Traditional versus Modern Biotechnology (Exam 2 coverage)
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Traditional (classical) biotechnology includes fermentation, breeding, and the production of antibiotics and vaccines. Fermentation involves using microbes like yeast and bacteria to produce foods and beverages through anaerobic respiration, including beer, wine, cheese, bread and yogurt. Breeding techniques like inbreeding and crossbreeding were used to selectively develop plant and animal varieties with desirable traits. Early methods discovered antibiotics produced by microorganisms and used vaccines containing weakened or killed pathogens to trigger immune responses without causing illness.
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Traditional versus Modern Biotechnology (Exam 2 coverage)
- 1. TRADITIONAL (CLASSICAL) BIOTECHNOLOGY Fermentation cellardoorfestival.com
- 2. recall: biotechnology •ancient •early history as related to food and shelter, including domestication ! •traditional (classical) •built on ancient biotechnology •fermentation promoted food production and medicine ! •modern •manipulates genetic information in organism •genetic engineering mitalee.wordpress.com
- 3. Grabbed from the PPT lectures of Professor/Dr. Arnold V. Hallare, (2013)
- 4. what to learn today… •overview of metabolism: aerobic and anaerobic respiration •Fermentation in Plants •Fermentation in Animals •Fermentation in Humans •traditional biotechnology: fermentation •virtual lab blog.leonardo.com
- 6. cellular respiration: a catabolic reaction •process of making ATP by breaking down organic compounds •exergonic •oxygen (O2) requiring •uses energy extracted from macromolecules (glucose) to produce energy (ATP) and water (H2O) enzymes 6O2 + C6H12O6 6CO2 + 6H2O oxygen glucose carbon dioxide water ADP + Pi ENERGY transfer between enzymes, other molecules ATP
- 7. stages of aerobic respiration • glycolysis: cytosol • krebs cycle: mitochondrial matrix • ETC: inner mitochondrial membrane You may watch a video here about GLYCOLYSIS: http:// www.science.smith.edu/departments/Biology/Bio231/
- 8. anaerobic respiration (fermentation): if oxygen is absent Glucose Pyruvic acid cellardoorfestival.com classes.midlandstech.com
- 9. organic acids instead of atp www2.bc.cc.ca.us
- 10. prokaryotes vs eukaryotes www.hns.org.uk
- 11. anaerobic respiration in plants www.ipm.iastate.edu In response to flooding stress www.vce.bioninja.com.au
- 12. anaerobic respiration in animals www.fashioncentral.pk www.vce.bioninja.com.au slow twitch versus fast twitch muscles
- 13. slow twitch and aerobic respiration • example: dark leg meat of chicken • Specialised for slow, sustained contractions over a long period for endurance • contain lots of myoglobin which acts as a store of oxygen • Respire aerobically
- 14. slow twitch works best in: or if you wanna try duathlon when you try running the bdm ultramarathon (160km)
- 15. lactic acid in meat? • fast twitch • example: pectoral flight muscles (chicken breast) • for producing rapid, intense contractions of short duration for rapid movement • do not have myoglobin so Respire anaerobically • can accumulate lactic acid and leads to fatigue thoughtchalk.com
- 16. fast twitch works best in: Provide the muscle power for rapid, fast movement e.g. a cheetah's burst of speed to catch a gazelle, or the gazelles burst of speed to escape the cheetah or to power up usain bolt’s legs in sprints omarmcknight.com
- 17. fermentation in ruminants sci.waikato.ac.nz www.tankonyvtar.hu
- 18. fermentation in humans •farting •When carbon dioxide is used as an electron acceptor, the product is either methane or acetic acid •Methane produced in our gut is produced by this process www.ausforces.com
- 20. fermentation : classical biotechnology •the use of microbes to enhance food flavor •the use of microbes to manufacture of beverages •the use of microbes to make the dough rise
- 21. products of fermentation: beer •An alcoholic beverage produced by the fermentation of sugar-rich extracts derived from cereal grains or other starchy materials •ancient biotechnology: beer brewing •Sumaria (4000 BC) •Sikaru •Egypt (3000 BC) •Zythum •India (2000 BC) •Sura •China (2000 BC) •Kiu www.nomad4ever.com
- 22. yeast in beer brewing •1680 Antonie van Leeuwenhoek Observed yeast in beer •1837 - Cagniard Latour decsribed that Microbe is responsible for alcoholic fermentation •1866 - Louis Pasteur stated that Yeast was responsible for alcoholic fermentation •1883 - Emil Christian Hansen Developed pure culture technique and Isolated pure cultures of brewing yeasts Weiss Ale Lager Lab Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company
- 23. microbes and beer brewing •malted barley Provides fermentable sugars, flavor, and color •hops Provides aroma and bitterness The Brewing Process Step Purpose Brewhouse Fermentation Lagering Starch Sugars Wort production Sugars Ethanol Flavor production Carbonation Flavor maturation Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company The Brewing Process Malt Mill Mash Tun Cereal Cooker Lauter Tun Brew Kettle Hot Wort Receiver Wort Cooler Brink Fermentation Aeration Lagering Hops Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company
- 24. microbes and beer brewing Yeast Metabolism During Fermentation Sugars Oxygen Glucose Pyruvate TCA Cycle Amino Acids Energy CO2 Ethanol Acetaldehyde Organic Acids Unsaturated Fatty Acids Sterols Amino Acids Esters Higher Alcohols VDK Sulfur Volatiles Membranes Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company
- 25. products of fermentation: cheese •cheese are ripened curds •milk is treated with lactic acid bacteria and rennin to coagulate proteins •curds + whey = milk •different microbes in the early and late stages of processing of cheese = different cheese characteristics idiva.com
- 26. types oTfy pcesh oef Cehseeese Acid Coagulated Fresh Cheese (lactic acid from bacteria) • no enzyme is used to finish the curd • Cottage and Cream Cheese Heat-Acid Precipitated Cheese (acid and heat precipitate/coagulate the protein and cause milk fat to curdle) • Add low amounts of acid to 75-100oC temp milk • High moisture and protein • Ricotta (Italy) Channa and Paneer (India) science of cooking
- 27. types oTfy pcesh oef Cehseeese Semi-hard Washed Cheese (washing cheese removes acid and lactose) • Acid and enzyme induced curdling • But removal of milk sugar and acid results in no fermentation results in a moist and less finished cheese • Gouda, colby, muenster, mozzarella … Hard Cheese (Low and High Temp) • Low moister makes a more dense hard cheese • Elevated temps and pressing drive off water • Cheddar, Romano, Parmesan, Swiss, science of cooking
- 28. swiss cheese and propionibacterium science of cooking Finishing Microbes Holy Cheese (cow)? – Propionibacteria: • Convert lactic acid to propionoic and acetic acid plus acetic acid and CO2. Also other flavors • Used to make Swiss Cheese • Need higher temps and time for bacteria to grow and produce • Growth requirements reflect origins of bacteria animal skin Lactic acid Propionoic acid + Acetic acid Carbon Dioxide (g) Finishing up…
- 29. Blue Cheeses – Based on Origen fungi and blue cheese Roquefort - France Cambreles- Spain Stilton- England Danish Blue Cheese Gorgonzola- Italy science of cooking Penicillium roqueforti and P. camberti BLUE = MYCELIA/ or growth filaments
- 30. making the cheese Non Starter– ripening Starter – acid producing 0 50 100 150 200 science of cooking Bacteria Growth Time (Days)
- 31. product of biotechnology: breads •biotechnology’s first utilization of microbes = bread making •Around 4000 BC, Egyptians used the living organism yeast to make bread •Airborne wild yeast accidentally got their bread dough, causing it to rise www.acebakery.com
- 32. the sourdough bread •microbe one (AEROBIC): yeast •makes carbon dioxide and bread will rise •microbe two: anaerobic: lactic acid bacteria •make lactic acid and acetic acid that give rich complexity of flavors www.weekendbakery.com foodists.ca www.rootsimple.com
- 33. products of fermentation: wine •after bread comes wine: 3000 bc •converts sugars in grapes into alcohol www.cell.com
- 34. making your wine http://www.chinookwines.com 1. harvest 2. processing 3. fermentation
- 35. making your wine http://www.chinookwines.com 4. maturation 6. bottling & corking 5. fining and filtration
- 36. products of fermentation: yoghurt •FERMENTED MILK RESULTING TO A SEMI-SOLID CURD •LACTIC ACID BACTERIA = PROBIOTICS •AIDS IN DIGESTION •ACID PRODUCED DURING FERMENTATION CAUSES THE PROTEIN TO COAGULATE •Lactococcus lactis, Streptococcus thermophilus and Lactobacillus bulgaricus www.wombourneshopping.co.uk
- 37. how to make yoghurt Making Yogurt in 4 Simple Steps 1. Start with Cow, Sheep, or Goat milk. 2. Heat milk to 80 °C. Two purposes: • destroy existing bacteria • condition the proteins = begins the denaturing process (a whey protein molecule binds to a casein molecule which disrupts the casein bundles allowing them to make short branched micelle chains) 3. Cool milk to 40 °C and innoculate with bacteria 4. Incubate at 30 °C to 45 °C Casein before heat pre-treatment: Casein after heat pre-treatment: Casein after acid: www.bnc.asn.au
- 38. bacteria in yoghurt Milk Yogurt Casein protein micelles Bacteria produce acid (bundles) 10-7 meters in diameter Fat globule Acid causes Casein bundles to fall apart into separate casein molecules. These rebind to each other in a network that traps water. = makes a gel
- 39. reading assignment… •try this virtual laboratory about pickling •https:// www.exploratoriu m.edu/cooking/ pickles/ picklelab.html kungfubistro.com fearlesseating.net
- 40. TRADITIONAL (CLASSICAL) BIOTECHNOLOGY breeding cellardoorfestival.com
- 41. what to learn today… •recall mendel’s heredity experiments •classical breeding in Plants •classical breeding in Animals blog.leonardo.com
- 42. RECALL: MENDEL’S HEREDITY EXPERIMENTS MEXAL, 2006
- 43. RECALL: MENDEL’S HEREDITY EXPERIMENTS F1 MEXAL, 2006
- 44. RECALL: NON-MENDELIAN HEREDITY MEXAL, 2006
- 45. WHAT IS PLANT BREEDING?
- 47. PLANT BREEDING TIMELINE 9000 BC First evidence of plant domestication in the hills above the Tigris river 1694 Camerarius first to demonstrate sex in (monoecious) plants and suggested crossing as a method to obtain new plant types 1714 Mather observed natural crossing in maize 1761-1766 Kohlreuter demonstrated that hybrid offspring received traits from both parents and were intermediate in most traits, first scientific hybrid in tobacco 1866 Mendel: Experiments in plant hybridization 1900 Mendels laws of heredity rediscovered 1944 Avery, MacLeod, McCarty discovered DNA is hereditary material 1953 Watson, Crick, Wilkins proposed a model for DNA structure 1970 Borlaug received Nobel Prize for the Green Revolution Berg, Cohen, and Boyer introduced the recombinant DNA technology 1994 FlavrSavr tomato developed as first GMO 1995 Bt-corn developed UNIVERSITY OF FLORIDA, 2008
- 48. FROM WILD TO DOMESTICATION
- 49. DOMESTICATION VS ! PLANT BREEDING • Domestication! • people try to control the reproductive rates of animals and plants! • NOTE: without knowledge on the transmission of traits from parents to their offspring! • Plant Breeding! • genetic analysis is used for the development of plant lines better suited for human purposes UNIVERSITY OF FLORIDA, 2008
- 50. FROM WILD TO DOMESTICATION
- 51. FROM WILD TO DOMESTICATION
- 52. CLASSICAL VS MODERN BIOTECHNOLOGY • CLASSICAL: ! • Plant Breeding and Selection Methods! • GOAL: to meet the food, feed, fuel, and fiber needs UNIVERSITY OF FLORIDA, 2008 of the world! • MODERN! • Genetic Engineering! • GOAL: to increase the effectiveness and efficiency of plant breeding!
- 53. ANIMAL BREEDING • Breeding animals to achieve certain characteristics in the offspring! • Natural method of improving plants and animals! • Scientists control the natural breeding process! • Examples: INBREEDING or CROSS HARRY, 2008 BREEDING!
- 54. CLASSICAL VS MODERN BIOTECHNOLOGY • Selection (Classical Breeding)! • Choosing a few parents with the desired traits with the intent of increasing the amount of desired qualities in the offspring! • Genetic Manipulation (Modern Breeding)! • Artificial means of producing desirable traits! • Genes can be moved from one species to another! • Gene splicing is the moving of hereditary characteristics from one organism to another often unrelated organism HARRY, 2008
- 55. INBREEDING • Mating 2 closely-related animals ! • parents to offsprings and sibling (25% inbred)! • uncle x niece (12.5% inbred)! • cousin x cousin (3.125% inbred)! • Examples: Close Breeding and Pure Breeding HARRY, 2008
- 56. THE INBREDS • CLOSE BREEDING:! • Mating animals that share common ancestors! • PURE BREEDING:! • Mating animals that are not related but of the HARRY, 2008 same breed!
- 57. CROSS BREEDING HARRY, 2008 • Mating animals of different breeds
- 58. TRADITIONAL (CLASSICAL) BIOTECHNOLOGY ANTIBIOTICS VACCINES www.icr.org
- 59. ANTIBIOTICS • a chemical substance (SECONDARY METABOLITE) produced by a microorganism that kills or inhibits the growth of another microorganism research.fuseink.com
- 60. SECONDARY METABOLITES IN ASPERGILLUS SPECIES research.fuseink.com
- 61. MODE OF ACTION research.fuseink.com
- 62. ANTIBIOTIC RESISTANCE medimoon.com www.ondineblog.com research.fuseink.com
- 63. VACCINES • Vaccination: deliberate stimulation of one’s immunity! • Work by mimicking what happens during natural infection without causing illness! • Use altered versions of viruses or bacteria to trigger an immune response! • Are the most effective means of controlling infectious diseases! • Not only protect those who get them, but they also help keep diseases at bay in the community (herd immunity) vaccineresistancemovement.org
- 65. HOW VACCINES WORK vaccineresistancemovement.www.cdc.org nfs.unipv.it org
- 66. ANTIBODIES AS VACCINES: ! PASSIVE IMMUNITY vaccineresistancemovement.popups.ulg.ac.be org
- 67. HOW VACCINES WORK vaccineresistancemovement.www.cdc.org nfs.unipv.it org
- 68. ANTIGENS AS VACCINES: ACTIVE IMMUNITY vaccineresistancemovement.org DISEASE VACCINE Antiviral vaccines Smallpox Attenuated live virus Yellow fever Attenuated live virus Hepatitis B Recombinant Measles Attenuated live virus Mumps Attenuated live virus Rubella Attenuated live virus Polio Attenuated live virus (Sabin) Polio Inactivated virus (Salk) Influenza Inactivated virus Rabies Inactivated virus
- 69. ANTIGENS AS VACCINES: ACTIVE IMMUNITY Antibacterial vaccines Diphtheria Toxoid Tetanus Toxoid Pertussis Acellular extract from Bordetella pertussis Meningococcal meningitis Capsular material from 4 strains of Neisseria meningitidis Haemophilus ínfluenzae Capsular material from Haemophilus influenzae type b type b (Hib) infection conjugated to diphtheria protein Cholera Killed Vibrio cholerae Plague Killed Yersinia pestis Typhoid fever Killed Salmonella typhi Pneumococcal pneumonia Capsular material from 23 strains of Streptococcus pneumoniae vaccineresistancemovement.org
- 70. WHOLE-KILLED VS LIVE-ATTENUATED vaccineresistancemovement.org • Whole-killed! • Killed by heat, chemical or UV irradiation! • more stable and safer than live vaccines! • can’t mutate back to their disease-causing state! • take several additional doses, or booster shots, to maintain a person’s immunity! • Live-Attenuated! • Made less pathogenic by passage in animals or thermal mutation! • Contain a version of the living microbe that has been weakened in the lab so it can’t cause disease! • elicit strong cellular and antibody responses and often confer lifelong immunity with only one or two doses
- 71. TOXOID AND SUB-UNIT VACCINES vaccineresistancemovement.org • TOXOID! • Used when a bacterial toxin is the main cause of illness! • inactivate toxins by treating them with formalin (detoxified” toxins = toxoids)! • Immune system produces antibodies that lock onto and block the toxin! • SUB-UNIT! • Instead of the entire microbe, subunit vaccines include only the antigens that best stimulate the immune system (EPITOPES)
- 72. ARE YOU READY FOR ! MODERN BIOTECHNOLOGY???
- 73. MODERN BIOTECHNOLOGY OVERVIEW OF CONCEPTS www.nist.gov
- 74. MODERN BIOTECHNOLOGY CONCEPTS • Involves gene manipulation and gene introduction! • Genetically-Modified Organisms (GMO)! • organisms with artificially-altered DNA! • APPLICATIONS! • Foreign gene is inserted to enable GMO to express the trait coded by the gene) = TRANSGENICS! • An existing gene is altered to make it express at a higher level or in a different way = FOR GENE THERAPY! • Gene is deleted or deactivated: to prevent the expression of a trait (e.g. delayed ripening) www.nist.gov
- 75. SOME COMMON GMOs www.nist.gov
- 76. THE GOLDEN RICE www.nist.gov www.goldenrice.org,
- 77. β!Carotene)Pathway)in)Plants) IPP) (Isopentenyl)diphosphate)) Geranylgeranyl)diphosphate) Phytoene)synthase! Phytoene) Phytoene)desaturase) ξ!carotene)desaturase)) Lycopene) Lycopene!beta!cyclase) β)!carotene) (vitamin)A)precursor)) Problem:! Rice!lacks! these!enzymes! Normal! Vitamin!A! Deficient! Rice! www.nist.gov www.goldenrice.org,
- 78. The$Golden$Rice$Solution$ β9Carotene$Pathway$Genes$Added$ IPP$ Geranylgeranyl$diphosphate$ Phytoene$synthase! Daffodil$gene$ Phytoene$ Phytoene$desaturase$ ξ9carotene$desaturase$$ Single$bacterial$gene;$ performs$both$functions$ Lycopene$ Lycopene9beta9cyclase$ Daffodil$gene$ β$9carotene$ (vitamin$A$precursor)$ Vitamin$A$ Pathway$ is$complete$ and$functional$ Golden! Rice! www.nist.gov www.goldenrice.org,
- 79. HOW TO MAKE INSULIN www.nist.gov muirbiology.wordpress.com1
- 80. HOW TO MAKE BT CORN www.nist.gov www.scq.ubc.ca)) www.scq.ubc.ca)
- 81. HOW TO MAKE THOSE?! ! GENETIC ENGINEERING www.nist.gov
- 82. METHODS IN GENETIC ENGINEERING • GENE ISOLATION! • TRANSFORMATION! • SELECTION AND REGENERATION! • CONFIRMATION OF EXPRESSION www.nist.gov Bacterium Bacterial chromosome Plasmid 2 1 3 4 Gene inserted into plasmid Cell containing gene of interest Recombinant DNA (plasmid) Gene of interest Plasmid put into bacterial cell DNA of chromosome (“foreign” DNA) Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of interest Protein expressed from gene of interest Copies of gene Protein harvested Basic research and various applications Basic research on protein Basic research on gene Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hormone treats stunted growth
- 83. GENE ISOLATION: BACTERIAL COLONIES www.nist.gov www.oxoid.com www.appletonwoods.co.uk us.bioneer.com
- 84. GENE ISOLATION: VIRUS CULTURE www.nist.gov www.oxoid.com www.appletonwoods.co.uk us.bioneer.com
- 85. GENE ISOLATION BY POLYMERASE CHAIN REACTION • What you will need:! • Primers! • PCR conditions and reaction mixture (optimized) www.nist.gov www.oxoid.com www.appletonwoods.co.uk
- 86. WHAT IS PCR? www.nist.gov www.oxoid.com www.appletonwoods.co.uk
- 87. STEPS www.nist.gov www.oxoid.com www.appletonwoods.co.uk www.austincc.edu
- 88. COMPONENTS • Buffer! • provide an optimal pH and monovalent salt environment for the final reaction volume! • MgCl2! • supplies the Mg++ divalent cations required as a cofactor for Type II enzymes, which include restriction endonucleases and the polymerases used in PCR! • dNTPs! • supply the “bricks” to synthesize a virtually unlimited amount of a specific stretch of double-stranded DNA (the individual DNA bases must be supplied to the polymerase enzyme)! • Primers and Taq polymerase www.nist.gov www.oxoid.com www.appletonwoods.co.uk www.austincc.edu
- 89. • DESIGNING YOUR PRIMERS! • http://www.ncbi.nlm.nih.gov/tools/primer-blast/! • http://bioinfo.ut.ee/primer3-0.4.0/ www.nist.gov www.oxoid.com www.appletonwoods.co.uk
- 90. • VERIFYING YOUR DESIGN! • http://www.basic.northwestern.edu/biotools/ OligoCalc.html www.nist.gov www.oxoid.com www.appletonwoods.co.uk
- 91. WHAT IS PCR? • How will it isolate your target gene?! • amplification! ! • How to make sure that you have isolated your gene correctly?! • sequencing www.nist.gov www.oxoid.com www.appletonwoods.co.uk
- 92. CHECKING AMPLIFICATION www.nist.gov www.oxoid.com www.appletonwoods.co.uk
- 93. VERIFYING AMPLICONS: Sequencing Then do BLAST and compare with NCBI (database) www.nist.gov www.oxoid.com www.appletonwoods.co.uk www.nature.com
- 94. T-VECTOR (TA) CLONING SEQUENCING Transformation was done in E. coli DH5 for blue-white selection on L-agar with 50μg/ml ampicillin plus 100μl100 mM IPTG and 20 ul 50 mg/ml X-gal (incubation, 37C for 12 hours max)
- 96. RESTRICTION ENZYMES: ! HindIII digestion 3.0kb 1.5kb 1.0kb Size of vector: 2.7kb! Size of Insert: 1.3kb
- 97. END OF EXAM COVERAGE expertelevation.com