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Topik Kuliah: Microbial Biotechnology1. Bioteknologi; definisi dan sejarahnya &Teknologi DNA
Rekombinan2. Bioremediation and biomass utilization3. Ethanol4. Microbial cell fuel5. Bioplastics produced by microorganisms6. Probiotics, Prebiotics and Synbiotics7. Biocatalysis in organic chemistry
1. Molecular diagnostics2. Vaccines and therapeutics agents3. Plant-growth promoting bacteria4. Microbial insecticides5. Microbial synthesis of commercial products6. Large-scale production of proteins from recombinant
microorganisms7. Regulasi & paten microb produk bioteknologi
Bacaan : Glazer AN & Nikaido H. 2007. Microbial Biotechnology, Fundamentals of Applied Microbiology, 2nd
Edition. Cambridge University Press. Cambridge.
IRM
ATW
What is biotechnology? • Biotechnology = bios (life) + logos (study of or
essence)– Literally ‘the study of tools from living things’
• CLASSIC: The word "biotechnology" was first used in1917 to describe processes using living organisms tomake a product or run a process, such as industrialfermentations. (Robert Bud, The Uses of Life: AHistory of Biotechnology)
• LAYMAN: Biotechnology began when humans began to plant their own crops, domesticate animals, ferment juice into wine, make cheese, and leaven bread (AccesExcellence)
What is biotechnology? • GENENTECH: Biotechnology is the process of
harnessing 'nature's own' biochemical tools to makepossible new products and processes and providesolutions to society's ills (G. Kirk Raab, FormerPresident and CEO of Genentech)
• WEBSTER’S: The aspect of technology concernedwith the application of living organisms to meet theneeds of man.
• WALL STREET: Biotechnology is the application ofgenetic engineering and DNA technology to producetherapeutic and medical diagnostic products andprocesses. Biotech companies have one thing incommon - the use of genetic engineering andmanipulation of organisms at a molecular level.
What is biotechnology?
• Using scientific methods with organisms to producenew products or new forms of organisms
• Any technique that uses living organisms orsubstances from those organisms or substances fromthose organisms to make or modify a product, toimprove plants or animals, or to developmicroorganisms for specific uses
What is biotechnology?
• Biotechnology is a multidisciplinarian in nature, involving input from
• Engineering• Computer Science• Cell and Molecular Biology• Microbiology• Genetics• Physiology• Biochemistry• Immunology• Virology• Recombinant DNA Technology Genetic manipulation
of bacteria, viruses, fungi, plants and animals, often for the development of specific products
What are the stages of biotechnology?
• Ancient Biotechnology• early history as related to food and shelter,
including domestication
• Classical Biotechnology• built on ancient biotechnology• fermentation promoted food production• medicine
• Modern Biotechnology• manipulates genetic information in organism• genetic engineering
Ancient biotechnology
• Paleolithic society – Hunter-gatherers Nomadic lifestyle due to migratory animals and edible plant distribution (wild wheat and barley) (~2 x 106 yrs.)
• Followed by domestication of plants and animals (artificial selection) People settled, sedentary lifestyles evolved (~10,000 yrs. ago)• Cultivation of wheat, barley and rye (seed
collections)• Sheep and goats milk, cheese, button and
meat• Grinding stones for food preparation• New technology Origins of Biotechnology
Agrarian Societies
History of domestication and agriculture
• Long history of fermented foods since people began to settle (9000 BC) (fervere –to boil)
• Often discovered by accident!
• Improved flavor and texture
• Deliberate contamination with bacteria or fungi (molds)
• Examples:•Bread•Yogurt•Sour cream•Cheese•Wine•Beer•Sauerkraut
Ancient biotechnology Fermented foods and beverages
• Dough not baked immediately would undergo spontaneous fermentation would rise
• Uncooked fermented dough could be used to ferment a new batch no longer reliant on “chance fermentation”
• 1866 – Louis Pasteur published his findings on the direct link between yeast and sugars CO2 + ethanol (anaerobic process)
• 1915 – Production of baker’s yeast –Saccharomyces cerevisiae
Ancient biotechnology Fermented foods and beverages
•Different types of beer•Vinegar•Glycerol•Acetone•Butanol•Lactic acid•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……..)
Classical biotechnology Industry today exploits early discoveries of the fermentation
process for production of huge numbers of products
• Substrate + Microbial Enzyme Product
• Examples:• Cholesterol Steroids (cortisone, estrogen, progesterone) (hydroxylation reaction -OH group added to cholesterol ring)
Classical biotechnology
Chemical transformations to produce therapeutic products
• Amino acids to improve food taste, quality or preservation
• Enzymes (cellulase, collagenase, diastase, glucose isomerase, invertase, lipase, pectinase, protease)
• Vitamins
• Pigments
Classical biotechnology
Microbial synthesis of other commercially valuable products
• Cell biology• Structure, organization and reproduction
• Biochemistry• Synthesis of organic compounds• Cell extracts for fermentation (enzymes versus whole cells)
• Genetics• Resurrection of Gregor Mendel’s findings 1866 1900s
• Theory of Inheritance (ratios dependent on traits of parents)• Theory of Transmission factors
• W.H. Sutton – 1902• Chromosomes = inheritance factors
• T.H. Morgan – Drosophila melanogaster
Modern biotechnology
Molecular Biology
• Beadle and Tatum (Neurospora crassa)• One gene, one enzyme hypothesis
• Charles Yanofsky colinearity between mutations in genes and amino
acid sequence (E. coli)• Genes determine structure of proteins
• Hershey and Chase – 1952 • T2 bacteriophage – 32P DNA, not 35S protein
is the material that encodes genetic information
Modern biotechnology
• 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
• DNA Revolution – Promise and Controversy!!!
• Scientific foundation of modern biotechnology • based on knowledge of DNA, its replication, repair and use of enzymes to carry out in vitro splicing DNA fragments
Modern biotechnology
• 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
• Vernon Ingram’s research on sickle cell anemia (1956) tied together inheritable diseases with protein structure
• Link made between amino acids and DNA
• Radioactive tagging experiments demonstrate intermediate between DNA and protein = RNA
• RNA movement tracked from nucleus to cytoplasm site of protein synthesis
Modern biotechnology
• DNA RNA ProteinTranscription 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
Modern biotechnology
What are the areas of biotechnology?
• Organismic biotechnology• uses intact organisms and does not alter genetic
material
• Molecular Biotechnology• alters genetic makeup to achieve specific goals
Transgenic organism: an organism with artificially altered genetic material
Recombinant DNA
• Recombinant DNA is a molecule that combines DNA from two sources
• Also known as gene cloning• Creates a new combination of genetic material • Human gene for insulin was placed in bacteria• The bacteria are recombinant organisms and
produce insulin in large quantities for diabetics • Genetically modified organisms are possible
because of the universal nature of the genetic code
Basic Cloning Process
•Plasmid is cut open with a restriction enzyme that leaves an overhang: a sticky end•Foreign DNA is cut with the same enzyme.•The two DNAs are mixed. The sticky ends anneal together, and DNA ligase joins them into one recombinant molecule.•The recombinant plasmids are transformed into E. coli using heat plus calcium chloride.•Cells carrying the plasmid are selected by adding an antibiotic: the plasmid carries a gene for antibiotic resistance.
• Recombinant DNA methods– Restriction enzymes
• Enzymes from bacteria• Used to cut DNA molecules in specific places• Enable researchers to cut DNA into manageable segments
– Vector molecule carrier of DNA fragment into cell– Transformation: uptake of foreign DNA into cells
• Restriction endonucleases
– recognize specific nucleotide sequences, and cleave DNA creating DNA fragments.
• Each restriction endonuclease has a specific recognition sequence and can cut DNA from any source into fragments.
• Because of complementarity, single-stranded ends can pair with each other.
– sticky ends» fragments joined together with DNA ligase
Types of Restriction endonuclease
Type I Type II Type IIIFunctions Endonuclease &
methylaseEndonuclease Endonuclease
Conditions ATP, Mb2+ Mg2+ ATP, Mg2+
Recognition sequences
EcoK: AACN6GTGCEcoB: TGAN8TGCT
Palindromic EcoP1: AGACCEcoP15: CAGCAG
Cutting sites At least 1000bp away
At or close to recog. seq
24-26 bp away
Restriction enzymes
Recognize 4-8 bp palindromic sequences. Most commonly used enzymes recognize 6 bp which occurs at a rate of 46=4096 bp. (44=256 bp; 48=65536 bp)
1. Highly specific2. Commercially available3. Require Mg2+ for enzymatic activity4. Compatible ends from different enzymes,
5’ GAATTC 3’3’ CTTAAG 5’
e.g. EcoRI site:
Recognition sequences
5’-CCCGGG-3’3’-GGGCCC-5’
5’-CCC-OH3’-GGG- p
p -GGG-3’OH-CCC-5’
+SmaI
blunt ends
Cohesive/sticky ends
Restriction sequences
Agarose: a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution (0.5%-2%)
- ve electrode + ve electrode
Agarose gel electrophoresis
Creating Recombinant DNA Molecules
• Cut DNA from donor and recipient with the same restriction enzymes
• Cut DNA fragment is combined with a vector• Vector DNA moves and copies DNA fragment of
interest• Vector cut with restriction enzymes • The complementary ends of the DNAs bind and
ligase enzyme reattaches the sugar-phosphate backbone of the DNA
Covalently join the DNA molecules with the base-pairing cohesive ends, or blunt ends, if the 5’-ends have phosphate groups.
DNA ligation
Cloning Vector Types
• For different sizes of DNA:– plasmids: up to 5 kb– phage lambda (λ) vectors: up to 50 kb– BAC (bacterial artificial chromosome): 300 kb– YAC (yeast artificial chromosome): 2000 kb
• Expression vectors: make RNA and protein from the inserted DNA– shuttle vectors: can grow in two different
species
Plasmid Vectors
• To replicate, a plasmid must be circular, and it must contain a replicon, a DNA sequence that DNA polymerase will bind to and initiate replication. Also called “ori” (origin of replication).
– Replicons are usually species-specific.– Some replicons allow many copies of the
plasmid in a cell, while others limit the copy number or one or two.
• Plasmid cloning vectors must also carry a selectable marker: drug resistance. Transformation is inefficient, so bacteria that aren’t transformed must be killed.
• Most cloning vectors have a multiple cloning site, a short region of DNA containing many restriction sites close together (also called a polylinker). This allows many different restriction enzymes to be used.
• Most cloning vectors use a system for detecting the presence of a recombinant insert, usually the blue/white beta-galactosidase system.
What are the benefits of biotechnology?
• Medicine• human• veterinary• biopharming
• Environment• Agriculture• Food products• Industry and manufacturing
What are the applications of biotechnology?
• Production of new and improved crops/foods, industrial chemicals, pharmaceuticals and livestock
• Diagnostics for detecting genetic diseases• Gene therapy (e.g. ADA, CF)• Vaccine development (recombinant vaccines)• Environmental restoration• Protection of endangered species• Conservation biology• Bioremediation• Forensic applications• Food processing (cheese, beer)
Monoclonal Antibodies
Molecular Biology
CellCulture
Genetic Engineering
Anti-cancer drugs
DiagnosticsCulture of plants from single cells
Transfer of new genes into animal
organisms
Synthesis of specific DNA
probes
Localisation of genetic disorders
Tracers
Cloning
Gene therapy
Mass prodn. of human proteins
Resource bank for rare human chemicals
Synthesis of new proteins
New antibiotics
New types of plants and animals
New types of food
DNA technology
Crime solving
Banks of DNA, RNA and proteins
Complete map of the human genome
Agricultural Applications
• Ti plasmid has been early successful vector.– nitrogen fixation
• introduce genes that allow crops to fix nitrogen– reduce need for fertilizer
– herbicide resistance• insert genes encoding for proteins making crops
resistant to herbicide– widespread herbicide use possible
Agricultural Applications
Insect resistance• insert genes encoding proteins harmful to insects
• Real promise - produce genetically modified plants with traits benefiting consumers– iron deficiency in developing countries
• transgenic rice
– increasing milk production• bovine somatotropin
Transgenicrice
“Golden rice”shown intermixedwith white ricecontain highconcentrationsof beta-carotene
Applications of Recombinant DNARecombinant DNA is used to:• Study the biochemical properties or genetic pathways of that
protein• Mass produce a particular protein (e.g., insulin)• Sometimes conventional methods are still the better choice• Textile industry can produce the dye indigo in E. coli by
genetically modifying genes of the glucose pathway and introducing genes from another bacterial species
Benefits of Biotechnology:1. Provide opportunities to accurately diagnose and
prevent or cure a wide range of infectious and genetic diseases.
2. Significantly increase crop yields by creating plants that are resistant to insect predation, fungal and viral diseases, and environmental stresses such as short-term drought and excessive heat.
3. Develop microorganisms that will produce chemical, antibiotics, polymers, amino acids, enzymes, and various food addiitives.
4. Develop livestock and other animal that have enhanced genetically determined attibutes.
5. Facilitate the removal of pollutants and waste materials from the environment.
Social Concerns and Consequences1. Will some genetically engineered organisms be harmful either to other
organisms or to the environment?2. Will the development and use of genetically engineered organisms
reduce natural genetic diversity?3. Should humans be genetically engineered?4. Will diagnostic procedures undermine individual privacy?5. Will financial support for molecular biotechnology constraint the
development of other important technologies?6. Will the emphasis on commercial success mean that benefits of
molecular biotechnology will be available only to wealthy nations?7. Will agricultural biotechnology undermine traditional farming practices?8. Will medical therapies based on molecular biotechnology supersede
equally effective traditional treatments?9. Will the quest for patent inhibit the free exchange of ideas among
research scientists?