BIOTECHNOLOGY

● 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

1. Glycolysis / substrate level phosphorylation (SLP)

● Breakdown of glucose (6-carbon) ⇒ pyruvic acid aka pyruvate
● Occurs in cytosol, w/ or w/o oxygen
 ● Reduction (gaining of electrons) of NAD+ to NADH
● Produces: pyruvate, ATP, NADH
● Pyruvic acid proceeds to either Krebs/tricarboxylic acid or the legit fermentation (if w/o O)
2. Krebs cycle / Tricarboxylic acid (TCA)
● Reduction of NAD+ to NADH, FAD to FADH2
● Produces: ATP, NADH, FADH2
3. Electron transport chain (ETC) / Oxidative phosphorylation
● Electrons pass along chain in stepwise fashion accdg to redox potential
● Occurs in the mitochondria (euks) or cell membrane (proks)
● NADH oxidized back to NAD+
● Produces: ATP
4. Fermentation ⇒ lactic, acetic acid, ethanol
● Uses pyruvate
● Oxidizes NADH back to NAD+ for glycolysis again
● Type of metabolism of C source where:
○ Energy is generated by SLP (glycolysis)
■ An organic molecule fxns as the final e- acceptor
○ Micro-organisms grown on large scale; even if final e- acceptor not organic cmpd
■ Fermentation of food, processing of micro-organisms
● Types of Ferm. Products
○ Cells (biomass) → yeast
○ Proteins, enzymes → cell components
○ Metabolites → process of producing energy
○ Biotransformation → steroids
● Industrial Ferm. Products
○ Aspergillus niger - citric acid
○ Saccharomyces cerevisiae - ethanol
○ Corynebacterium glutamicum produces amino acid - glutamate & lysine
○ Penicillium - penicillin
○ Xanthomonas campestris - xanthan gum

Metabolism Overview: fueling rxn ⇒ biosynthetic rxn ⇒ polymerization ⇒ assembly

Fueling rxns Which produce Biosynthetic rxns Polymerization Assembly
Glycolysis NADH/FADH Fatty acids Lipids Cell membrane
TCA cycle Precursor mat’ls Cytosol
Sugars Polysaccharides
ETS ATP Polyribosomes
CO2 units Amino acids Proteins Nucleoid
Nucleotides DNA/RNA

● Cellular metabolism → overview of respiration and fermentation

○ Aerobic respiration → requires oxygen; last acceptor in cycle is oxygen
○ Anaerobic respiration → doesn’t require oxygen; last acceptor isn’t oxygen
● Metabolites
○ Secondary metabolite → no clear fxn for growth purposes, e.g. antibiotic
○ Primary metabolite → needed for growth and survival
■ E.g. lactic, acetic, ethanol, alcohol, amino acids
● 3 RNA kinds
○ Ribosomal RNA (rRNA)
○ Transfer RNA (tRNA)
○ Messenger RNA (mRNA)
● All processes dictated by DNA aka what enzymes are encoded ⇒ “DNA is the blueprint of life”
 The Molecular Biology Story

Gregor Mendel (botanist) ● Father of modern genetics

● Cross pollinated plants to examine inheritance patterns: Principles of Inheritance /
Mendelian Genetics
● Dominant & recessive genes, homozygous & heterozygous genes
Miescher (biochemist) ● isolated nuclei of WBCs aka nuclein now nucleic acid
Flemming (cytologist) ● threadlike bodies during cell division aka chromosomes
Sutton (cytologist ) ● chromosomes were carriers of units of heredity aka genes
● Ultracentrifuge was developed
Griffith ● Transformation expt’s
○ used pneumonia-causing bacteria (Streptococcus pneumoniae)
○ Heat killed virulent strain + live non-virulent strain = virulent strain
Avery, MacLeod, ● DNA, polysacch, RNA compared
McCarty ● Transforming principle: DNA or nucleic acid
Weaver ● First used term molecular bio
Astbury ● used term molecular bio
Hershey & Chase ● The Blender Exp’t, showed the DNA was introduced into bacterial cell, DNA
infected bacteria vs protein
○ Sulfur-labeled protein & phosphorus-labeled DNA
● Further showed that DNA held genetic info
○ Transduction → involves DNA entering bacterium thru virus
○ Transformation → no virus involved
Franklin ● X ray diffraction data: periodicity & helical
Pauling & Corey ● Suggested triple helix structure
Watson & Crick ● Double helix structure
Messelson & Stahl ● DNA replication

Race to Double Helix

● Timeline
○ 1958: Franklin passed away at 37
○ 1962: Crick, Watson, Wilkins won Nobel Prize
○ 1968: Watson released The Double Helix
● Franklin’s early life
○ Scholarship to Cambridge, xray crystallography
○ During war, studied coal, gas masks
○ Got research position in Paris lab (4 yrs)
■ Perfected techniques of xray diffraction
■ Exceeded safe levels of radiation
○ Got position in King’s College, London (head: Randall)
■ Initially to create xray diffraction for protein, then DNA
■ Physicist Wilkins, PhD student Gosling
● Watson
○ Initially wanted to work w Wilkin at King’s, initially working on viruses
○ Invited to Cavendish at Cambridge (head: Bragg)
○ Met physicist-crystallographer Crick, initially working on proteins
● Sequence
○ Franklin discovered 2 DNA forms (A drier, B wetter), X shape meant helix, Photo 51 of B
○ Watson & Crick: model-building, proposed 1 strand LOL failure, forbidden by Bragg
○ Wilkins sharing data w Watson & Crick
○ Pauling Jr. moved into Cavendish, Pauling Sr. proposes 3-stranded helix
○ Wilkins shows Photo 51 to Watson: helix, 10 units per twist, symmetry aka antiparallel
■ Astbury: four bases stacked like pennies
■ Chargaff: equal A & T, G & C
○ Watson: specific pairings
○ Prob: unpublished data
■ Bragg & Randall w Nature: W&C, Wilkins, Franklin & Gosling DECEPTION
○ Franklin moved to Birkbeck College, London
■ Headed virus research lab, virus structure & located infectious element
■ Partner Sir Klug won Nobel Prize

The Genetic Material

● PROKARYOTES: closed circular DNA
○ Chromosomal DNA → one big circle, can stretch out, contains most of the genes
○ Plasmid DNA
■ extrachromosomal DNA
■ Replicate independently of chromosomal DNA
■ Some engineered plasmids for research e.g. pUC18
■ *cryptic plasmids: naturally-occurring but found to have no apparent fxn
■ Usually carry useful genes but not essential for growth (metal, antibiotic resistance)
○ Circular DNA found in nucleoid
○ Proteins assoc w prok DNA:
■ DNA polymerase → enzyme producing DNA thru polymerization
■ RNA polymerase → enzyme producing RNA in transcription
■ Histone-like proteins
■ Helicase → enzyme allowing denaturation
● EUKARYOTES: linear DNA
○ DNA found in nucleus
○ Made up of chromosomes
○ Have histone proteins → basic, contrary to acidic DNA
○ Structure = histone core with DNA coiled around it = nucleosome
● DNA structure
○ Specificity of base pairings: A w/ T (2 H bonds), C w/ G (3 H bonds)
○ Anti-parallel strands, double helix
■ Complementary strands
■ Stability from parallel stacking of base pairings in the center, not H bonds
■ 10 base pairs per helical turn
○ Separation of strands aka denaturation → heat or enzyme helicase
○ Parts:
■ Nitrogen bases (A, G, C, T) → heterocyclic aka w/ rings
● Purine bases (A, G) → double ring
● Pyrimidine bases (C, T) → single ring
■ Phosphate group → negative charge (highly acidic, nucleic acid)
■ 5-carbon sugar → deoxyribose, ribo sugar
○ Nucleoside: 1 nitrogen base + ribo sugar (deoxyribose)
○ nucleotide : nucleoside + phosphate grp
○ Gene → segment of DNA strand containing code for particular protein
 ○ Properties
■ Stability
■ Mutability
■ Replication
■ Transcription
■ Can transform

CENTRAL DOGMA

Gene products: RNA, proteins, traits & phenotypes

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

II) Transcription (5’ to 3’ direction)

● “Rewriting” or encoding of DNA to mRNA
● mRNA correspondence to DNA: adenine & uracil, guanine & cytosine
● Steps:
○ RNA polymerase binds to promoter (before gene)
○ RNA polymerase uses DNA template strand to make new complementary RNA molec
○ RNA polymerase: enzymes that transcribe DNA into RNA
○ Transcription ends when terminator is reached

● For prokaryotes: can occur simultaneously w/ translation since no venue separation

○ Transposons AKA jumping genes (have enzyme that allows them to insert into DNA)
● For eukaryotes: transcription in nucleus, translation in cytosol + mRNA processing needed
○ RNA splicing (removing excess AKA introns, exons remain)

III) Translation (5’ to 3’ direction)

● mRNA travels to ribosome in cytosol
○ Ribosomes: made of proteins and rRNA
● Ribosome attaches to start codon (methionine), will travel along mRNA from 5’ to 3’ end
 ● tRNA kinds are combined to amino acids, and contain anti-codons (complementary)
● TADA polypeptides formed
● NOTE: wobble hypothesis
○ 61 diff types of tRNA needed for 61 anticodons?? But most only have 40
○ So wobble: unconventional base pairing in the their dbase of the codon

Genetic Engineering & Molecular Cloning

● Genetic engineering / manipulation
○ Taking a gene from cell and inserting into DNA fragment (AKA cloning vector) + transferring this vector w/
insert into another cell (AKA transformation)
○ OR taking a gene from a cell + modifying + returning it back
○ Products: insulin, HGH, antibiotics, improved vaccines, better plants
● Important enzymes
○ Restriction enzymes → found in bacteria, can do sequence-specific cutting in DNA
○ DNA ligase → can connect DNA
● Plasmid vector
○ when you cut plasmid DNA, you can insert fragments
○ cohesive ends: cut ends have specific pattern, fragment matches
● Molecular Cloning / gene cloning / recombinant DNA technology (whole process)

○ 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

POLYMERASE CHAIN REACTION (PCR) / PCR amplification

● Basically in vitro replication (in test tube)
● DIFF: in PCR, only small specific portion is replicated multiple times
● Steps:
○ Denaturation thru heat
○ Taq polymerase (enzyme from Thermus aquaticus: extremophile) → heat-resistant, so can survive
denaturation and can remain
○ Put in thermocycler (thermocycling: quick temp changes)
○ Annealing temp will allow primer to bind
○ Then new temp again: optimal for in vitro replication
○ Repeat rinse repeat = thousands of copies!!! Easily!!!

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