Topik Kuliah: Microbial Biotechnology 1. 2. Bioteknologi; definisi dan sejarahnya &Teknologi DNA Rekombinan Bioremediation and biomass utilization 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 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. 2007. Microbial Biotechnology, Fundamentals of Applied Microbiology, 2nd Edition. Cambridge University Press. Cambridge. Microbial Biotechnology Biotechnology: Definition & History Recombinant DNA Technology 1
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Topik Kuliah: Microbial Biotechnology1.
2.
Bioteknologi; definisi dan sejarahnya &Teknologi DNARekombinanBioremediation and biomass utilization
3.4.5.6.7.
8.9.10.11.12.13.
EthanolMicrobial cell fuelBioplastics produced by microorganismsProbiotics, Prebiotics and SynbioticsBiocatalysis /biosensor
Molecular diagnosticsVaccines and therapeutics agentsPlant-growth promoting bacteriaMicrobial insecticidesMicrobial synthesis of commercial productsLarge-scale production of proteins from recombinant
IRM
ATW
microorganisms14. Regulasi & paten microb produk bioteknologi
Bacaan : Glazer AN & Nikaido H. 2007. MicrobialBiotechnology, Fundamentals of Applied Microbiology, 2nd
Edition. Cambridge University Press. Cambridge.
Microbial Biotechnology
Biotechnology:Definition & HistoryRecombinant DNA
Technology
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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 beganto plant their own crops, domesticate animals,ferment juice into wine, make cheese, and leavenbread (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.
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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
••••••••••
EngineeringComputer ScienceCell and Molecular BiologyMicrobiologyGeneticsPhysiologyBiochemistryImmunologyVirologyRecombinant DNA Technology Genetic manipulationof bacteria, viruses, fungi, plants and animals, often forthe development of specific products
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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 Nomadiclifestyle due to migratory animals and edible plantdistribution (wild wheat and barley) (~2 x 106 yrs.)
• Followed by domestication of plants and animals(artificial selection) People settled, sedentarylifestyles 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
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• Long history of fermented foods since peoplebegan to settle (9000 BC) (fervere –to boil)
• Often discovered by accident!
• Improved flavor and texture
• Deliberate contamination with bacteria orfungi (molds)
• Dough not baked immediately would undergospontaneous fermentation would rise
• Uncooked fermented dough could be used toferment a new batch no longer reliant on“chance fermentation”
• 1866 – Louis Pasteur published his findings onthe direct link between yeast and sugars CO2 +
ethanol (anaerobic process)
• 1915 – Production of baker’s yeast –Saccharomyces cerevisiae
Ancient biotechnology
Fermented foods and beverages
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•Different types of beer•Vinegar•Glycerol•Acetone•Butanol•Lactic acid•Citric acid•Antibiotics – WWII (Bioreactor developed for largescale production, e.g. penicilin made by fermentationof penicillium)
•Today many different antibiotics are produced bymicroorganisms•Cephalosporins, bacitracin, neomycin,tetracycline……..)
Classical biotechnology
Industry today exploits early discoveries of the fermentationprocess for production of huge numbers of products
• Beadle and Tatum (Neurospora crassa)• One gene, one enzyme hypothesis
• Charles Yanofsky colinearitybetween 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 geneticinformation
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 vitrosplicing DNA fragments
Modern biotechnology
• DNA RNA ProteinTranscription Translation
Genetic code determined for all 20 amino acids byMarshal Nirenberg and Heinrich Matthaei and GobindKhorana – Nobel Prize – 1968
• 3 base sequence = codon
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Modern biotechnology• Breaking the Genetic Code – Finding the CentralDogma
• An “RNA Club” organized by George Gamow (1954)assembled to determine the role of RNA in proteinsynthesis
• 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 demonstrateintermediate between DNA and protein = RNA
• RNA movement tracked from nucleus to cytoplasm site ofprotein synthesis
Modern biotechnology
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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 artificiallyaltered genetic material
Recombinant DNA
• Recombinant DNA is a molecule that combinesDNA 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 andproduce insulin in large quantities for diabetics
• Genetically modified organisms are possiblebecause of the universal nature of the genetic code
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Basic Cloning Process
•Plasmid is cut open with a restrictionenzyme that leaves an overhang: asticky end•Foreign DNA is cut with the sameenzyme.•The two DNAs are mixed. Thesticky ends anneal together, and DNAligase joins them into onerecombinant molecule.•The recombinant plasmids aretransformed into E. coli using heatplus calcium chloride.•Cells carrying the plasmid areselected by adding an antibiotic: theplasmid carries a gene for antibioticresistance.
• 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
Type I Type II Type III
Functions Endonuclease &methylase
Endonuclease Endonuclease
Conditions 2+ATP, Mb
2+Mg
2+ATP, Mg
Recognitionsequences
EcoK: AACN6GTGC
EcoB: TGAN8TGCT
Palindromic EcoP1: AGACC
EcoP15: CAGCAG
Cutting sites At least 1000bpaway
At or close torecog. seq
24-26 bp away
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• Restriction endonucleases
– recognize specific nucleotide sequences, and cleaveDNA creating DNA fragments.
• Each restriction endonuclease has a specific recognitionsequence and can cut DNA from any source intofragments.
• Because of complementarity, single-stranded endscan pair with each other.– sticky ends
» fragments joined together with DNA ligase
Types of Restriction endonuclease
5’ GAATTC 3’3’ CTTAAG 5’
e.g. EcoRI site:
Recognition sequencesRecognize 4-8 bp palindromic sequences. Most commonly
used enzymes recognize 6 bp which occurs at a rateof 46=4096 bp. (44=256 bp; 48=65536 bp)
5’-CCCGGG-3’3’-GGGCCC-5’
p -GGG-3’OH-CCC-5’
5’-CCC-OH +3’-GGG- p
SmaI
blunt ends
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Restriction enzymes1. Highly specific2. Commercially available3. Require Mg2+ for enzymatic activity4. Compatible ends from different enzymes,
Restriction sequences
Cohesive/sticky ends
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- ve electrode + ve electrode
Restriction digestion
Agarose gel electrophoresisAgarose: a polysaccharide derived from seaweed, whichforms a solid gel when dissolved in aqueous solution (0.5%-2%)
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Agarose gelelectrophoresis
Creating Recombinant DNAMolecules
• Cut DNA from donor and recipient with thesame restriction enzymes
• Cut DNA fragment is combined with a vector
• Vector DNA moves and copies DNA fragment ofinterest
• Vector cut with restriction enzymes
• The complementary ends of the DNAs bind andligase enzyme reattaches the sugar-phosphatebackbone of the DNA
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Covalently join the DNA moleculeswith the base-pairing cohesiveends, or blunt ends, if the 5’-endshave phosphate groups.
DNA ligation
Recombinant DNA molecules
Restriction Endonucleases
Cloning Vector Types
• For different sizes of DNA:–
–
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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 proteinfrom the inserted DNA– shuttle vectors : can grow in two different
species
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Plasmid Vectors
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To replicate, a plasmid must be circular, and itmust contain a replicon , a DNA sequence thatDNA polymerase will bind to and initiatereplication. Also called “ori” (origin ofreplication).
– Replicons are usually species-specific.– Some replicons allow many copies of the
plasmid in a cell, while others limit thecopy number or one or two.
Plasmid cloning vectors must also carry aselectable marker : drug resistance.Transformation is inefficient, so bacteria thataren’t transformed must be killed.Most cloning vectors have a multiple cloningsite , a short region of DNA containing manyrestriction sites close together (also called apolylinker). This allows many differentrestriction enzymes to be used.Most cloning vectors use a system for detectingthe presence of a recombinant insert, usuallythe blue/white beta-galactosidase system.
What are the benefits ofbiotechnology?
• Medicine
• human
• veterinary
• biopharming
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Environment
Agriculture
Food products
Industry and manufacturing
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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)
MonoclonalAntibodies
CellCulture
GeneticEngineering
Anti-cancer drugs
Diagnostics
Culture of plantsfrom single cells
Transfer of new
genes into animalorganisms
specific DNA
probes
Localisation ofgenetic disorders
Tracers
Synthesis of
Gene therapy
Cloning
Mass prodn. ofhuman proteins
Resource bankfor rare humanchemicals
Newantibiotics
Synthesis
of newproteins
New types ofplants andanimals
New typesof food
DNAtechnology
Crime solvingMolecular
Biology
Banks ofDNA, RNAand proteins
Completemap of thehumangenome
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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 plantswith traits benefiting consumers
– iron deficiency in developing countries• transgenic rice
– increasing milk production• bovine somatotropin
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Transgenicrice
“Golden rice”shownintermixedwith white ricecontain highconcentrationsof beta-carotene
Transgenic Rice
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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 andintroducing genes from another bacterial species
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Benefits of Biotechnology:
1. Provide opportunities to accurately diagnose andprevent or cure a wide range of infectious and geneticdiseases.
2. Significantly increase crop yields by creating plantsthat are resistant to insect predation, fungal and viraldiseases, and environmental stresses such as short-termdrought and excessive heat.
3. Develop microorganisms that will produce chemical,antibiotics, polymers, amino acids, enzymes, andvarious food addiitives.
4. Develop livestock and other animal that have enhancedgenetically determined attibutes.
5. Facilitate the removal of pollutants and waste materialsfrom the environment.
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Social Concerns and Consequences
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7.8.
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Will some genetically engineered organisms be harmful either to otherorganisms or to the environment?Will the development and use of genetically engineered organismsreduce natural genetic diversity?Should humans be genetically engineered?Will diagnostic procedures undermine individual privacy?Will financial support for molecular biotechnology constraint thedevelopment of other important technologies?Will the emphasis on commercial success mean that benefits ofmolecular biotechnology will be available only to wealthy nations?Will agricultural biotechnology undermine traditional farming practices?Will medical therapies based on molecular biotechnology supersedeequally effective traditional treatments?Will the quest for patent inhibit the free exchange of ideas amongresearch scientists?
