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

Microbial Biotechnology

Biotechnology:Definition & HistoryRecombinant DNA

Technology

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

Restriction digestion

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

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

Recombinant DNA molecules

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

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

Transgenic Rice

Bovine Somatotropin

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?