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Topik Kuliah: Microbial Biotechnology 1. 2. Bioteknologi; definisi dan sejarahnya &Teknologi DNA Rekombinan Bioremediation and biomass utilization 3. 4.

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Presentation on theme: "Topik Kuliah: Microbial Biotechnology 1. 2. Bioteknologi; definisi dan sejarahnya &Teknologi DNA Rekombinan Bioremediation and biomass utilization 3. 4."— Presentation transcript:

1 Topik Kuliah: Microbial Biotechnology Bioteknologi; definisi dan sejarahnya &Teknologi DNA Rekombinan Bioremediation and biomass utilization Ethanol Microbial cell fuel Bioplastics produced by microorganisms Probiotics, Prebiotics and Synbiotics Biocatalysis /biosensor Molecular diagnostics Vaccines and therapeutics agents Plant-growth promoting bacteria Microbial insecticides Microbial synthesis of commercial products Large-scale production of proteins from recombinant IRM ATW microorganisms 14. Regulasi & paten microb produk bioteknologi Bacaan : Glazer AN & Nikaido H Microbial Biotechnology, Fundamentals of Applied Microbiology, 2 nd Edition. Cambridge University Press. Cambridge. Microbial Biotechnology Biotechnology: Definition & History Recombinant DNA Technology 1

2 2 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 in 1917 to describe processes using living organisms to make a product or run a process, such as industrial fermentations. (Robert Bud, The Uses of Life: A History 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 make possible new products and processes and provide solutions to society's ills (G. Kirk Raab, Former President and CEO of Genentech) WEBSTER’S: The aspect of technology concerned with the application of living organisms to meet the needs of man. WALL STREET: Biotechnology is the application of genetic engineering and DNA technology to produce therapeutic and medical diagnostic products and processes. Biotech companies have one thing in common - the use of genetic engineering and manipulation of organisms at a molecular level.

3 3 What is biotechnology? Using scientific methods with organisms to produce new products or new forms of organisms Anytechniquethatuseslivingorganismsor substances from those organisms or substances from those organisms to make or modify a product, to improveplantsoranimals,ortodevelop microorganisms 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

4 4 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 10 6 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

5 5 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  CO 2 + ethanol (anaerobic process) 1915 – Production of baker’s yeast – Saccharomyces cerevisiae Ancient biotechnology Fermented foods and beverages

6 6 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

7 7 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

8 8 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 – 32 P DNA, not 35 S 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

9 DNA  RNA  Protein TranscriptionTranslation Genetic code determined for all 20 amino acids by Marshal Nirenberg and Heinrich Matthaei and Gobind Khorana – Nobel Prize – base sequence = codon 9 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

10 10 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

11 11 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

12 Type IType IIType III Functions Endonuclease & methylase Endonuclease Conditions 2+ ATP, Mb 2+ Mg 2+ ATP, Mg Recognition sequences EcoK: AACN 6 GTGC EcoB: TGAN 8 TGCT Palindromic EcoP1: AGACC EcoP15: CAGCAG Cutting sitesAt least 1000bp away At or close to recog. seq bp away 12 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

13 5’ GAATTC 3’ 3’ CTTAAG 5’ e.g. EcoRI site: Recognition sequences Recognize 4-8 bp palindromic sequences. Most commonly used enzymes recognize 6 bp which occurs at a rate of 4 6 =4096 bp. (4 4 =256 bp; 4 8 =65536 bp) 5’-CCCGGG-3’ 3’-GGGCCC-5’ p -GGG-3’ OH-CCC-5’ 5’-CCC-OH + 3’-GGG- p SmaI blunt ends 13 Restriction enzymes 1. Highly specific 2. Commercially available 3. Require Mg2+ for enzymatic activity 4. Compatible ends from different enzymes, Restriction sequences Cohesive/sticky ends

14 14 - ve electrode + ve electrode Restriction digestion Agarose gel electrophoresis Agarose: a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution (0.5%-2%)

15 15 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

16 16 Covalently join the DNA molecules with the base-pairing cohesive ends, or blunt ends, if the 5’-ends have phosphate groups. DNA ligation Recombinant DNA molecules

17 Restriction Endonucleases 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 17

18 18 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

19 19 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 Cell Culture Genetic Engineering Anti-cancer drugs Diagnostics Culture of plants from single cells Transfer of new genes into animal organisms specific DNA probes Localisation of genetic disorders Tracers Synthesis of Gene therapy Cloning Mass prodn. of human proteins Resource bank for rare human chemicals New antibiotics Synthesis of new proteins New types of plants and animals New types of food DNA technology Crime solving Molecular Biology Banks of DNA, RNA and proteins Complete map of the human genome

20 20 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

21 21 Transgenic rice “Golden rice” shown intermixed with white rice contain high concentrations of beta-carotene Transgenic Rice

22 22 Bovine Somatotropin Applications of Recombinant DNA Recombinant 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

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24 24 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.

25 25 Social Concerns and Consequences Will some genetically engineered organisms be harmful either to other organisms or to the environment? Will the development and use of genetically engineered organisms reduce natural genetic diversity? Should humans be genetically engineered? Will diagnostic procedures undermine individual privacy? Will financial support for molecular biotechnology constraint the development of other important technologies? Will the emphasis on commercial success mean that benefits of molecular biotechnology will be available only to wealthy nations? Will agricultural biotechnology undermine traditional farming practices? Will medical therapies based on molecular biotechnology supersede equally effective traditional treatments? Will the quest for patent inhibit the free exchange of ideas among research scientists?

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