BC 180 Notes PDF

BIOCHEM 180: Biochemical Engineering ● Agricultural/aquatic: biotechnology for A1: Introduction to Biotechnology plants / animals 1.1. Biotechnology vs other fields ● Biotechnology: application of sciences to create different products or solve a spores ○ Genetically problem ● Biochemical ○ Bt-...

BIOCHEM 180: Biochemical Engineering ● Agricultural/aquatic: biotechnology for A1: Introduction to Biotechnology plants / animals 1.1. Biotechnology vs other fields ● Biotechnology: application of sciences to create different products or solve a spores ○ Genetically problem ● Biochemical ○ Bt-modified organisms: pesticidal engineering: chemical engineering in biological sciences to create products from raw materials and sciences: cats and zebrafish (GloFish) ○ Transgenic salmon overproducing growth hormone ● Bioremediation: contribute to reduction develop processes for achieving this ● Pharmaceutical altered drug development (without the use of living of environmental pollution ● Genomics: application of bioinformatics to analyze genomes organisms) ● Medical: whole spectrum of human medicine 1.2. History of Biotechnology ○ Using ● Ancient (pre-1800) ○ Cheese: adding rennet (enzyme found in the stomach of calves) smallpox and with drug or targeted drug therapy via gene therapy or genetically ○ Crossbreeding modifying stem cells ○ Penicillin ● Modern (1945–present): manipulation of genetic material ○ Consumer genomics: direct to consumer genomic testing ● Regulatory: control 1.3. Fields of Biotechnology vector variation ○ Regenerative medicine: treatment rabies microorganisms disease and response e.g. precision medicine ● Classical (1800–1945) ● Microbial: to track other genetic traits genetic ○ Beer: yeast against predisposition to ○ Pharmacogenomics: influence of to sour milk ○ Vaccine SNPs manipulation e.g. cloning into of a quality assurance and A2: Classic Techniques of Molecular Biology and Recombinant DNA Technology 2.1. Classical paradigm and tools of molecular biology ■ Restriction fragment length polymorphism (RFLP): differences among DNA sequences at sites recognized by REs (application: DNA barcoding e.g., ● Paradigm species identification) ○ Genetics: genes & function ● along the MstII site ○ Biochemistry: proteins & function ○ Molecular biology: proteins Normal cell: two bands ● & Sickle cell anemia (characterized by a missense mutation): one genes band along the MstII site ● Tools ○ Recombinant DNA/Molecular ○ Restriction enzymes ■ Essential in cutting foreign DNA ■ Cuts unmethylated DNA sequences that palindromic in are character along an approximately 6 bp recognition site ■ Type I: random (>1kbp from RS); ATP-dependent ■ Type II: within RS; ATP-dependent ■ Type III: ~25 bp from RS; non-ATP-dependent ■ Frequency of n cutters: once / 4 bp n ○ Agarose gel electrophoresis ■ Essential for visualization of differently sized DNA fragments clones ■ DNA fragment: gene of interest ■ Vector: where target gene is inserted; self-replicate can and have selectable markers ● Plasmid: 50bp–20kb ● Cosmids: <45kb, lambda phage into E. coli ● BACs: 100–300kb (not used necessarily for molecular cloning; if used, confirmed via screening blue-white such that recombinant BACs are white) ● YACs: up to 1 Mb (more applied in organismal sequencing) ● Essential elements ○ ori ○ ampR ○ MCS of any point mutation in human DNA by analyzing the PCR products using a primer that forms a 3’ mismatch ○ Promoter ■ Polylinker: inserted DNA fragments with multiple recognition sites; can be Assembly PCR method for the assembly of large DNA oligonucleotides from shorter fragments with overlaps Methylation-specific PCR used to detect genes or sequences with DNA methylation after bisulfite treatment using methylated and unmethylated primers Microfluidic Chip PCR allows rapid, real-time amplicon sampling and quantitation w/o need for probes PCR for DNA fingerprinting uses noncoding regions (minisatellites) as basis to determine the probable identity of an individual PCR diagnostics for disease/pathogens PCR tests can find evidence of disease in the earliest stages of infection PCR in genotyping identifies the differences in DNA sequence among individuals or populations synthetically inserted via PCR ■ Expression cloning ● Transformation: heat-shock electroporation, etc. ● Transfection: calcium PO4, liposome, etc. ○ PCR ○ DNA sequencing ○ Blotting PCR Multiplex PCR Nested PCR Colony PCR Loop-mediated isothermal amplification (LAMP) assay Allele-specific PCR uses several primer sets to amplify several DNA sequences simultaneously uses two sets of primers (inner and outer) to reduce non-specific binding method for rapidly screening colonies grown on selective media to verify that the desired genetic construct is present or to amplify a portion of the construct a single-tube technique for amplification of DNA, using “looped” primers at constant temperature without thermal cycler, & low-cost alternative to detect certain disease permits the direct detection 2.2. Recombinant Protein Technology ● Natural (expensive, can have negative effects) vs recombinant (cheaper, safer, abundant) ● Vector selection ○ Compatible with host system: prokaryotic vs eukaryotic ○ Contains elements such as: ■ Inducible e.g. lac (on/off switch operon) constitutive promoters ■ Ribosome binding sites vs ■ Tags (competitor) or thrombin ○ Expression vectors for E. coli ■ pBAD: inducible (protease) by ● Thrombin cleaves sequence between arabinose ■ Lac operon target protein and system: its tag inducible by IPTG ● Insert ■ pET: produce recombinant typically strain BL(DE3) with T7 promoter adjacent ■ pGEX: encodes GST gene, useful for affinity activity usually associated with a turnover rate or amount needed for Unit = 0.1 optical density increase in a 1 cm ○ Basic strategies ■ addition of affinity handle cuvette at 650 nm at 37°C and pH 6.2 (e.g. tags) ■ addition of sequences that aid secretion, precipitation, ○ Lactate dehydrogenase assay ■ Increase in A340 observed in 1 minute detection ■ addition of protein that help recombinant protein to refold and stay soluble ○ Column affinity chromatography bead = ligand immobilized on bead (e.g., for His tag, glutathione for GST) ■ GST-tagged eluted ■ Unit: quantitative measure ■ 1 ● Protein purification and detection Ni2+ ○ Unit activity measurement stoichiometric binding chromatography ■ Affinity ● Recognition of target proteins of to lac be in-frame protein up to 50% of total cell protein, host bacteria should with proteins glutathione ■ NADH absorbs light at 340 nm ○ Immunoaffinity chromatography ■ MAbs in stationary phase that will bind protein target ■ However, may leach off the column and/or inactivated/bound non-specific proteins be by ○ Recombinat DHFR = ○ Scaled GST-DHFR-His up fermentation and purification 3.3. Pathway Design ● Even A3: Metabolic Engineering 3.1. Brief Background ● San Francisco: biotechnology capital of the world ● Genentech: leading pharmaceutical in San Francisco, California; pioneer of biotechnology in 1976 ○ Academic/government institution ○ Pharmaceutical companies 3.2. Metabolic Engineering ● Cells can serve as sustainable “factories” for beneficial biomaterials ○ Ex: insulin derived from pancreas glands of pigs (contaminated by other proteins) vs. recombinant insulin (highly defined and high purity) ● Purposeful modification for enhanced production of a desired material ○ Produce novel material ○ Lessen cellular expenditure ○ Decrease byproducts ○ Degrade undesirable material ● Strategies and procedures ○ Pathway design ○ Systems biology ○ Synthetic biology ○ Strain Optimization before designing pathways, identification of (renewable) source of raw bio-materials is done ● Identifying a biotransformation pathway ○ Retrosynthesis: process of “deconstructing” target molecule into available starting materials ○ Chemical reactions/processes ○ Enzymatic processes ● Computational predictions can now be used to design the pathway 3.4. Systems Biology ● Challenges with proceeding with designed pathway ○ Not all organisms have all the genes needed in the pathway ○ Not all are suitable production strains ○ Not all genes can be expressed heterologously ● Interrogation systems of complex through biological large-scale quantification of numerous biomolecules ○ DNA–mRNA–protein–metabolites ● Possible scenarios: ○ Overproducer; large-scale (ideal) ○ Incompatible to lab conditions ○ Imbalanced flux in pathway ○ Accumulation of product is toxic ○ Incomplete pathway in one 3.5. Synthetic Biology (Strain Optimization) ● Use of “workhorse” strains ● Perform genetic manipulation ○ Knock-in/out genes ○ Modifying promoters or adding repressors ○ Taking a plasmid with high copy number ● Flux optimization ● Culture optimization ○ pH (buffer) ○ Reverse engineering ○ Metabolic rewiring ● Note however that there is not one solution to a given biotransformation ● Exciting areas for metabolic engineering ○ Bioremediation ○ Drug delivery ○ Protein engineering 3.7. Protein Engineering ● Purposeful modification of peptides and proteins for enhancement of existing or endowment of novel features ○ Temperature ○ Physicochemical properties ○ Aeration ○ Structure ○ Osmotic pressure (salt) ○ Function ○ Carbon source ○ Feed material concentration 3.6. Adaptive Laboratory Evolution ● Multiple cycles are necessary to screen ● Structural Biology: structure relate to function; where protein engineering is anchored ○ Ex: Akt kinases ■ PH synthetic strains = very low efficiency of domain: allows its recruitment to the plasma evolution membrane (via phospha- ● “Brute force” approach for screening tidylinositols) greater number of strain variants ■ Kinase domain: catalyzes ● Process: protein ○ Unoptimized candidate strain is via ATP/substrate binding passaged until better-performing “evolved” strains are obtained ○ Structure-based engineering does not always work as planned ○ Apply positive/negative pressure ■ Positive: feeds the strain ■ Negative: kills the strain phosphorylation ● Tools ○ Sequence optimization ○ Fittest strain is isolated ○ Non-natural aa incorporation ○ Genome sequencing ○ Directed evolution ○ Bioconjugations making it less soluble and long-lived in bloodstream 3.8. Sequence Optimization ■ A-chain C-terminus Asn is ● Physicochemical / structural properties ○ Solubility: increase hydrophilicity degraded via deamination by adding charged amino acids hence the need for Asn → Gly substitution ○ Proteolytic degradation: prevent trypsin degradation by lysine and ● Multiple rounds of random mutagenesis arginine mutations ○ Redox 3.9. Directed Evolution sensitivity: remove to allow screening a large mutational landscape cysteine residues and accumulate desired beneficial mutations ● Case study: Insulin therapies ● However requires a functional screen ○ Types: ■ Active, short-lived monomer i.e., ■ Inactive, long-lived hexamer peptides/proteins ○ Insulin lispro activity-based which assay for mostly only attach to other proteins without activity ■ Inversion into Lys-Pro in the an B-chain C-terminus results into its clashing ● Hence affinity based evolution platforms were developed where genetic material is connected to the protein with the Arg of the other ○ Phage display B-chain and opens up the ○ Yeast display surface ○ mRNA display ■ Prevents the dimerization ● This allows screening of protein-protein of insulin, locking it into binding the mutational variants monomeric form, making it fast-acting ○ Insulin glargine ■ Introduction of two Arg in the B-chain C-terminus ■ Increases pI from 5.4 to 6.7, precipitating it out at the physiological pH and interactions and enriching ● Essential for antibody drug research ○ Frances H. Arnold: directed evolution of enzymes ○ George P. Smith / Sir Gregory P. Winter = phage display peptides and antibodies ● Case study: Peginesatide of ○ Small peptides were found to mimic erythropoietin (EPO) in its ability to control hematopoiesis signaling ○ Some amino acids are conserved which are essential for binding to ○ More engineering strategies are terminals are modified as it may inhibit receptor binding & signal ○ Benchmark GLP-1 analogs: ■ Dimerization increase concentration of peptide amino acid e.g. PEG incorporation existing chemical moieties within a peptide/protein ○ Amino acid bioconjugation e.g. N-hydroxysuccinimide → (NHS) esters or Cys → Maleimide ○ Peptide modification: primarily use tags to introduce into the receptor ■ Unnatural 3.11. Bioconjugations Lys protease degradation stick to blood proteins and enhance receptor binding 3.10. Non-natural Amino Acids ● Downside of tag-and-modify ○ Introduction of large, artificial sequences ○ Stochastic/random modification without precision ● Amino acid can also be chemically synthesized, in addition to ribosomal synthesis ● Hence, click reactions are developed ○ Click tags are very small, generally smaller than peptide more access to more functionalities and expansion of features ● Case study: GLP-1 analogs insulin, stabilize it against DPP4 cleavage ● Chemical or enzymatic modification of done to produce the final drug ■ Charged introduced to an amino acid to liraglutide and semaglutide the EPO receptor ○ Peptide ○ It is short-lived; cleaved by a ○ To optimize GLP-1, a sugar was ■ Peptides = 2 kDa (<15 aa) allows receptor protease DPP4 ■ EPO = 34 kDa ● This utilization by binding to GLP-1 that acts increasing similar to glucose motifs; produces high yield and selectivity products carbon-hetero bond by formation reactions ○ Carolyn R. Bertozzi / Morten Meldal / K. Barry Sharpless = development of click chemistry ○ Cohen and bioorthogonal chemistry and Boyer: restriction ● Case study: Antibody-drug conjugates (ADCs) use enzymes of for recombinant DNA technology ● While genetic engineering leverages the ○ Chemotherapy (efficient killing of catalytic machinery cancer cells but also kills normal manufacture cells) vs immunotherapy (highly engineering specific enabling to cancers cells but inefficient killing) of the cell to proteins, goes the metabolic one step further, cell to manufacture enzymes to tune the cell's own catalytic ○ ADCs have been developed to machinery. Furthermore, metabolic link cytotoxic drug to an antibody, engineering empowers the cell to carry resulting out to high specificity, variety of chemical enables the cell to make proteins, 3.12. Synthesis / Summary metabolic engineering enables the cell ● Biochemistry vs chemical biology ○ Biochemistry: study biomolecules in their natural biological context biology: biomolecules wider reactions. Whereas genetic engineering efficient killing of cancer cells ○ Chemical a study by chemical alterations of to make vitamins chemotherapeutics small-molecule (5), antibiotics, (6), drugs, other industrial introducing polymers, dyes, fuels (7), and other to create specialty chemicals. All that is required is a host cell with the necessary starting more biology 3.13. Biology as a Basis for Biochemical materials Engineering genetic material coding for the pathway ● Since DNA sequence completely defines an RNA, which subsequently completely and the necessary DNA enzymes. ● Complexities of cellular manufacturing defines a protein, then any desired ○ Metabolic flux: rate of turnover of protein can be manufactured by cellular molecules through a metabolic machinery pathway; ○ Via large-scale fermentation given gene is inserted to host metabolically engineered pathways suffer flux imbalances; solved by modulating expression of enzymes, directed evolution, control of spatial ○ Genomics (1976 - emergence of organization ○ Metabolic Genentech) burden: increased levels of non-essential proteins robs the cell of its building blocks ○ Genetic instability: ○ Convergence ● Organism selection ○ Prokaryotic expression system is genetically not fruitful for some eukaryotic engineered are less stable than molecules plasmid-free non producer cells; membrane proteins) caused by loss of and mutations for production of products such ● Future of biochemical engineering as ○ Genetic engineering: production individual proteins of individual metabolic pathways in cells design flux, pull - decrease, block remove competing pathways) synthetic genome ○ Directed is reproduced by chemical starting and continuous evolution ● Tools ■ Complete genetic system with digitized DNA sequence in a computer ○ Genome editing (classical vs CRISPR/Cas9) ○ Static vs dynamic regulation ○ Omics tools (NGA for DNA, RNA; LC-MS for proteins, metabolites) Engineering and Functional Food Research ○ Genome-scale models (GEMs) ○ Machine learning ● The Three Revolutions biology strain and combinatory) created the first cell with a ○ Molecular for ○ Push-pull-block (push - increase ■ In 2010, J. Craig Venter Metabolic and (knowledge-based design, evolutionary cells = synthetic life 3.14. acid 2-propanol/acetone ○ Future: engineering entire new synthesis, itaconic ● Strategies engineering: introduction hydrophobic ○ There are “non-model” organisms in plasmid DNA vectors ○ Metabolic (e.g. ● Applications (1953 elucidation of DNA structure) - ○ 1979: synthetic human insulin expressed in E. coli 3.15. Functional Food ● Foods that offer health benefits beyond their nutritional value ○ Long-term pronounced consumption Purification effects by are long-term (mostly disease prevention function) strongly associated with metabolic syndrome (diabetes) ○ Cyanidin 3-o-glucoside (Cy3G): black soybean seed coat extract prevented body weight gain and increase in white adipose tissues; elevates the intracellular cAMP levels, which induces beige adipocyte phenotypes ○ Anthocyanin-rich foods: bignay, baligang or lipote, duhat ○ Purple sweet potato is rich in 3,4,5-tricaffeoylquinnic acid ○ Artemisinin: effective antimalarial agent ○ Flavonoids: phenolic compounds (apigenin-7-glucoside) in olives on leukemia ■ Cell differentiation therapy (HL60: cancerous version common progenitor) ○ Protein quantitation (A280) = ○ DHFR enzyme assay ● Inactive proteins may require ○ post-transcriptional modifications ● Natural products of ● Analysis of protein extract ○ molecular chaperones ○ Hypertrophic adipocytes also 4.1. Protein Analysis and Purification Beer’s law ○ Examples: Yakult, green tea ● Obesity A4: Protein Analysis, Expression, and myeloid ○ cofactors/protein partners ● Good purification scheme = good purification level & yield ○ High degree of purification but poor yield leave little protein for experiments ○ High yield with low purification leaves many contaminants in the fraction 4.2. Expression Systems ● Bacterial systems ○ Grow quickly but has difficulty expressing large proteins; cannot perform glycosylation and handle S-S rich proteins ○ Alternatives: ■ Yeast (Pichia pastoris e.g., used to infect sex cells) ■ Virus (Baculovirus)–insect cell system ■ Mammalian cell (optimal) ● Yeast cells ○ Single-celled eukaryotes, but need not be integrated into similar to bacteria in reproduction ■ S. cerevisiae ■ Stable: ■ Pichia pastoris: induced by methanol using alcohol quickly ■ Gene ○ Selection viruses with ● DHFR gene makes cells ○ Plasmid into baculovirus DNA and in multiple rounds of increasing MTX ○ Can express large proteins but levels to select for grows very slowly cells ● Mammalian cell line and highest expression can be achieved correctly vectors: with DHFR gene and GOI ○ Optimal: all necessary processes adenoviruses to ● Gene is expressed difficult-to-express protein with low level of risk ○ Eukaryotic resistant MTX undergoes lysogenic cycle for (MTX) selection dsDNA genomes (insect larval host) ○ Used locations within ■ Methotrexate ● Baculovirus into different chromosomes allows expression of large proteins protein integrates random and for expression oxidase (AOX) ○ Insect (e.g., electroporation) ○ Strains: ○ Grows the genome human retroviruses, ○ Generally used to test the function of a protein in vivo rather than produce proteins in SV40 ■ Promoter: CMV or vaccinia ● Best expression systems for: virus ■ Selectable marker gene: ○ Large proteins (>100kD): eukaryote DHFR ○ Cells: derived from CHO cell line ○ Small proteins (<30 kD): essential: yeast, prokaryote ○ Transfection ■ Transient: large amounts for plasmid introduction; foreign gene ○ Glycosylation baculovirus or mammalian cells ○ High yields, low cost: E. coli, ■ Characteristics change with subsequent yeast ○ Post-translational ● Primary vs continuous ○ Primary only last at most 20 4.3. Cell Culture vitro passage modification: yeast, baculovirus, eukaryotes ● In may analysis of multicellular weeks, have moderate consistency, but need higher cost eukaryotes to optimize culture conditions ● Types: ○ Primary: finite life span ○ Continuous: abnormal, immortal ○ Continuous have relatively culture costs, but subject to genetic drift ○ Primary can become continuous ● Maintenance ○ Growth environment: 37C, 5% via transformation ● Continuous cells (Cell lines) CO2 ○ Growth media: dependent on cell ○ Have inherent gene mutations that type ○ General medium requirements: bulk ions, trace elements, sugars, amino acids, vitamins, choline, inositol, serum, antibiotics allow escape of cell proliferation regulation processes ○ Makes them useful “workhorses” in most laboratories ● Stem cell lines ○ Have the ability to self-renew or ● Primary cells ○ Most closely represent, as it is directly taken from, tissue ○ Advantages ■ Avoids ethical objections contrast to in vivo ■ Provides relevant results differentiate to various cell lines ● Cell culture transfection ○ Introduction of foreign DNA into eukaryotic cells, which may be stable or transient ○ Methods than cell lines ■ Calcium chloride + DNA = ■ Cost-effective calcium PO4 complex will ○ Disadvantages ■ Limited growth potential and eventually die be endocytosed into the host cell, but low efficiency ■ Lipofection vesicles uses to lipid transiently introduce DNA, about 30-40% efficiency modification: uses electronic pulses ● Recombinant proteins, gene resistance, and enhanced properties ○ Goals ○ Transgenic animals can be used ■ Expression of protein product on for commercial production ■ Analysis of effect of DNA cell function using large-scale e.g. recombinant proteins in the milk of cows and sheep, rhAT in transgenic goats, ANDi the first transgenic primate reporter genes carrying GFP gene ○ Common reporter genes ■ GFP: green under UV light ○ Bt–modified organisms allow for ■ Luciferase: produces light insecticidal ■ lacZ conferring human toxicity gene: encodes beta-Gal, appear as blue colonies ■ CAT ○ Golden produce gene: confers resistance to chloramphenicol Genetically specific insertion of a gene ■ Electroporation 4.4. ○ Genetic modified effects rice without engineered beta-carotene, to niacin, iron, essential minerals, etc and thus improve vitamin A content ■ Rice lacks four enzymes to organisms (GMO) produce beta-carotene, the precursor of vitamin A ● Also known as transgenic organisms ○ Fruits e.g. virus-resistant papaya, ○ Organism with altered genetic material using engineering ○ DNA from different sources are combined into one (chimera) to mosaic-resistant squash, enhanced tomato, herbicide-resistant non-browning apple create a new set of genes ● Traditional breeding vs genetic engineering ○ Traditional: A5: Genome Editing hybrid cross of domesticated and wild; transfer of genes cannot be controlled 5.1. Vaccine Production sugar beet, ● Traditional method: making recombinant ○ Studying of genetic diseases and gene to produce recombinant protein as vaccine efficacy of treatments ● Classic ways of genome editing ● Kariko & Weissman: discovered that ○ Site-directed mutagenesis base-modified mRNA can be used to ○ Cassette mutagenesis block ○ Use of viral vectors activation of inflammatory reactions ● mRNA ● Site-directed mutagenesis vaccines: delivery via lipid ○ PCR-based technique of changing nanoparticles ○ Allows DNA at a desired position e.g. transport through cell membranes SNP, missense, deletion, etc. ○ Determines ○ Protects RNA from being broken down make them last longer in the body) subtilisin have reduced activities ● Cassette mutagenesis ○ Variety of mutations already introduced to gene of interest ○ Limitation: need for low temperature ● Use of viral vectors ○ Random ○ Bivalent vaccines variants of mRNA very rapidly for in the other diseases 5.2. Genome editing ● Eukaryotic model organisms ○ Saccharomyces cerevisiae ○ Drosophila melanogaster ○ Caenorhabditis elegans 5.3. Analysis of Gene Function ● RNAi or post-translational gene silencing currently in clinical trials ○ Mus musculus (anywhere genome) vs targeted insertion ● Advantage: you can make different ● Knockout mice DNA ○ Example: site-directed modified nanoparticles with PEG (which vaccines of sequence change on function ○ Moderna and Pfizer: used lipid ● mRNA effect ○ Produces siRNAs that silence complementary sequence ○ Degradation of mRNA blocks gene functions ○ Function: immune response against foreign RNA ● Synthetic siRNAs ○ Limitation: only transient outcome targets mRNA, produces a as it only allowing continuous transcription of the DNA sequence; that cleaves and degrades mRNA, silencing the gene siRNA-based treatments should be continuous ● Gene disruption/knockout ○ Mutated gene is introduced to embryonic stem cell ○ Normal gene “knocked out” by foreign gene via homologous recombination RNA Interference small non-coding RNA (e.g., miRNA, siRNA, shRNA, piRNA) blocks post-transcriptional expression of gene by binding to mRNA and prevents the protein from being translated microRNA (miRNA) encoded by cell genome in a naturally occurring process small interfering RNA (siRNA) derived from exogenous viral dsRNAs introduced to cells via injection or delivery by vectors e.g., modified viruses short hairpin RNA (shRNA) precursor of siRNA which is one strand of RNA that is folded over, creating a dsRNA with hairpin loop on one side PIWI-interacting RNA (piRNA) specific to the germline, protecting genome integrity from transposable elements messenger RNA (mRNA) ssRNA that contains genetic info for protein translation Dicer cleaves dsRNA to ~20-25 nt-long siRNA (if shRNA, hairpin loop is removed) RNA-induced silencing complex (RISC) enzyme complex that binds siRNA, attaching to the guide strand of the siRNA Guide strand binds to its complementary sequence in mRNA Argonaute enzyme that is part of RISC ● Gene knock-in ● Genome editing vs genetic engineering ○ Genome editing: in situ alteration fused to the DNA-cleaving domain of FokI endonuclease of DNA sequence ○ DSB DNA repair mechanisms: NHEJ and HDR ● Genome editing mechanisms ○ Zinc finger nucleases (ZFNs): zinc finger DBD anchored Transcription Activator-like effector nucleases (TALENs) variable DNA-binding domains of bacterial proteins called TALEs (which can recognize a single, unique DNA base) is fused to FokI CRISPR-Cas9 simple customizability provide advantages over ZFNs and TALENs Protospacer pieces of invading foreign DNA that are incorporated into a CRISPR locus Protospacer adjacent motif (PAM) a 3-bp long sequence required for Cas9 to recognize and cleave the target DNA sgRNA scaffold two sequences, identical and complementary to the target, with extra sequences included on their 5′ ends to engineered nuclease FokI which makes a nonspecific DS break ○ TALENS gene editing - single nucleotide DBD resolution: TALENs (have longer recognition sequences than ZFNs) anchored to FokI Genome Editing Gene targeting by homologous recombination incorporation of homologous sequences into the target leads to the “switching out” of the endogenous gene for an altered version Genome editing similar to gene targeting but with much higher efficiency, establishing the desired mutations in more cells in any given experiment Nonhomologous end joining (NHEJ) DNA repair mechanism where broken dsDNA ends are resealed directly; could result to mutations due to several bases being deleted or added Homology-directed repair (HDR) DNA repair mechanism where dsDNA damage is fixed by copying from a homologous template; allows addition of specific changes to target site zinc finger nucleases (ZFNs) DNA-binding “zinc finger” domains of transcription factors (which can recognize a specific nucleotide triplet) is ○ Medical applications (gene knockout) ○ Industrial applications CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein 9) ● CRISPR ○ Variants: can induce gene silencing or enhance transcription CRISPR-Cas9 bacterial system which act as an adaptive immune defense against invading viruses viral DNA enters bacterial cells upon infection by a virus CRISPR locus region of bacterial genome where viral DNA is inserted tracrRNA associates with transcribed RNA product to orient the Cas9 protein and RNAse RNAse cleaves the RNA transcript into short segments crRNA recognizes and guides Cas9 to complementary viral DNA Cas9 (or Cpf1) RNA-guided endonuclease that destroys viral DNA CRISPR arrays library of short viral DNA segments used to transcribe RNA which help recognize and destroy viral DNA when re-exposed to viruses single guide RNA (sgRNA) synthesized RNA construct that mimic tracrRNA & crRNA which can target a gene of interest by introducing along with a Cas9 construct ○ Potential uses: population suppression or replacement ○ Gene drive mosquitoes engineered to fight malaria will pass resistance to Plasmodium to its offsprings A6: Plant Tissue Culture 6.1. Animal Cell Culture Animal Culture Plant Culture can only do specialized function of the organ it is extracted from can differentiate into any type of cell require wide range of nutrients can grow under limited number of nutrients tend to degrade after several cell cycles can undergo unlimited number of cell cycles genetic manipulation and production of secondary metabolites artificial micropropagation of plants 6.2. Plant Tissue Culture ● Culture of any part of the plant in artificial media, in aseptic conditions, under controlled environments ● Fundamental ability of plants ○ Totipotency: potential to develop into an entire plant ○ Dedifferentiation: ● Genome-edited crops ● Gene drives ○ Systems of biased inheritance mature cells capacity of return to to meristematic condition ○ Competency: potential to develop in a particular way, giving directionality to differentiation ● Factors affecting plant tissue culture ○ Culture media ○ Organ ○ Callus ○ Cell suspension ○ Protoplast ● Callus formation ○ Environmental factors ○ Callus: disorganized cell mass ○ Genetics derived from somatic tissues and ○ Explant source induced ○ Aseptic conditions hormone application ● Basic components of plant culture media ideal for plant cell suspension in obtaining secondary metabolites P, K, S, Fe ■ Microelements: B, Co, Cu without cell walls sugars, vitamins, complex compounds substances: ● Protoplast culture ○ Protoplast: “naked” plant cells I, Mn, Mo, Zn ○ Support via regenerate into new plants and ■ Macroelements: Ca, Mg, N, compounds: explants ○ Friable (loose, watery): do not ○ Mineral source ○ Organic from ○ Used for DNA transformation and production of hybrid plants agar, ■ Somatic hybridization agarose, gellan gums ■ Cybridization ○ Hormones: auxins, cytokinins ● Hormonal control of organ formation ○ Auxin: root development ● Micropropagation ○ Tissue culture accompanied by soil culture (acclimatization) ○ Cytokinin: shoot development ○ Ratio dictate plant development ■ A > C: root 6.4. Practical Applications and Recent ■ A = C: callus Advances ■ A < C: shoot ● Ancient seeds brought to life ○ Silene 6.3. Types of Plant Tissue Culture ● Types ○ Seedling ○ Embryo stenophylla regenerated from placental tissue of fossil fruit in a Siberian permafrost for over 30,000 years ○ Most ancient, viable, multicellular living organism ○ Abscisic acid (ABA): hormone which activate the ABFS-ABRE ● Endangered/rare species conservation plant stress pathway ○ Ex situ conservation ○ Editing of OsWRKY24/53 gene ○ In vitro regeneration regions which could be negative regulation of ABA-responsive ● Production of artificial seeds ○ Seed: encapsulated single single knockout of OsWRKY24 were somatic embryo ○ Somatic embryogenesis: process where embryo-like structures are produced using somatic tissues ○ Synthetic seed technology: artificially encapsulating somatic ○ Bioplastics ○ Anti-cancer drugs ● Genetic engineering ○ Agrobacterium-mediated: use of microbial vector A. tumefaciens ○ Biolistic-mediated: gene gun with DNA/gold or tungsten particles 6.5. Utilizing Drought-Response WRKY Genes for Improvement of Abiotic Stress Tolerance in Rice highly negative drought-inducible, homologous regulators genes during dehydration response of rice however this led to growth defects ○ OsWRKY53 does not appear to play a role in long term drought response (wild-type-like) prevent stress ● Production of secondary metabolites Two more drought-tolerant than wild-type ○ Foregoing growth to efficiently embryos in protective coating ● Hypothesis: ● Double knockout mutants as well as encode the