Biotech applications

Gene therapy: Engineered cells make protein that replaces defective protein. Hard to target and express correctly, might die from immune rejection.
Pharmaceuticals: Insulin, growth hormone, tissue plasminogen activator, recombinant vaccines.
Environmental cleanup: Organisms can degrade pollutants, extract heavy metals.
Agriculture: Use less pesticides and herbicides. Use Ti plasmid to genetically engineer plants. Golden rice has Vitamin A genes from daffodils and bacteria.
Diagnosis + forensics

Important figures

Mendel: Peas, mendelian genetics.
Griffith: Attenuated culture of bacteria that lost ability to cause disease. Transforming principle.
Avery, Macleod, McCarty: Transforming principle is DNA.
Hershey, Chase: Genetic material is DNA, not protein.
Chargaff: A = T. G = C.
Watson, Crick, Franklin, Wilkins: B form shape of DNA.
Berg: Recombinant bacteriophage.
Cohen, Boyer: Recombinant plasmid, founded Genentech, recombinant insulin.
Kary Mullis: PCR.
Fred Sanger: Structure of protein, insulin, chain termination sequencing.

Model Organisms

DH5 alpha, JM109, JM110, HB101

E. coli: $, -, K12 derivatives, easy to manipulate, fast.
B. Subtilis: $, +, easy to change, fast, many strains.
Yeast: $, easy to manipulate.
Insect cells: $$, fast, not as easy to modify.
Mammal cell culture: $$, Hela immortalized cancer cells.
Mus musculus (Mouse): $$$, IRB, short life cycle, 2 year life span, 2 week generational span, many transgenic mouse lines.
Primates: $$$$, Rhesus, baboons, chimps.

Recombination

Genes from different sources.

Genetic engineering: Led to development of biotech.
GOI: Gene of interest.
RT: Reverse transcriptase.
ORF: Open reading frame. The region between a start codon NTG and one of the three possible stop codons.
Cloning: Inserting gene into vector to make products. Requires vector, restriction enzymes, ligase, transformation. Use selectible markers to identify and remove nonrecombinant cells.
Recombinant cells: Might have disrupted genes from inserting a GOI into them. Use X-gal marker (blue white screening) to detect disrupted genes.
LacZ + X-gal: Lactose triggers LacZ operon in E. coli. Lac Z makes an enzyme that cleaves lactose into glucose and galactose. It also contains a multiple cloning site. If the LacZ gene is intact, the cleaving enzyme is produced, and 89 X-gal turns the colonies blue. If recombination occurred, and the LacZ gene was disrupted, the colonies remain white.
Ethidium bromide: Fluorescent dye used in gel electrophoresis.
cDNA production: mRNA + RT -> cDNA (only exons). RT is error prone.
RFLP: Restriction fragment length polymorphism. Many fragment lengths can be seen with gel electrophoresis. Used to make genomic or cDNA libraries.
Star activity: Nonspecific activity of restriction enzymes. A restriction enzyme may cut at different recognition sites.
Multiple cloning site: Site on one gene where many restriction enzymes can cut in one area.
PCR: Polymerase chain reaction. Quicker and more selective than cloning because it doesn't require a vector or transformation.
Nucleid acid hybridization: Detects a gene sequence, useful for separating recombinant from nonrecombinant. Denature / unzip DNA and introduce a single stranded complementary probe. Artificial dye intensifies desired gene that is complementary to the probe. Use filter paper + photographic film to see which colonies on plate have the GOI.
Old sequencing method: Chain termination w/ dideoxy chain terminators (ddNTP) -> arylamide gel electrophoresis -> blotting paper -> x-ray film. Fluorescent colour, Sequence read from graph of waves.
Blotting: Southern (DNA), Northern (RNA), Western (Protein), Southwestern (DNA binding protein).
Computer analysis: Scans for protein sequencing.
Microarray analysis of gene expression: Analyze thousands of genes' expressions at the same time. Fluorescent spots show parts of cDNA with mRNA activity / amount in tissue. Compares amount of mRNA from normal sample + abnormal sample (healthy vs sick) by converting mRNA -> sscDNA that hybridizes with cDNA probes on the microarray chip and lights up in different colours.
Laser-Capture microdissection: Distinguish healthy cells from tumor cells from images. Can use to collect samples for microarray analysis.

Plasmid design

Build in bacteria.

Types: Cloning vectors, sequencing, expression to make a protein.
Bacterial origin of replication (pUC ori): Low copy # (1-12) or high copy # (100-600).
Selectable markers for prokaryotes: Ampicillin, tetracyclin, chloramphenicol, kanamycin, neomycin (in eukaryotes), lac Z, suicide genes (helicase / replication inhibitor).
Reporter genes: Green / yellow / red fluorescent protein, luciferase, chloramphenicol acety transferase.
Bacterial promoters: Lac, T7 bacteriophage (easy to control), araBAD (arabinose inducing promoter)
Mammalian promoters: SV40 (simian virus), CMV (cytomegalavirus), HSV (herpes virus thymidine kinase)
Plant promoters: TMV (tobacco mosaic virus), CamV35 (cauliflour virus)
Yeast promoters: AOXI (methanol).
Protein tags: Histidine (bind metal), glutathione-S-transferase (GST), maltose binding protein, thioredoxin, epitopes (Cam bind antibodies to them. Myc, V5, XPRESS, FLAG).

Expression influencers

Promoter: May be inducible.
Post transcription factors: Codon usage (different codons are used to make the same amino acid in different organisms), splicing and poly A (in eukaryotes), RBS (in prokaryotes).
Post translation factors: PEST sequence, folding (chaperone, disulfide bond, unusual nucleic acids), glycosylation (N linked lys/ars in eukaryotes, O linked thr/ser in prokaryotes), phosphorylation, acetylation, fatty acid (C-term or N-term), secretary signals (NLS in eukaryotes, sec-signal in prokaryotes).

Purify recombinant protein

Clone into expression vector, grow bacteria to log phase, induce expression, lyse bacterial cells depending on cell components' solubility and solvent conditions.

Soluble cell components: Column purification, wash, elution (with competitor), dialysis.
Insoluble cell components: Wash with detergent (can denature), solubilization, dialysis.
Solvent conditions: Temp, pH buffering, ionic strength (solutes), hydrophobicity (glycerol, PEG), reducing agents (disulfide bonds, mercaptoethanol / DTT)
Detergents: Weak (Triton x 100, tween 20), strong (urea, guanidine HCl), ionic (permanent, sodium dodecyl sulfate)

Industrial production

Most biotech products are not proteins.

Bulk fermentation: Ethanol, lactic acid preservative / bioplastic, amino acids, antibiotics, vinegar, CO2, H gas, food (cheese).
Biotransformation: Antibiotic, cortisone / stigmasterol.

PCR

Depends on synthetic oligonucleotides and heat stable polymerase.

Taq: Has different extension rates and proofreading.
Designing primers: Consider reaction conditions. Determine Tm melting temperature (50% hybridization, want it ~2-6° higher than annealing temperature, want similar Tm for forward + reverse primer), length (~20), %GC vs AT.
Simple Tm equation: (#G+Cx4°C)+(#A+Tx2°C)-7°C.
Complex Tm equation: 64.9+41*(#G+C-16.4)/(total#N). Only for ~16 G+C.
Avoid in primer design: Primer dimer (primers hybridize with themselves), hairpin (self complementary, folds and builds on itself)
Optional primer design: GC clamp (3' ends in G or C for more stability and efficient polymerization), 50% GC, 50% AT.
Make forward primer + reverse primer: Forward (from start codon, same as coding strand), reverse (ends with stop codon, same as template strand). Reason is because primer goes from 5' to 3'.
Quantitative PCR / Real Time PCR: See how much material was there.
SYBR green dye method: Ct is the threshold cycle from the beginning to exponential fluorescence. Find Ct from "PCR cycles vs fluorescence" graph, then use a "starting amount vs Ct graph" to find the starting amount of DNA.
Taqman probe method: Like a primer but with one fluorescent end and one Q (quencher) end that prevents fluorescence. Insert the actual primer and elongate, polymerase removes the probe by proofreading activates fluorescence.

PCR uses

Forensics: 0.1% gene / 3 million base difference from person to person. Short tandem repeats are microsatellite clusters. FBI CODIS system assesses 13 short tandem repeats to identify people. Samples can be degraded by fire, bleach, water, and UV light. PCR was used in the Narborough Village murder case to narrow down one suspect. The Golden State killer was found from his relatives on a lineage site.
Paternity: Bryce Cleary has 19 kids from sperm donation. Donor #2757 has 27 girls and 19 boys. Donald Cline has >99.
Identifying the unknown: Soldiers, 9/11 victims.
Identifying the living: Tsunami survivors, Holocaust victims' families, Bobby Dunbar.
Identifying commercial products: Chesapeake crab meat, product labeling.

Synthetic Oligonucleotides

Made in 3' to 5' direction without polymerases. Can use for microarray analysis.

Khorana, Nilenberg, and Halley: Discovered the codon / triplet genetic code and synthetic oligonucleotides.
McBride and Caruthers: Developed the phosphoamidite method and formed the Applied Biosystems company.

How to form synthetic oligonucleotides

Coupling (Attach last nucleotide to silica support)
Deprotection (Remove blocking group)
Coupling (2nd nucleotide, 3rd, 4th....)
Capping (Add protective group to mutant nucleotides to prevent extension)
Stabilization (oxidize to phosphodiester), repeat 2-5
Final deprotection
Phosphorylate the 5' end
Elute and purify

RNA Sequencing

Old methods

Sanger / chain termination sequencing
Maxam-Gilbert / chain degradation sequencing: Uses toxic chemicals. Separates A+G, G, C, and C+T.

Next gen sequencing: by synthesis / deep sequencing containing a lot of contig fragments.

Illumina sequencers: MiSeq, HiSeq, and NovaSeq. Image based method, 300-600 bases per read, uses flow cells. Prepare samples by extracting DNA, attaching adapters (primer binding site + capture), primers on flow cell capture the template and form a bridge, cluster flow cell, then transfer to sequencer. Add base, imaging, chemistry to remove fluorescence from last base. 4, 2, or 1 colour chemistry sequencing.
Oxford nanopores: Embedded in lipid membrane. The current changes based on the base entering the pore. High and biased error rate, but long reads and can sequence RNA.
Pacific Bioscience sequencing: Bases flow in and out of a well with the template, bases bind to template, and take a photo.
Crawling: Adjacent fragments are formed and ligated in order.
Next gen contigs: Contigs overlap at random spots. Can use to compare single nucleotide polymorphs.
Metagenomics: Sequence human microbial flora. Can be done by RNA sequencing (mRNA -> cDNA).
Pyrosequencing: dNTP is added and detected by Luciferase light in a flow cell with DNA fragments attached to a bead.
Recombineering: The current state of cloning. Use polymerase and ligase to join oligonucleotude fragments. Can replace oligonucleotide to account for different codon usage. .

RNA Technology

Types of RNA

tRNA

Transfer amino acids to a polypeptide chain.

rRNA

Forms ribosomes.

microRNA

Regulatory, gene dosage compensation. Gene silencing in plants and fungi by methylation turns off genes and genes around it. 20-40% of human genes have microRNA partners.

Xist: Long RNA inactivates X chromosome by methylation.
piRNA: Suppresses transposons.

TERC RNA

Telomerase template.

RNAi

Knock down / downregulate gene expression. Silencing, Ago -> RISC + siRNA complex -> cut / slice mRNA to silence gene expression. Easiest to design. Most off target effects. Least versatile. Cannot introduce mutations.

CRISPR

Knock out gene expression. Charpentier and Duodna won Nobel Prize. Cas9 endonuclease cuts at a specific 3 base pair PAM region. sgRNA forms ribonucleoprotein with Cas9. sgRNA must be designed to work with DNA sequences next to PAM sequences. Cas9 nickase reduces off target effects. Nonhomologous end joining and hemology directed repair (with donor DNA) are used to rejoin strands / fix dsDNA splicing. Most versatile. Dead Cas9 can have effector proteins attached for different functions. Can be too big for viruses. Dr. He Jianku genetically modified twin girls to delete the CCR5 HIV coreceptor. T7 nuclease assay / surveyor assay amplifies + hybridizes + cleaves mismatches in DNA sequence.

TALEN

Knock out gene expression. Artificial restriction endonuclease proteins that don't bind to pallindromes. Cleavage domain (nuclease) of Fok1 area on protein is flanked by 33-35 amino acid repeats (DNA binding domain) on protein. Combination of repeats correspond to DNA bases. Same as CRISPR but better specificity and least off target effects. Can have effector proteins attached for different functions. Most difficult to design. Proteins are bigger. Double cloning work because they must be done in pairs.

Ribozymes

RNA that catalyzes a reaction. Can bind to and cleave a specific target. Smaller ribozymes (hammerhead or hairpin). Use unusual nucleotides (phosphorothioate, nucleotide analogs) to prevent RNA degradation. Naturally occurring ribozymes were meant to cleave themselves.

Aptamer

Oligonucleotides with 3D structure that bind with high affinity to targets. Randomly generate thousands of RNA sequences, use SELEX high throughput screening method to screen for type of activity (Does it bind? Cleave?).

Genetic Modification of Eukarotes

Genetic engineering, transgenics.

Get DNA or RNA into a bacterial cell by transformation (with CaCl2, CaPO4 precipitation, or electroporation).
Get DNA or RNA into a eukaryotic cell by transfection (Liposomal agents / soaps / vesicles containing DNA, electroporation, gene gun for plant cell walls by ballistic projection, CaPO4 precipitation, aptamers, modified adeno-associated viruses, microinjection). Transient transfection is taking in a gene without integrating it.

Distinguish modified organisms from unmodified.

Selection: By antibiotic. Insert GOI by homologous recombination and grow in ganciclovir + geneticin. Neor neomycin resistance gene allows cells to survive in geneticin. Cells with thymidine kinase make ganciclovir toxic.
Cell sorting: By fluorescent activated cell sorting (FACS). Gives cell # and sorts cells on quadrant chart.

Methods

Long Terminal Repeats: Use as homologous region to modify virus. Can make infectious recombinant viruses fast. The problem is that the gene can be inserted anywhere any number of times.
Cre-Lox recombination: Cross breed cre individual with lox individual. Cre recombinase targets palindromic recognition site. Function depends on orientation of lox gene (from bacteriophage). Can invert, delete, or translocate a gene between lox regions.
Microinjection (Germ line): Cage female mice together, sync cycles and fertility hormones, produce 30-40 eggs, mate and fertilize, flush oviducts. Egg has 2 pronuclei, inject recombinant DNA into nucleus. Screen progeny by southern blotting. Dolly.
Blastocyst / STEM cell method: Donor blastocyst becomes embryonic STEM cells (some are transfected) that become blastocysts, inject them into the mother, observe possibly mosaic progeny.

STEM cells

Embryonic: From plastocyst, from IVF in humans.
Fetal: From HEK 293 fetal cell line or from donated placenta.
Adult: Limited variability, potency, and replication cycles. Bone marrow.
Potency: The ability to differentiate into other cell types. Totipotent / omnipotent / pluripotent -> multipotent / oligopotent -> unipotent / monopotent.

Animal GMOs

Applications: Female goats can make milk with gene product such as Atryn anticoagulant protein, produce world supply, and lower cost. AquAdvantage salmon overexpresses growth hormone. Fish with vitellogin promoter near GFP acts as biosensor and responds to estrogen mimics.
Lab grown beef: Use animal myeloid STEM cells (mosa meat, memphis meats). Lean, less animals used. Requires power, growth media, and fetal bovine serum.
Meat substitution: Not really biptech. Beyond burger made of mung bean, pea, potato starch. Less calories, same amount of fat, 0 saturated fat, less methane used, has beef extract for flavour and colour.
Benefits: Increases available food because of disease resistance, increase nutrition, make humanized tissues and organs.
Concerns: Ecological (plants) and economic (public trust, "rubbery chicken" syndrome).

Transgenic humans

Germline modification: Dr. He (CCR5), genetic conditions (down syndrome, muscular dystrophy, cystic fibrosis).
Somatic corrections: STEM cells + targeted delivery to tissues. Jesse Gelsinger died from too high of a dosage and anaphylactic shock.

Plant GMOs

By traditional breeding, mutation breeding (mutagens + x rays), totipotent STEM cells (apical meristem, root tip, immature embryo), callus culture (mass of cells on solid support), suspension culture (liquid), Ti plasmid (Agrobacterium sp).

Direct introduction into callus / suspension culture from protoplasts.
Floral dip (treat with surfactant, infect with Agrobacterium).
Particle bombardment (colloidal metal coated with DNA can target organelles).
CRISPR, siRNA, and gene splicing are common (epigenetics).

Benefits: Lower pesticides (from cry protein) and herbicides (glyphosate), fungal resistance (protect against tropical race in bananas), viral resistance (protect against papaya ringspot virus by viral coat protein in papaya), increase nutrition (golden rice, vitamin A), pharmaceuticals (vaccine banana), phytoremediation (bioremediation), novel products (arctic apple).
Concerns: Requires approval from USDA, FDA, EPA. No human health concequences, Neor drug resistance removed, spread in ecosystem is rare, ecological (lower carcinogenic herbicide), economic exploitation (10 companies control 75% market share).

Immunotherapy

Clonal selection: B cell becomes plasma (secrete antibodies) or memory cells by antigen.
Hybridoma: Immunize 1 mouse, isolate B cells and fuse with myeloma cells (immortal cancer cells) to form hybridoma. Immunoglobulin genes + recombination + mutation = billions of antibodies. Use to produce commercial antibodies.
Applications: Diagnostics (COVID-19, strep, pregnancy), antivenom, treating disease, cancet treatment (modify immunoglobulin to deliver toxin, radioisotope, or metal).
Anti PD-L1: Prevents PD-L1 on tumor cells from deactivating T cells.

Legal review

FDA, VSDA, EPA, NSF / NIH guidelines. Center for Biologics Evaluation & Research.

Clinical trials phases

Safety and dosage.
Effectiveness.
Is it better than available treatment?
Phase 3+, post marketing surveillance (acute lymphoblastic leukemia, factor IX hemophilia, AAV therapy for retinal dystrophy).