B.tech biotechnology ii elements of biotechnology unit 1 biotechnology
Traditional versus Modern Biotechnology (Exam 2 coverage)
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TRADITIONAL (CLASSICAL) BIOTECHNOLOGY Fermentation cellardoorfestival.com
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Transcript of Traditional versus Modern Biotechnology (Exam 2 coverage)
TRADITIONAL (CLASSICAL)
BIOTECHNOLOGY
Fermentation
cellardoorfestival.com
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 engineeringmitalee.wordpress.com
Grabbed from the PPT lectures of Professor/Dr. Arnold V. Hallare, (2013)
what to learn today…• overview of metabolism:
aerobic and anaerobic respiration
• Fermentation in Plants
• Fermentation in Animals
• Fermentation in Humans
• traditional biotechnology: fermentation
• virtual labblog.leonardo.com
recall: metabolism
academic.pgcc.edu
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)
6O2 + C6H12O6 6CO2 + 6H2O oxygen glucose carbon dioxide water
enzymes
ADP + Pi
ENERGY transfer between enzymes,
other molecules
ATP
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/
anaerobic respiration (fermentation): if oxygen is absent
classes.midlandstech.com
Glucose Pyruvic acid
cellardoorfestival.com
anaerobic respiration in plants
www.vce.bioninja.com.auwww.ipm.iastate.edu
In response to flooding stress
anaerobic respiration in animals
www.vce.bioninja.com.auwww.fashioncentral.pk
slow twitch versus fast twitch muscles
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
slow twitch works best in:
or if you wanna try duathlon
when you try running the bdm ultramarathon (160km)
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
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
omarmcknight.com
or to power up usain bolt’s legs in sprints
fermentation in ruminants
sci.waikato.ac.nz
www.tankonyvtar.hu
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
fermentation in microbes
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
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
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
Lab Lager
Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company
Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company
microbes and beer brewing• malted barley
Provides fermentable sugars, flavor, and color
• hops Provides aroma and bitterness
The Brewing Process
Brewhouse
Fermentation
Lagering
Step Purpose
Starch Sugars
Ethanol Sugars
Carbonation Flavor maturation
Wort production
Flavor production
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
Fermentation Brink
Aeration
Lagering
Hops
Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company
microbes and beer brewingYeast Metabolism During Fermentation
Sugars Oxygen
Amino Acids
Glucose
Pyruvate
TCA Cycle
Energy CO2
Ethanol
Acetaldehyde
Organic Acids
Amino Acids
Unsaturated Fatty Acids Sterols
Esters
Higher Alcohols
VDK
Sulfur Volatiles
Membranes
Kindly provided by Tom Pugh and David Ryder of Miller Brewing Company
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
types of cheeseTypes of Cheese
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
types of cheeseTypes of Cheese
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
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
Carbon Dioxide (g) Acetic acid
Propionoic acid
+
Finishing up…
Blue Cheeses – Based on Origen
Roquefort - France Cambreles- Spain
Danish Blue Cheese
Stilton- England
Gorgonzola- Italy
fungi and blue cheese
science of cooking
Penicillium roqueforti and P. camberti BLUE = MYCELIA/ or growth filaments
making the cheese
science of cooking
Bac
teria
Gro
wth
Time (Days)
0 50 100 150 200
Starter – acid producing
Non Starter– ripening
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
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 www.rootsimple.comfoodists.ca
products of fermentation: wine
• after bread comes wine: 3000 bc
• converts sugars in grapes into alcohol
www.cell.com
making your wine
http://www.chinookwines.com
1. harvest
2. processing
3. fermentation
making your wine
http://www.chinookwines.com
4. maturation
6. bottling & corking
5. fining and filtration
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
how to make yoghurtMaking Yogurt in 4 Simple Steps
1. Start with Cow, Sheep, or Goat milk.
Casein before heat pre-treatment:
Casein after heat pre-treatment: Casein after acid:
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
www.bnc.asn.au
bacteria in yoghurtMilk Yogurt
Bacteria produce acid Casein protein micelles (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
reading assignment…
• try this virtual laboratory about pickling
• https://www.exploratorium.edu/cooking/pickles/picklelab.html
kungfubistro.com
fearlesseating.net
TRADITIONAL (CLASSICAL)
BIOTECHNOLOGY
breeding
cellardoorfestival.com
what to learn today…
• recall mendel’s heredity experiments
• classical breeding in Plants
• classical breeding in Animals
blog.leonardo.com
RECALL: MENDEL’S HEREDITY EXPERIMENTS
MEXAL, 2006
RECALL: MENDEL’S HEREDITY EXPERIMENTS
F1
MEXAL, 2006
RECALL: NON-MENDELIAN HEREDITY
MEXAL, 2006
WHAT IS PLANT BREEDING?
WHY BREED PLANTS?
PLANT BREEDING TIMELINE9000 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 Mendel�s 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
FROM WILD TO DOMESTICATION
DOMESTICATION VS !PLANT BREEDING
UNIVERSITY OF FLORIDA, 2008
• 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
FROM WILD TO DOMESTICATION
FROM WILD TO DOMESTICATION
CLASSICAL VS MODERN BIOTECHNOLOGY
UNIVERSITY OF FLORIDA, 2008
• CLASSICAL: !
• Plant Breeding and Selection Methods!
• GOAL: to meet the food, feed, fuel, and fiber needs of the world!
• MODERN!
• Genetic Engineering!
• GOAL: to increase the effectiveness and efficiency of plant breeding!
ANIMAL BREEDING
HARRY, 2008
• 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 BREEDING!
CLASSICAL VS MODERN BIOTECHNOLOGY
HARRY, 2008
• 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
INBREEDING
HARRY, 2008
• 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
THE INBREDS
HARRY, 2008
• CLOSE BREEDING:!
• Mating animals that share common ancestors!
• PURE BREEDING:!
• Mating animals that are not related but of the same breed!
CROSS BREEDING
HARRY, 2008
• Mating animals of different breeds
TRADITIONAL (CLASSICAL)
BIOTECHNOLOGY
ANTIBIOTICS & VACCINES
www.icr.org
ANTIBIOTICS• a chemical substance (SECONDARY METABOLITE)
produced by a microorganism that kills or inhibits the growth of another microorganism
research.fuseink.com
SECONDARY METABOLITES IN ASPERGILLUS SPECIES
research.fuseink.com
MODE OF ACTIONresearch.fuseink.com
ANTIBIOTIC RESISTANCE
research.fuseink.comwww.ondineblog.com
medimoon.com
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
VACCINES
vaccineresistancemovement.org
ANTIBODIES AS VACCINES: !PASSIVE IMMUNITY
vaccineresistancemovement.orgpopups.ulg.ac.be
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
ANTIGENS AS VACCINES: ACTIVE IMMUNITY
vaccineresistancemovement.org
Antibacterial vaccines
Diphtheria Toxoid
Tetanus Toxoid
Pertussis Acellular extract from Bordetella pertussis
Meningococcal meningitis Capsular material from 4 strains of Neisseria meningitidis
Haemophilus ínfluenzae type b (Hib) infection
Capsular material from Haemophilus influenzae type b 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
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
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)
ARE YOU READY FOR !MODERN BIOTECHNOLOGY???
MODERN BIOTECHNOLOGY
OVERVIEW OF CONCEPTS
www.nist.gov
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
SOME COMMON GMOs
www.nist.gov
THE GOLDEN RICE
www.nist.gov www.goldenrice.org,
www.nist.gov www.goldenrice.org,
β!Carotene)Pathway)in)Plants)
IPP)
Geranylgeranyl)diphosphate)
Phytoene)
Lycopene)
β)!carotene)(vitamin)A)precursor))
Phytoene)synthase!
Phytoene)desaturase)
Lycopene!beta!cyclase)
ξ!carotene)desaturase))
Problem:!Rice!lacks!
these!enzymes!
Normal!Vitamin!A!�Deficient�!
Rice!
(Isopentenyl)diphosphate))
www.nist.gov www.goldenrice.org,
The$Golden$Rice$Solution$
IPP$
Geranylgeranyl$diphosphate$
Phytoene$
Lycopene$
β$9carotene$(vitamin$A$precursor)$
Phytoene$synthase!
Phytoene$desaturase$
Lycopene9beta9cyclase$
ξ9carotene$desaturase$$
Daffodil$gene$
Single$bacterial$gene;$performs$both$functions$
Daffodil$gene$
β9Carotene$Pathway$Genes$Added$
Vitamin$A$Pathway$
is$complete$and$functional$
Golden!Rice!
HOW TO MAKE INSULIN
www.nist.gov
muirbiology.wordpress.com1
HOW TO MAKE BT CORN
www.nist.govwww.scq.ubc.ca)
www.scq.ubc.ca))
HOW TO MAKE THOSE?!!
GENETIC ENGINEERING
www.nist.gov
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
Protein harvested Copies of gene
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
GENE ISOLATION: BACTERIAL COLONIES
www.nist.gov www.oxoid.com www.appletonwoods.co.uk us.bioneer.com
GENE ISOLATION: VIRUS CULTURE
www.nist.gov www.oxoid.com www.appletonwoods.co.uk us.bioneer.com
GENE ISOLATION BY POLYMERASE CHAIN REACTION
www.nist.gov www.oxoid.com www.appletonwoods.co.uk
• What you will need:!
• Primers!
• PCR conditions and reaction mixture (optimized)
WHAT IS PCR?
www.nist.gov www.oxoid.com www.appletonwoods.co.uk
www.nist.gov www.oxoid.com www.appletonwoods.co.uk www.austincc.edu
STEPS
www.nist.gov www.oxoid.com www.appletonwoods.co.uk www.austincc.edu
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
• 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
• VERIFYING YOUR DESIGN!
• http://www.basic.northwestern.edu/biotools/OligoCalc.html
WHAT IS PCR?
www.nist.gov www.oxoid.com www.appletonwoods.co.uk
• How will it isolate your target gene?!
• amplification!
!
• How to make sure that you have isolated your gene correctly?!
• sequencing
CHECKING AMPLIFICATION
www.nist.gov www.oxoid.com www.appletonwoods.co.uk
VERIFYING AMPLICONS: Sequencing
www.nist.gov www.oxoid.com www.appletonwoods.co.uk www.nature.com
Then do BLAST and compare with NCBI (database)
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)
SELECTION MARKER
www.nist.gov
RESTRICTION ENZYMES: !HindIII digestion
1.5kb1.0kb
3.0kb
Size of vector: 2.7kb!Size of Insert: 1.3kb
END OF EXAM COVERAGE
expertelevation.com
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