Metabolism & Genetics PDF

# Metabolism - Catabolism (cat destroy things) - Energy is harvested - break down - Big -> small - Anabolism - Energy is stored / synthesized - Small -> big - Oxidation - Loss of e- - Reduction - Gain of e- ## LEO the lion goes GER - **Redox reactions** = potential energy...

# Metabolism - Catabolism (cat destroy things) - Energy is harvested - break down - Big -> small - Anabolism - Energy is stored / synthesized - Small -> big - Oxidation - Loss of e- - Reduction - Gain of e- ## LEO the lion goes GER - **Redox reactions** = potential energy transfer of 1e- from 1 -> another | Autotrophs | Energy Source | C source | | ----------- | ----------- | ----------- | | Photoautotroph | sunlight | CO2 | | Chemoautotroph | inorganic chemicals (CH₂S, S, Fe) | CO2 | | Heterotrophs | | | | Photoheterotroph | sunlight | organic compounds | | chemoheterotroph | organic compounds (sugar) | organic compounds | # Enzyme - Enzymes - Proteins - Catalyst: speed up reactions (reduces activation time / less energy used) - Affected by: temp., pH, concentration of substrate - **Activation site:** Where substrate binds to enzyme. Very specific to substrate - **Substrate:** specific to enzyme ## Coenzymes - NAD / FAD - Synthesized from vitamins (helps substrate bind tightly) ## Cofactor - Fe, Mg, Zn, Mn - Inorganic ions (metals) ## Competitive Inhibition of Enzymes - Blocks / inhibits substrate from binding to active site. - **Competitive inhibitor** - Can't bind to active site. ## Non competitive Inhibition of Enzymes - Binds to site other than active site - distorting active site - **Active site** is distorted - Can't bind to active site. - **Temperature** - 37°C / 98.6°F (optimal activity) - Avg. body temp - **pH:** 7 @ body (neutral bodies pH) - **Substrate concentration:** Keeps going ↑ until all enzymes are activated. 2 enzymes = max @ z/ enzymatic activity stops going ↑ # Glycolysis - Break down (apart) = Glucose -> Pyruvate/ Pyruvic Acid - The break down of glucose -> pyruvate ## 4 Important Events: 1. Substrate level phosphorylation: transfer of phosphate groups from ATP -> glucose 2. Breaking of a 6-carbon molecule (glucose) -> 2 3-carbon molecules. 3. Transfer of 2e- -> wenzyme NAD 4. Capture of energy in ATP ## Payoffs: 1. 1 glucose / spent 2 ATP 2. Got back 2 NADH + HATP 3. Net gain 2 ATP + pyruvate ## Location: - Wl or wio Oz present can happen - Both aerobic + anaerobic respirations. - Cytoplasm # Fermentation - Wlo the presence of Oz - Pyruvic acid metabolizes ## Types - **Alcoholic Fermentation** - Saccharomyces / yeast - Product: 2 ethanol + 2 CO2 + 2 ATP - makes beer - **Lactic Acid Fermentation** - Strep to wccus / Lactobacillus / Bacillus - Product: 2 lactate + 2 АГР - makes yogurt # Krebs Cycle: - Wl presence of 02 - Includes: TCA cycle, citric acid cycle ## Products: - O2 (wastes) - NADH (e- carriers / major prod. being prod.) - FADH - ATP - Aerobic respiration - pyruvate enters this cycle # Electron Transport Chain (ETC): Bacteria - Occurs in the cell membrane ## Info: 1. - O2 is the final e- acceptor 2. - O2 combines wl Hydrogen to form H2O (Wastes prod.). 3. - Lots of ATP produced - 38 (40) ATP produced ## Calculation: - 1 glucose = 10 NADH + 2 FADH = 30 ATP + 4 ATP = 34 ATP # Oxidative Phosphorylation - Transfer of e- from high energy to low energy - Wl each transfer energy in the form of ATP is captured - High energy -> low energy level. - Wl each transfer energy is being released - released energy captured as ATP - bits like a slide # Chemiosmosis - Conversion of ADP -> ATP using ATPase enzyme complex. - Cell membrane # Summary: - **Glycolysis** - Cytoplasm - Glucore -> pyruvate - No 02 - + 2 ATP - W/ breakdown of glucore + 2 ATP - **Kreb Cycle** - Cytoplasm - 02 - Pyruvate -> NADH + FADH (e- carriers) - + 2 ATP - **ETC** - Cell membrane - 02 - ATP - + 34 ATP - **Fermentation (N/O Oz + cytoplasm)** - **Lactic** - Pyruvate - lactic acid - Strep tococcus, Lactobacillus / bacillus - **Alcohol** - Pyruvate -> ethanol + CO2 - Sacchromyces (yeast) # Protein Structures - **Peptide linkage:** Formation of peptide bond + removal of H2O - Chemical/ Molecular structure - **Primary Structure:** Polypeptide strand - Multiple strands of peptide - AA in order. - **Secondary Structure:** helix + pleated sheets - Wl 3 polypeptide strands. - **Tertiary Structure:** Folded helix + pleated sheet - Active sites - **Quarternary Structure:** 2+ polypeptides in folded states # Binary Fission - Cell elongates and DNA is replicated. - Cell wall and plasma membrane begin to constrict. - Cross-wall forms, completely separating the two DNA copies. - Cells separate. - Mother cell -> 2 daughter cell - Exact replica of 1st cell - Budding: another form of cell division - Small new cells develop @ surface # 4 Phases of Bacterial Growth (Curved) - Lag: Preparing for growth - no growth yet - Log: Exponential growth / ↑ population - Stationary: Growth / death of cells - Death: No more nutrients / population ↓ - Happens @ optimal conditions: pH,Temp, Oz, Salinity, Nutrients - Pure culture only. ## How do we measure bacterial growth? - **Serial Dilution** | Dilution | | | | ------- | -------- | -------- | | original inoculum | 1 ml | 9 ml broth in each tube | | 1:10 | 1 ml | 1 ml | | 1:100 | 1 ml | 1 ml | | 1:1000 | 1 ml | 1 ml | | 1:10,000 | 1 ml | 1 ml | | 1:100,000 | 1 ml | 1 ml | - **Calculation: Number of colonies on plate x reciprocal of dilution of sample = number of bacteria/ml** ## Serial Dilution Methods - Using a super concentrated solution wl cultures - Diluting it ↓ by series of 10 fold - Do until necessary - Count the colomes in medium - Diluting a glass of juice ul water - Less strong & diluted # Standard Plate Counts - Add to medium + let grow - count colonies ## Direct Microscopic Counts: Counting Chamber - Can't distinguish dead to alive - Cells must be evenly distributed ## Most Probable Number (MPN) - Based on chemical reactions, few bacteria in culture - Can't do standard plate counts - Could + water quality studies (recalls for prod.) - Measures small amounts of microbes we don't want # Alternative Method: Turbidity- Spectrophotometer - Light source -> Light -> Spectrophotometer -> Absorbance - Cloudiness of sample (not counting) # Factors that affects growth - **pH** - **Acidophiles:** - Extremophiles - Not pathogenic - **Neutrophiles** - **Alkaliphiles** ## pH scale | pH | Description | | ----------- | ----------- | | 0 | Stomach acid | | 1 | Lemon juice | | 2 | Grapefruit juice | | 3 | Wine | | 4 | Tomato juice | | 5 | Urine | | 6 | Milk | | 7 | Pure water | | 8 | Seawater | | 9 | Milk of magnesia | | 10 | Household ammonia| | 11 | Household bleach | | 12 | Oven cleaner | | 13 | | | 14 | | - **Temp** - **Psychrophiles:** -10-20℃ - **Psychro trops:** 0-30℃ - **Mesophites:** 10-50°C (most active, our body temp) - **Thermophiles:** 40-716 - **Hyperthermophiles:** 65-110℃ # 02 - **Obligate aerobe:** Only grows wl presence of Oz - **Facultative anaerobe:** Wl or w/o Oz present - **Obligate anaerobe:** W/o presence of Oz # Salinity/Salt # Nutrients - **Carbon** - Ex) glucose - **Nitrogen:** (organic + inorganic sources) - Ex) ammonia / AA - **Sulfur + phosphorus** - Ex) sulfer AA + inorganic phosphate - **Trace elements:** very small amounts; used as cofactors for enzymes (micronutrients) - **Vitamins:** small amounts; used as coenzymes # Culturing Bacteria - **Streak Plate Method** - Isolating pure culture - Types of Media ## 1. Natural Media - Using the exact envi. of where microbe lives / was found - Ex) lake water, ocean water, soil - Don't know the components of media - Sterilized ## 2. Synthetic Media - Media made in lab - Provides optimal growth (we know the exact components & concentrations of media) ## 3. Complex Media - Cross btw natural & synthetic media - Ex. of ingredients: peptone, yeast extract, Nacl - Know the components of media but not the concentrations of the ingredients. ## 4. Selective Media - Only allowing certain microbes to grow are to: - Absence of critical nutrient - Presence of inhibitor (antibiotics, salt) - Allows us to grow what we want to grow - Ex) S. aureus - add more salt ## 5. Differential Media - Contains substances that cause certain bacteria to look different from others. - pH: Phenol red - Presence of `S. aureus & Saccharomyces` - media red -> yellow - Fermentation of mannitol -> acid prod. # Nucleic Acids - Many nucleotides - polynucleotide chain (nucleic acid) - Consists of: 1. Pentane sugar 2. Phosphate 3. Organic base (Nitrogenous base) - A - adenine - T - thymine - G - guanine - C - cytosine - DNA -> RNA T -> u - uvacil - Pure as silver - Purines - A,G - Pyrimides: T, U, G # Gene: - Basic unit of heredity - Ordered sequence of nucleotides located in a particular position (locus) on a particular chromosome -> encodes specific functional product. - Ex) protein - one gene = one enzyme = RNA molecule # Allele: - Genes wl dit. info. @ the same locus. - Ex. prokaryote -> mutation -> particular position / location # Mutation: - Permanent alteration in DNA - Change nucleotide sequence in DNA = change into. in DNA - Can't be fixed - permanent - Will get copied over + over again - too big mutation can be fatal / deadly ## Transfer of Info Train: DNA -> RNA -> Protein - **Replication** - DNA polymerase: ATGC - TACO - DNA -> DNA - **Transcription** - RNA polymerase: ATGC - UACG - DNA -> RNA - **Translation** - mRNA: messenger RNA - tRNA: transfer RNA - rRNA: ribosomal RNA - AUG / GCU / UAC / UGA - Met - Ala- Tyr - (stop) - RNA -> Protein # Replication: DNA -> DNA - Leading - Lagging - Helicase proteins - DNA polymerase - RNA plymerase - Okazaki fragments - DNA ligase - Replication fork - point @which 2 strands of DNA separate for replication - DNA polymerase - enzyme that synthesizes new ompementary DNA - Okazaki Fragment - short DNA fragments - DNA ligase - deals any nicks made in DNA synthesis & assists in DNA repair - Helicase - unwinds double strand DNA - Origin of replication - single location on the chromosome where DNA synthesis starts # Transcription: DNA -> RNA - RNA polymerase - Uracil instead of Thymine: AT GC -> UACC ## Transcription of RNA 1. RNA polymerase binds to the promoter sequence + opens the DNA double helix. 2. Assembles ribonucleotides (supplied as triphosphastes: ATP, GTP) into a strand of RNA 3. Ribonucleotide is inserted into the growing RNA strand following he rules of base paining: G, C, A, U 4. Synthesis of the RNA proceeds in the 5'-73' direction - until meets termination sequence then releared # Types of RNA Synthesized - Messenger RNA (mRNA): Will later be translated -> a polypeptide - Transfer RNA (tRNA): RNA molecules that carry AA to the growing polypeptide - Ribosomal RNA (rRNA): Used in building of ribosomes machinery for synthesizing proteins by translating MRNA # Types of Mutations: - May be neutral, bereficial, harmful. - **Spontaneous Mutation:** Occur in the absence of a mutagen. - Rate: 1 in a million - Mutagen: agent that causes mutations - Rate: 10-1000x more frequent - **Base Substitution:** Change in one base. - Pt mutation - Ex) Normal: ATGCTA - Substitution: AТССТА - **Missense Mutation:** Result in change in amino acid. - Ex) Normal: AUG GGC YUU AAA UGA - Met-Gly-phe- Lys - Mutation: AUG GGC UCU AAA UGA -> Met - Gly-Ser-Lys - **Nonsenpe Mutation:** Results in a nonrenre codon. - Results in a stop codon to be made. - Ex) Normal: AUG Goc uun AAA UGC CCA UAA -> Met-Gly-phe-Lys-Cys-pro - Mutation: AuG GCC Yuu AAA UGA -> Met-Gly-phe-Lys - **Frameshift Mutation:** Insertion / deletion of one or more nucleotide pairs (lethal) - Ex) Normal: AUG GCA UCG A45 UGA -> Met-Ala-Ser-Lys - Mutation: AUG CAUCG A GUGA -> Met - His- Arg-Ser-? # Ames Test: - A carcinogen test that test for cancer cells. - Mutant bacteria expored to a mutagenic substance, may cause a reversion - Carcinogens - mutant mutagen, original phenotype (no mutation) - Higher the revertants = more carcinogenic - If a microbe starts growing w/o a needed nutrient that means it had a mutation -> cancer # Polymerase Chain Reaction (PCR) - Lab technique that resembles DNA replication - Ures 3 temperatures: - Denaturing (95°C) - replication J- fork - targets specific components - Annealing (40-50°C) - binding (Diners) - Extension (72°C) - new strands forms - Purpose - Amplification of DNA - Make a lot of DNA from a small sample - Applications: - Forensics (identification), Relationships, Detection: disorders, pathogens - Pros: Don't need much sample & fast (<1hr) - Cons: Don't distinguish btw alive / dead # Genetic Code - Codons: 3 letter abbreviation for AA - Start codon: AUG (Methionine / Met) - Stop codons: UAA, UAG, UGA (terminator codons) # Translation - tRNA - Each AA is joined to correct tRNA by aminoacyl-tRNA synthetase. (20 dif synthetase = 20 dif AA). - Each has active sites for specific tRNA + AA combo. - Synthetare catalyzes a covalent bond btw them = aminoacyl-tRNA / activated AA # Translation is like cooking a dish wl recipe. - mRNA is the reape that tells the vibosome (chef) how to assemble the ingredients (AA) to create the final dish (protein) - mRNA: reape - tRNA: chefs (finds the ingredients) - rRNA: kitchen & utensils (place where recipe come together) # Gene Expression + Regulation - A switch to turn things off / on - Operon: 3 components 1. **Operator:** Region of DNA adjavent to structural genes that control transcription. - Ex) Light switch - Switch controls whether light is on / off 2. **Promoter:** Start site for transcription - Ex) Power source - provides energy to light bulb - needs to be connected for light bulb to work 3. **Structural Genes:** Encodes proteins (enzymes) - Ex) light bulb - Represents actual otuput (the prod.) that is created when on ## Operon - Control region structural genes: I P O Z Y A - DNA - regulatory promoter operator gene ## Constitutive: - Expressed @ fixed rate - Always on ## Induction: - Increases transcription of genes - Turned more up when it is needed more ## Repression: - Decreases transcription of genes - Too much in system so it is turned off # Gene Expression - Inducible Operon: Lactose Metabolism - RNA polymerase - I - P - O - Z - Y - A - Transcription, repressor mRNA, translation, Active repressor protein, allalactose (inducer) - Inactive repressor protein, transacetylase, permease, b-galactosidase ## Absence of Lactose: - Process is always off - Repressor protein binds to promoter region blocking it - Results: Prevents the enzyme needed for lactose metabolism from being prodund. No enzyme = no metabolism. ## Presence of Lactose (noncompetitive inhibition) - Wl the presence of allolactore (inducer) it prevents the repressor from its function - Results: enzyme can be created - enzyme metabolism occurs (only when there is no more glucose present - a last resort) # Catabolite Repression: - All glucose consumed -> microbe starts using lactose as energy source # Gene Expression - Repressible operon: Trytophan Synthesis - RNA polymerase - I - P - O - E - D - C - B - A - Transcription, repressor mRNA, translation, inactive repressor protein, polypeptides comprising the enzymes for tryptophan synthesis. - Active repressor protein - The synthesis of tryptophan is constantly on because our body needs it to function. When there is too much tryptophan, however, our system it can lead to bad effects, meaning we need to stop it. - An active repressor protein is used to repressred the synthesis of tryptophan. # Feedback Inhibition - Non competitive inhibition. - The end product inhibits the 1st enzyme in the biosynthetic pathway - Active site is distorted -> substrate cant bind to it. - End product produced inhibits the entire pathway. - Binds to another site @enzyme # Catabolic Activator Protein (CAP) regulator - c AMP: cyclic AMP, no glucose causes an ↑ in cAMP - Absence of glucose + presence of lactose -> induce transcription ## Lactose present / no glucose - Promoter lacl - DNA - CAP-binding site: cAMP - Inactive CAP -> active CAP ## Lactose not present / No glucose - Promoter lacl - DNA - CAP-binding site - Inactive CAP - Operator lacz ## CAMP levels - cAMP levels↓ - does not activate CAP - No transcription (if glucose is present) - Glucose = No lactose metabolism - Lactose + glucose present # Types of Gene Transfer - Gene transfer: refers to the movement of genetic into. btw organisms. - Recombination: combining of genes (DNA) from 2 dit. cells - **Vertical Gene Transfer:** When genes pass from parents -> offspring Cmutation) - **Horizontal Gene Transfer:** Pass genes to other microbes of their same generation - **Lateral Gene Transfer:** Transformation (Griffiths experiment), transduction, conjugation # Transformation: - <1 gene transferred - A change in an organisms characteristics bc of transfer of genetic into. - Naturally picking up DNA info. from surrounding (ex) Griffiths experiment, antibiotic resistence - Naked DNA (free floating DNA) - DNA that's been released from organism after cell is lysed. DNA is no longer incorporated into chromosomes. - **Competence factor** - released into medium & facilitates entry of DNA into cell. - Cell needs to want the DNA bc it can be lethal - Recipient cell - chromosomal DNA - Degraded unrecombined DNA - DNA fragments from donor cells - Only similar genes is transtered - a very small amt is transtewed. - Non pathogenic bacteria picked up info. on how to be pathogenic from the lyred pathogenic bacteria. # Transduction: - 2-5 genes transferred - A method of transteming genetic material using a bacteriophage (a virus that can infect bactena.) - **Phage:** Composed of a core of nucleic acid covered by protein coat - Attaches to receptor site on cell wall ## Stages: 1. Bacteriophage infection of a host bacterium initiates the lytic cycle 2. Chromosome is broken -> fragments can be picked up + package wl phage DNA 3. Particles released & infect another bacterial cell. 4. Host acquires genes that were brought along (transducted) from previous cells. - You can think of transduction like getting an injection: the humans are the bacteria. - Bacteriophage = injeciton # Conjugation - 10 genes - Genetic into. is transferred from 1 cell -> another - Requires contact btw donor & recipient cells. - Transfers much larger quantities of DNA. - Into. is transferred in the form of plasmids (extra dna - antibiotics resistance) ## Transfers of F plasmids: - Circular, double-stranded DNA molecules - **Types:** - F+ cells: contain F (fertility) plasmids & makes an F pilus - F- cells: lack F plasmids - F pilus (sex pilus): conjugation pilus - bridge which attaches to F-cell * Donor cell has F plasmid - F+ * Recipient cell does not have F- * F+ creates & attaches to F- * F+ creates copy of it self * Copy is sent to F- * F- becomes F+ bc it has the blueprint # Types of Plasmids - **F plasmids/ Fertility plasmids** - **Resistance plasmids:** Carry genes that provides resistance to various antibiotics (inorganic substances) - **Virulence plasmids:** Carry genes that cause disease (toxin genes) - **Tumor-inducing plasmids:** Cause tumor formation in plants. - **Some plasmids contain genes for catabolic enzymes** # Transposon: - Jumping genes - Mobile genetic sequence - Ability to move from one location to another (transposition) - Transposase: enzyme that allow it to jump + resistance genes (antibiotics). - Segments of DNA that move from one region of DNA to another. - Contain insertion sequences for cutting & resealing DNA (transposase). - Complex transposons carry other genes ## Ex: - ACTTACTG ATCAGTAAGT AT - TGAATGACTA TA GTCATTCA - Transposase gene - ACTTACTGAT ATCAGTAAGT - TGAATGACTA TAGTCATTCA - Inverted repeat, Tn5, kanamycin resistance, IS1, IS1 ==End of OCR==