Professional Documents
Culture Documents
● Biotechnology
○ uses biological systems/living organisms/derivatives to make products/processes for a specific use
○ basically, use of living organisms to make useful materials thru transformation
● Types of Biotech
○ Ancient
■ often discovered by accident or common practice, e.g. beer and wine
■ 4000 BC: wine vinegar making, fermentation
● First: juices from grapes fermented in presence of yeast
■ 500 BC: cheese making
○ Traditional
■ Beer → yeast Saccharomyces cerevisiae converts sugar into alcohol
■ Rice wine → mold Aspergillus oryzae (koji) converts starch to sugar + yeast S. cerevisiae converts sugar
to alcohol
■ Vinegar → bacteria Acetobacter turns ethanol from wine to acetic acid
■ Nata de coco → bacteria Acetobacter produces the cellulosic nata
■ Cheese
● protease enzyme chymosin (from calf intestine rennet) for milk curdling
● camembert: mold penicillium camemberti
● roquefort: mold penicillium roqueforti
● yogurt/yakult: bacteria Lactobacillus producing lactic acid
● kimchi: Lactobacillus producing lactic acid
○ Modern
■ Makes living organism perform a specific process to make a specific product
■ Genetic eng’g → makes changes in genes to improve quality/quantity of cell products
● Biotech Products
○ Environmental (gray) → use of microbes to feed on solid waste, to remove toxic wastes (bioremediation)
○ Aquatic (blue)
○ Agricultural (green) → adjust nutritional content, produce herbicide/drought resistant, improve yield of
animal products
■ Hybrid seed technology
● just cross pollination, no genetic modification whatsoever, uses pure lines
● Char A + char B = chars A & B
■ Tissue culture
● Get tissue from plant, put in petri dish, grow in test tube
■ Mutation breeding
● Exposing seeds to physical/chemical mutagen e.g. UV, radiation
● Before: random i.e. genes could actually worsen
● Now: can focus mutation on desired genes
○ Medical (red)
■ Vaccine → either dead/inactivated or live attenuated virus to elicit immune response
■ Antibiotics
● treat infections from bacteria e.g. penicillin
● However, many not naturally occurring, needed natural mutations before / chemical synthesis
● E.g. insulin, human growth hormone, TPA, interlekins, urogastrone, DNAse I
■ Enzymes → in detergents, digestive aids like protease papane in papaya
● Protease: degrades protein
● Amylase: degrades starch
■ Also to develop mat’ls that could help detect/diagnose diseases
CENTRAL DOGMA
I) Replication
● Denaturation of strands (enzyme helicase or artificial heat)
● Crucial: DNA polymerase III
● Meselsohn & Stahl Exp’t → proof that 2 parent strands serve as template
○ Bacteria grown in medium containing N15 isotope
○ Cells then transferred to medium containing N14
○ Samples collected, dissolved in cesium chloride, centrifuged = gradient
○ So after 2 gens: N14 & 15, N14 & 14
○ Operons in prokaryotes: clustered genes, transcribed together
○ No operons in eukaryotes, genes transcribed 1 at a time
● Steps:
○ Helicase enzyme unzips double stranded helix
○ Leading strand (continuous rep.): DNA polymerase 3 adds new nucleotides to free 3’ end
○ Lagging strand (discont. rep.):
■ RNA primase: attaches to DNA and synthesizes short RNA primer
■ DNA polymerase 3: adds new nucleotides (Okazaki fragments) to free 3’ end of RNA primer
■ DNA polymerase 1: replaces DNA 3, removes RNA primer and replaces it with DNA
■ Ligase enzyme: connects
○ R. enzyme cuts plasmid vector → insert fragment of new DNA → D. ligase connects
= TADA recombinant DNA molecule in gene construct
○ “Cloning” : introduced to host bacteria, replicate by themselves
● Bacterial Transformation
○ Gene construct is introduced into bacterial host cell (AKA transformed/GM/recombinant bacteria)
○ GM bacteria: has new DNA, can produce new protein
○ STEPS
i. Entry (TWO TYPES) → some cells will have plasmids
● Calcium chloride heatshock protocl
○ Expose & incubate bacteria in CaCl to damage the bacteria’s cell membrane ⇒ competent cells
(aka weakened aka accept new DNA more easily)
○ Add ligase to competent cells
○ Put in ice then 42 deg ⇒ bacteria are “shocked” and gulp in DNA
○ Put culture medium then incubate in normal temp for “healing”
● Electroporation / electrotransformation
○ Electroporator → high voltage instead of heatshock
ii. Selection → to select only the cells with plasmids
● Medium contains antibiotic
● If cell w/ plasmid, antibiotic-resistant! So will survive yay
iii. Screening → to select only plasmids w/ inserts
● Blue and white screening: uses beta galactosidase
● If w/ insert: blocked, can’t produce B gal = WHITE color
GM PLANTS
● Tissue culture! Bacteria containing plasmid
○ Biolistic / gene gun → bombards cells for plant transformation
○ Agrobacterium tumefaciens → tumor inducing (Ti) bacteria, insertion of small segment of DNA from
plasmid into plant cell
● Bt protein / toxin (Bacillus thuringenesis)
○ Bt toxin = pro-toxin (before toxin)
○ When eaten by corn borer, toxin activated by its basic ph gut + has affinity allowing it to harm digestive
tract & enter intestine lining
○ So toxin is toxic to corn borer; corn borer dies after eating Bt corn
○ Bt corn = corn borer resistant
● Modified EPSPS (mEPSPS gene & protein)
○ Plants normally have enzyme EPSPS since it’s essential to growth but this can be killed by herbicide
○ Herbicide can no longer attach thus resistant
○ mEPSPS = herbicide resistant
● Golden rice
○ Genes for encoding enzymes daffodil phytoene synthase or lycopene cyclase: necessary to convert to
beta-carotene (these genes from Eudovora species)
○ Plant can synthesize beta carotene in endosperm
○ Golden rice endosperm now w/ beta carotene = converted by human body to vitamin A
● Papaya: delayed ripening
○ RNA molecules inhibit gene expression or translation
○ Interference RNA binds to mRNA, is recognized as foreign, mRNA is degraded, ribosome cannot move =
no translation
○ No ethylene production = delayed ripening
● GM plants info
○ Purposes
■ Improve plant growth, survival, crop yield
■ Improve quality of crop, marketability
■ Develop plants that produce pharmaceutical substances (AKA biopharming)
○ Characteristics
■ Extra gene, produces extra protein
■ All cells have the new gene!
■ Other genes are same old same old
GM ANIMALS
● In vitro fertilization! → introduce it to a fertilized egg, insert back in womb, fertilization
● Undifferentiated cell (zygote to blastula)
○ Can still develop into any kind of cell
○ Starting blastula, diff. will start (epithelial / muscle / nerve / etc)
● Differentiation
○ Diff genes will be expressed → diff proteins → diff phenotypes
● Gene regulation
○ Cells have all genes, but diff genes are “on” thus diff genes are expressed in the end
● Timeline (nuclear transfer)
○ 1952: nucleus from frog blastocyst into enucleated oocyte → whole frog [undiff is guds]
○ 1975: nucleus of fully diff. Xenopus cell into enucleated cell → whole tadpole [diff is guds]
○ 1989: nuclei from sheep blastocyst [mammal undiff gud]
○ 19xx: nuclei from sheep diff. cell [mammal diff gud]
○ 1997: Dolly
■ breast epithelial cell (S1) into enucleated oocyte (S2)
■ culture cells until blastocyst (undiff)
■ Implant into surrogate ewe (S3)
■ TADA clone of S1
● Clones → ~technically~ not genetically modified, no change is introduced (same cells as donor, so no
modification whatsoever)
GM FOOD
● GM food → if GM organism or cells are used
○ e.g. Bt corn, soybean, flavr savr, GM potato, papaya, eggplant, herbicide tolerant canola plant, squash
● Biosafety Regulations → tested by developer & scientists in nutrition, toxicology, allergenicity
● Eaten GM food? → Broken down into proteins, no diff. bet. GM & non-GM amino acids
● Fear?
○ Widespread use of addtl proteins: disturbs balance
○ GMO “contamination”
○ “Accumulation” of GM product in body & env’t
○ Fear from inaccurate conditions/concentrations/applications
MODERN BIOTECH
● Stem Cell Fraud
○ Still ~10 years from real improvements for conditions like cerebral palsy, ALS, etc.
○ Cells sent by con men: dead or dying = fragments/debris, would be very dangerous if injected
○ No medical papers!! No clinical trials!! No basis whatsoever
● Killing Cancer
○ Injecting polio into glioblastoma (brain tumor)
○ Polio: seeks out a receptor that can attach to cancer cells, can’t cause paralysis bec can’t reproduce in
normal cells ony in cancer cells
○ Polio kinda alerts immune system; starts damage but immune system actually does most damage
● Regenerating Life
○ Burns (2nd and 3rd deg) → Skin patch from foreskin of babies, body doesn’t reject it, has collagen++, no
scarring
○ Osteoarthritis → carticel cartilage implant for loss of cartilage, cartilage cells taken from other knee and
injected to other knee
○ Bone fracture (collarbone) → plate & experimental synthetic putty (osteogenic protein 1 AKA OP 1),
same protein that stimulates regrowth of bone
○ Tissue eng’g → polymer: regrow ear and thumb, non-invasive cell therapy: use stem cells to regen liver