1 2. Introduction to Molecular Biotechnology 2. Introduction to Molecular Biotechnology Bi7430 Molecular Biotechnology Definition of biotechnology History of biotechnology Fundamentals of molecular biotechnology Basic concept of rDNA technology Methods of gene transfer Main fields of biotech applications Positive aspects, concerns and consequences Outline biotechnology („biotech") bios – techne – logos Kalr Ereky, 1917 – „biotechnology is a process by which raw materials could be biologically upgraded into socially useful products“ Definition of biotechnology „any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use“ (The United Nations Convention on Biological Diversity, 1992) 2 a story that began long time ago 10,000 B.C. neolitic revolution cultivation and domestications 8,000 B.C. fermented bread (ancient Egypt) 8,000 B.C. cheese making (the Middle East) 6,000 B.C. wine production (Egypt and the Middle East) 5,000 B.C. brewing (ancient Egypt) develo pe d without any knowled g e about existence of cells or enzymes History of biotechnology 1665 Robert Hooke - term the cell 1675 Anton Van Leeuwenhoek - the father of microbiology 1839 Matthias Schleiden, Theodore Schwann and Rudolf Virchow - the cell theory History of biotechnology 1822-95 Louis Pasteur – germ theory, pasteurisation, vaccines 1859 Charles Darwin - evolutionary theory 1866 Gregor Johann Mendel - laws of inheritance 1869 Johann Miescher - discovery of DNA 1900 rediscovery of Mendelism History of biotechnology 3 1902 Walter Sutton - chromosome theory of heredity 1910 Thomas Morgan - genes are carried on chromosomes, basis of modern genetics (Nobel Prize in 1933) 1928 Frederick Griffith - bacterial transformation History of biotechnology 1944 Oswald Avery – DNA the genetic carrier 1952 Joshua Lederberg – conjugation and plasmids (Nobel Prize in 1958) 1953 James Watson nad Francis Crick, Maurice Wilkins and Rosalind Franklin – structure of DNA (Nobel Prize in 1962) 1967 Hargobind Khorana, Marshal Nirenberg, Robert Holley nucleotides carry the genetic code (Nobel Prize in 1968) History of biotechnology 1970s biotechnology recognized as scientific discipline (interlin k of chemical engineerin g, microbi ol o g y nad biochemis tr y ) traditional biotechnology – based on fermentation development focused on process technology (biorea ct or design, upstream, downstream ) History of biotechnology 4 1970s biotechnology recognized as scientific discipline (interlin k of chemical engineerin g, microbi ol o g y nad biochemis tr y ) traditional biotechnology – based on fermentation development focused on process technology (biorea ct or design, upstream, downstream ) biotransformation component natural strains - far from optimum difficult to optimise induced mutagenesis and selection (chemica l mutagens, UV radiat i on) limite d by inherite d propert i es of the strain History of biotechnology Available strain Available strain Adopted strain MOLECULAR BIOTECHNOLOGY REVOLUTION 1973 Stanley Kohen and Herbert Boyer - development of recombinant DNA technology History of biotechnology g e n e t i c e n g i n e e r i n g p r o v i d e d t h e m e a n s t o c r e a t e , r a t h e r t h a n m e r e l y i s o l a t e , h i g h l y p r o d u c t i v e s t r a i n s MOLECULAR BIOTECHNOLOGY REVOLUTION 1973 Stanley Kohen and Herbert Boyer - development of recombinant DNA technology 1976 Herbert Boyer and Robert Swanson 1978 production of human insulin in E. coli by Genentec (recombin ant "human" insulin approved by FDA 1982) 1981 production of recombinant growth hormone 1987 production of recombinant tissue plasminogen activator used to dissolve blood clots during myocardial infarction 1980-83 about 200 small biotechnological companies founded in US History of biotechnology 5 History of biotechnology MOLECULAR BIOTECHNOLOGY REVOLUTION 1974 Rudolf Jaenisch - first transgenic mammal (a mouse) 1982 first recombinant animal vaccine approved 1983 engineered Ti plasmid – plant transformation 1988 Kary Mullis - PCR method (Nobel Prize in 1993) 1994 first genetically engineered food aproved by FDA (tomato) a n i m a l s a n d p l a n t s b e c a m e t a r g e t s t o a c t a s n a t u r a l b i o r e a c t o r s History of biotechnology MOLECULAR BIOTECHNOLOGY REVOLUTION 1995 first genome sequenced (bacterium Haemophilus influenzae) 1996 complete eukaryotic DNA sequence 1996 commercial planting of GMO crops begins 1997 Ian Wilmut – nuclear cloning of a mammal 1998 first antisense drug approved by FDA 1999 Drosophilia genome sequenced 2000 Arabidopsis genome sequenced 2000 development of „golden rice“ 2001 human genome sequenced 2009 first drug produced in genetically engineered animal (a goat) THE ORGANISM? Molecular biotechnology classical biotechnology based on selective breeding molecular (modern) biotechnology („mol biotech“) is revolutionary scientific discipline based on gene manipulation the ability to transfer specific units of genetic information from one organism to another recombinant DNA (rDNA) technology modern genetic engineering enable create rather then isolate highly productive organisms 6 Basic concept of rDNA technology isolate gene(s) of interest modify gene(s) protein engineer ing (Lecture 3) metabol i c engineerin g (Lecture 4-5) ligate gene(s) into a vector transform host organism Techniques of DNA transfer transformation and transfection direct methods electropora ti on (2.5 kV, 5 ms) chemical transformat i on (CaCl2 ) heat shock (42°C) micro-inject ion biolistic deliver y - „gene gun“ liposomal transfecti on indirect methods transduction (bacter io ph a g e) viral and bacteri a l „infection“ Basic concept of rDNA technology isolate gene(s) of interest modify gene(s) protein engineer ing (Lecture 3) metabol i c engineerin g (Lecture 4-5) ligate gene(s) into a vector transform host organism select transformed cells culture host organism application of gene product 7 Molecular biotechnology M O L E C U L A R B I O T E C H N O L O G Y m i c r o b i o l o g y ( b i o ) c h e m i s t r y g e n e t i c s e n g i n e e r i n g i m m u n o l o g y m o l . b i o l o g yc e l l b i o l o g y m e t a b o l i c e n g i n e e r i n g C R O P S D R U G S V A C C I N E S D I A G N O S T I C S L I V E S T O C K p r o t e i n e n g i n e e r i n g Main fields of application white - industrial biotechnology (Lecture 6) p r o d u c t i o n o f f i n e c h e m i c a l s p r o d u c t i o n o f p r o t e i n s / e n z y m e s green - agricultural biotechnology (Lecture 7) t ra n s g e n i c p l a n t s a n d a n i m a l s b i o f e r t i l i z e r s a n d b i o p e s t i c i d e s red - medical biotechnology (Lecture 8-10) d e v e l o p i n g n e w va c c i n e s a n d d r u g s t i s s u e e n g i n e e r i n g a n d r e g e n e ra t i v e t h e ra p i e s m o l e c u l a r d i a g n o s t i c s a n d p h a r m a c o g e n o m i c s c e l l a n d g e n e t h e ra p y grey - environmental biotechnology (Lecture 11) b i o s e n s i n g a n d b i o r e m e d i a t i o n blue - marine and aquatic Pros and cons safety and ethical concerns of molecular biotechnology Do we have a right to move genes, creating new life forms ... „playing God“? Will transgenic organisms be harmfull to other organism or environment? Should humans be genetic a l ly engineer e d? positive aspects of molecular biotechnology opportunit i es to accurate l l y diagnose, prevent and cure a wide range of infectious and genetic diseases increase crop yield and resistence to insects and diseases, environment al stress (e.g., drought, heat, cold) develo p microorg an isms that produce chemicals in sustainabl e manner facilit at e removal of pollutants and waste materia ls from environment