f h
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The Birth of Genentech
excited by the discovery of Boyer and Cohen,
Swanson contacted Boyer
Boyer agrees to give him ten minutes
meeting lasted 3 hours
1976, Genentech was born
http://www.gene.com/
G h h Pi
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Genentech: the Pioneers
initially faced skepticism from academic and business communities,
however..
1977, produced first human protein (somatostatin) inE. coli
1978, genes for human insulin cloned
1979, human growth hormone cloned
1980, Genentech raised 35 million with IPO
(stock price went from initial $35 to $88 after one hour)
1999- Roche purchases all of Genentechs shares ($2.1 Billion)
2000-Genentech ranks 32 in Fortune Magazines list of 100 best
companies to work for in America
Cl i I li
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Cloning Insulin
Recombinant Pharmace tical Markets
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Recombinant Pharmaceutical Markets
world wide biotech market for recombinant therapeutics:
US$55.5 Mrd in 2007; growth ~40% per annum since 1995
growth will be accelerated by:
genomics, proteomics, bioinformatics
modern platform and enabling technologies:
combinatorial libraries, molecular evolution
improved and safer expression systems
forecast for the market share of
therapeutic antibodies
in 2010 ~US$ 24 billion 59 109 313 7121.336
2.2062.958
3.5674.700
24.000
1995 1996 1997 1998 1999 2000 2001 2002 2003 2010
Recombinant Proteins
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Recombinant Proteins
therapeutics (vaccine, antibodies, cytokines, growth factors,
blood substitutes, enzymes and peptides) for treatment of:
infectious diseases of humans and animals
tumor diseases
autoimmunity
allergies
cardiovascular diseases
inflammation and woundhealing
neurological disorders
diagnostics
enzymes
Types of Biologicals in Development
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Angiogenesis Inhibitors
Antisense
Clotting factors
Soluble receptors
Growth factors
Signaling Proteins
Genetherapy
MoAbs
Vaccines
Other
Recombinant proteinsGrowth hormones
Interferons
Interleukins
Immune-based therapy
9
25
5
59
98
83
12
6
3
4
17
Source: PhRMA 2000. Survey of New Medicines in Development, Biotechnology
Types of Biologicals in Development
5
20
6
4
Drug Discovery Process
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Source: Jones-Grizzle and Draugalis, DICPAnn Pharmacotherapy, 1991.
Drug Discovery Process
DISCOVERY
PLATFORMS
PRODUCT
DEVELOPMENT
Target Identification
Toxicology
In Silico Modeling
Pre-Clinical Development
Compound Screening
Lead Optimization
Human Trials
Target Validation Computational Chemistry
Animal Studies
The Magic Triangle of HTS
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The Magic Triangle of HTS
Costs
reagens
consumables
instrumentation
Speed time/well
wells/day
screens/year
Quality
few false positives
few false negatives
S/N, H/L, Z`-factor
H T S
Making Drugs is hard to do
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Making Drugs is hard to do..
HOW MUCH DOES IT COST?
roughly $ 1000 million
That`s more than it cost to build Queen Mary 2
HOW LONG DOES IT TAKE?
from inception to market is between 10-15 years
It took NASA less time to put a man on the moonTHE ODDS OF SUCCESS?
one in 5,000 developing drugs make it to market
Exactly the likelihood of making a hole-in-one
during any given round of golf.
Fortune Magazine 11-72005 p.77
Time Lines and Costs for Pharmaceuticals Development
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Time Lines and Costs for Pharmaceuticals Development
5000
500
50
5
s
u
b
s
t
a
n
c
es
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 years
drug discovery drug optimization clinical efficicay registration/approv.
50 150 450 500 Mio $
screening
optimisation
toxikology
phase I
phase II
phase III
approval
beeinflubarer Bereich
250 Mio $
Leading Products in the Protein Therapeutics Market (US$mn)
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Leading Products in the Protein Therapeutics Market (US$mn)
1,400MAbAbbott LaboratoriesHumira
1,543InterferonsBiogen IdecAvonex
1,808EPORoche/ChugaiNecoRecormon
2,288CSFAmgenNeulasta
2,455EPOAmgenEpogen
2,506InsulinNovo NordiskNovolin
3,273EPOAmgenAranesp
3,324EPOOrtho BiotechProcrit/Eprex
3,334MAbGenetechRituxan
3,542MAbJahnson&Johnson/
Schering Plough
Remicade70
2005
Sales
Protein
Class
CompanyBrand Name
Global Protein Therapeutic MarketArrowhead Publishers 2007
The greatest Pharmacompanies (2005, in billion Euro)
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The greatest Pharmacompanies (2005, in billion Euro)
0 10 20 30 40
Pfizer, USA
GlaxoSmithKline, GB
Sanofi-Aventis, F
Novartis+Chiron, CH
Johnson & Johnson, USA
AstraZeneca, GB
Merck & Co., USA
Roche, CH
Abbott, USA
Wyeth, USA
Bristol-Myers Squibb, USA
Lilly, USA
Amgen, USA
Bayer+Schering, D
Boehringer Ingelheim, D
Takeda, J
Schering Plough, USA Teva+Ivax, Israel
Daiichi Sankyo, J
Eisai, J
WirtschaftsWoche
Page 51
08/06
And the most dynamic Pharmacompanies (2005, in %)
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And the most dynamic Pharmacompanies (2005, in %)1. Boehringer Ingelheim, D
2. Amgen, USA
3. Teva+Ivax, Israel
4. Novo Nordisk, DK
5. Roche, CH
6. Otsuka, J
7. Merck KGaA, D
8. Novart is+Chiron, CH
9. AstraZeneca, GB
10. Abbott, USA
11. Sanofi-Aventis, F
12. Daiichi Sankyo, J
13. Takeda, J14. Lilly, USA
15. Eisai, J
16. Bayer+Schering, D
17. GlaxoSmithKline, GB
18. Astellas Pharma, J
19. Wyeth, USA20. Schering Plough, USA
21. Johnson & Johnson, USA
22. Merck & Co., USA
23. Bristol-Myers Squibb, USA
24. Pfizer, USA
-10 0 10 20 30
WirtschaftsWoche
Page 51
08/06
Expression Platforms
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MicrobesE.coli & Yeast
Expression Platforms
transgenic animalscell lines
Transgenic plants
Fermentation Production Platforms
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Fermentation Production Platforms
1
Advantages
cGMP, cGLP compliant
biologically contained
upscaling for:
E. coli
yeast animal cells
insect cells
plant cells high cell density
reproducibility
yield optimization
Limitations
initial investment
expert staff required engineering & infrastructure
demands
Fermenatation Facility
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y
Cell Separation and StreamlineTM IMAC
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p
Downstream Processing of Recombinant Proteins
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g
Bacterial Expression Platform
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1
Advantages
rapid cloning and expression
yields: 100-1500 mg/L
low cost
proven technology (FDA)
Limitations
most complex proteins are inactive
no eukaryotic post-translationalmodification
poor post-translational assembly
many proteins are mis-folded, costly
re-folding is required: poor yields
endotoxins
P. pastoris Microbial Expression Platform
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p
1
Advantages
commercial systems
yields: >100mg/L
low cost, defined medium
fermentation ready
proven technology
rapid fermentation
Limitations
intracellular proteins often inactive
tedious clone generation
hyper-glycosylation is possible
MeOH use (toxic, explosive)
Mammalian Cell Expression Platform
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1
Advantages
active complex proteins
more authentic glycosylation
well-defined system
FDA approval
Limitations
expensive infrastructure
high media costs
poor assembly of complex
proteins (sIgA)
long development time
viral contamination
oncogene contamination
Transgenic Animal Expression Platform
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1
Advantages
active complex proteins
accurate glycosylation
yields up to 40g/L milk
herds can be bred
Limitations
very slow (years for cloning)
upscale slow (breeding)
BSE and other pathogens
cloning very labor intensive and yet
to be optimised
host protein contamination
expensive: 100K to 300K$/animal
public ethical concerns
Transgenic Plant Expression Platform
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1
Advantages
low initial investment
medium time scale (months)
unlimited scale-up potential
faster than transgenic animals
very low costs
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Production of recombinant proteins in:
intact plants or Bioreactors
Field Trials with GM Plants
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R & D
product developmentprotein engineering
research &
development
small scale pharmaceutical
production in plant cells
Molecular Farming,upscaled production
large scale production
purification
QC, FDA,
marketing
product/technology
transfer
Traditional Breeding and Genetic Modification (GM)
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humanity has been shaping its environment for millenia
wheat, rice, corn, grape are all the product of breeding
GM permits introduction of desirable traits
all our staple crops are GM through plant breeding
traditional breeding is at its limit
GM gives us new opportunities
Our Food Supply
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world population today is ~ 5 billion
this figure will double by 2030
agricultural land is ~ 1.4 billion hectares
decreasing by erosion, salinization and urban growth
it will be ~ 50% less by 2030
~10 billion people to
feed on half the land
The Worlds most important Crops
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How can we obtain better Crops ?
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1. selection
2. breeding
3. hybridization
4. cloning
5. grafting
6. radiation mutagenesis
7. chemical mutagenesis
8. gene splicing
9. genomics/gene expression
10. tissue culture
Major Inputs in Crop Production
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breeding (hybrids)
mechanization
fertilizers
irrigation
crop protectants
Cro
pproductivity/impro
vement
past present future
information
biotechnology
smart breeding
Plant Biotechnology Platforms and Areas of Interest
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enabling technologies: molecular biology, genetics, biochemistry, cell biology
gene expression, promoters, targeting signals
transformation & regeneration technologies
engineering of input traits:
biotic (pathogen) and abiotic stress (salt, drought, frost, UV, herbicides)
metabolic engineering (carbohydrates, starch, lipids, shapes & colours)
engineering of output traits:
product quality (shelf life, nutraceuticals, functional food)
production of enzymes and speciality chemicals, silk fibre monomers
production of diagnostics, therapeutics, blood substitutes and vaccines
phytoremediation
Engineering Viral Resistance inNicotiana tabacum
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virus infected wild type virus resistant transgenic lineexpressing a molecularpathogenicide
Fungal Resistance
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fungus infected
wild type
fungus resistant
transgenic line
Insect Resistance
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anti-feedant proteins
Bacillus thuringensis d-endotoxins
Bt transformed cotton untransformed cotton
Increase Use of Transgenic Crops
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from one million acres to more than ninety million acres in six years
Soybeans 54%* Cotton 61%*
Corn 25%*
*32 % intended 2002
*74% intended 2002 *71% intended 2002
*= per cent of 2001 actual acres *= percent of intended 2002 acres, USDA
The Future of Biotechnology
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TOOLS
PRODUCTS
TARGETS
Monoclonalantibodies
RecombinantDNA
PCR
Gene therapy
Humanized
monoclonals
Genomics
Combinatorialchemistry
Molecularmodeling
Antisensemolecules
Kinases
Largemolecules
Hormones
Smallmolecules
Ex vivo celltherapy
Liposomaldrugs
Oralavailability
Anemia In vitrodiagnostics
In vivodiagnosticsCancer
CNS disorders AlzheimersHormone
deficiencies Inflammation
VirusesInfections
Targeteddelivery
Blood celldisorders
In vivo genetherapy
Past Present Future
Hot Jobs in Biotechnology
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Rational protein design and in silico structure resolution
High throughput screening (genomics, proteomics, imaging)
Large-scale cell culture
Process engineering and scale-up development
Protein purification and downstream processing
cGMP and validation & regulatory affairs
Bioinformatics
Combinatorial chemistry & biology
Corporate development