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Microbiology and genetics, Chapter 1: Structure of prokaryotic cells,…
Microbiology and genetics
Chapter 1: Structure of prokaryotic cells
General structure of prokaryotic and eukaryotic cells.
Prokaryotic cell (e.g. Bacteria) contains cell membrane, cytoplasm, ribosomes (fewer, smaller), nucleoid (single circular chromosome is not bound by nuclear membrane)
Eukaryotic cell (e.g. fungus) contains cell membrane, cytoplasm, ribosomes (many, larger), nucleus (linear chromosomes are bound by a nuclear membrane), membrane-bound organelles.
Bacteria
Unicellular organisms. single celled, most divide by binary fission. after cell division, two daughter cells are formed. Bacteria sizes vary tremendously, but most are less than 1 mm in diameter. Bacterial cell shapes (morphologies) are also varied.
external structures
Appendages
Flagella
Flagella confer motility to certain cells. Flagella are made from a protein called flagellin. They are slender appendages that can rotate 360 degrees. Number and arrangement of flagella (flagellation) can vary among cells.
Monotrichous: single flagellum at one end. Amphitrichous: single (or several) flagella at both ends. Lophotrichous: several flagella at one end. Peritrichous: Flagella dispersed over the cell surface.
Motile cells are able to respond to external stimuli: chemical stimuli - chemotaxis. Positive chemotaxis (e.g. moving towards a food source). Negative chemotaxis (e.g. moving away from a toxin)
Pili
Do not confer motility
Long, slim, tubular projections (few per cell) that facilitate DNA transfer between bacteria cells (in the process called conjugation) - F (sex) pili. Only present in Gram negative bacteria.
Fimbriae
Do not confer motility
Short, fine, hair-like bristles (many per cell) that facilitate adhesion of the cell to surfaces or to other cells. Common in Gram negative bacteria, also observed in Gram positive bacteria and Archaea.
Glycocalyx
Capsule
Tightly bound to cell wall; difficult to wash away. Defined boundary. Protective structure, thick and glue-like, thus presents bacteria from ingestion and destruction by white blood cells (human immune system).
Slime layer
Less tightly bound to cell wall; easily washed away. Thinner, less defined. Prevents desiccation. Traps nutrients near the cell.
Streptococcus mutans forms slime layers which helps the bacteria adhere to tooth enamel.
Dental carries
It is the demineralisation of tooth surface by streptococcus mutans bacteria. This bacterium plays a major role in tooth decay. It metabolises sugars and convert them to lactic acid. The acid renders the tooth enamel vulnerable to decay. Left untreated, this disease can lead to pain, tooth loss, infection and even death.
Bacterial glycocalyx is a surface coating the cell walls of many bacteria. Glycocalyx vary in thickness and composition but are mostly polysaccharide in nature.
cell envelope structures
Cell wall
Peptidoglycan (or murein) is a polymer. It consists of glycan chains made from alternating sugar molecules: N-acetylglucosamine (NAG) and N-acetylmuramic (NAM) molecules linked by Beta 1->4 linkages. NAM molecules from opposite chains are joined by short peptides of 4 amino acids. Cross link formation is catalysed by transpeptidase enzymes. Penicillin, which is an antibiotic, can act as an inhibitor of these enzymes, preventing peptidoglycan formation.
Mode of action of 2 anti-bacterial compounds
Penicillin inhibits formation of peptidoglycan, which is necessary for bacterial cell wall formation. The bacteria faces osmotic stresses from the surroundings and can easily burst as their cell membranes offer little protection.
Lysozyme breaks the glycosidic bonds in the glycan chains, therefore destroying existing peptidoglycan. Bacterial cell walls are destroyed and the cells eventually die.
Two groups of bacteria based on differences in cell wall structure is distinguished through gram staining procedure.
Gram positive bacteria: stain blue/ purple. Gram negative bacteria: stain red/ pink.
The thick peptidoglycan layer encasing Gam positive bacteria traps crystal violet dye, so the bacteria appear purple. Gram-negative bacteria have much less peptidoglycan located between the plasma membrane and the outer membrane, so they do not retain the crystal violet dye and so exhibit the red counterstain, usually safranin dye.
Gram positive bacteria
Thick peptidoglycan layer (60% of cell wall; 20-80 nm). Outer membrane absent. Composed of peptidoglycan, teichoic acid, lipoteichoic acid, mycolic acid and polysaccharides. Thin periplasmic space. More permeable to molecules than Gram negative bacteria
E.g. S. aureus, Bacillus cereus, Streptococcus mutans
Gram negative bacteria
Thin peptidoglycan layer (10-20% of cell wall; 8-11 nm). Outer layer is present, together with periplasmic space protects against antibiotics and enzymes. Composed of lipopolysaccharides, lipoprotein, peptidoglycan, porin proteins. Has a thick periplasmic space. Is less permeable then Gram positive bacteria.
E.g. E. coli, P. aeruginosa, Samonella enterica
Cell membrane
Selectively-permeable and flexible lipid bilayer surrounding the cytoplasm. Composed of 30-40% phospholipids and 60-70% proteins. Cell membranes of archaea contains unique unbranched hydrocarbons rather than fatty acids. The cell membrane regulates the movement of nutrients into the cell and discharges waste. It is also a site for energy reactions, nutrient processing and synthesis. The cell membrane is also involved in secretion of metabolic products (enzymes and toxins) into the extracellular environment.
Cell envelop
Bacterial cell envelope consist of the cell wall and cell membrane. Bacterial cell walls are strong, semi-rigid casing. Cell wall is primarily made up of peptidoglycan. Cell wall provide structural support and protects cells against changes in osmotic pressure.
internal structures
Cytoplasm
It is a semi-fluid substance (70-80% water). A solvent for nutrients. Site for synthesis and biochemical activity. It holds chromosomes, ribosomes, granules, plasmids etc.
Ribosome
Ribosomes are sites of protein synthesis. They are made from rRNA and proteins. In prokaryotes: ribosomes are free-floating in the cytoplasm or attached to cell membrane. Each ribosome consists of 2 part: a large subunit and a small subunit
Prokaryotic ribosome are 70s ribosomes: made of a 50s subunit and a 30s subunit.
Eukaryotic ribosomes are 80s ribosomes: made of a 60s subunit and a 40s subunit.
Nucleoid/chromosome
Nucleoid (or nuclear region) is the area where the bacterial chromosome is located. Single bacterial chromosome is circular and condensed. It contains genes that are necessary for growth and reproduction.
Plasmids are additional DNA. It is also a single chromosome and circular. They can replicate independently. The genes they contain are not necessary for call basic growth and metabolism, but may confer protective traits like antibiotic resistance and toxin production
Endospore
Endospores are formed when a cell develops a thick wall around their genome and a small portion of the cytoplasm (highly-hydrated). Endospores are formed when cells are exposed to environmental stress (heat, desiccation, radiation chemicals etc.). They can remain dormant for extended periods and are highly resistant (especially to heat). When conditions are improved, they can germinate and return to normal cell division. Two genera of rod-shaped bacteria are known to produce endospores: Bacillus, Clostridium.
Bacterial morphology: shape (appearance) of the bacterial cell
Coccus (spherical), Bacillus (rod), Coccobacillus (short rod), Vibrio (comma-shaped rod), Spirillum (helical, rigid rod), Spirochete (spring-like, flexible rod)
Vibrio look like curved rods. Gently curved at one end. E.g. Vibrio cholerae.
Spirillium are helical shaped, fairly-rigid bodies. 1-20 helical turns. E.g. Helicobactor pylori.
Spirochete are helical shaped, flexible bodies. 3-70 helical turns. E.g. Treponema pallidum.
Bacterial Arrangement: Bacterial pattern of arrangement (after cell division). Depends on the plane of division and whether daughter cells remain attached.
Cells in pairs; diplo-. Cells in chains; strepto-. 4 cells in a cube; tetrad. Cells in a cube; sarcina. Irregular cluster; Staphylo-
Chapter 7: Methods of microbial control
Introduction to Microbial control
'Contamination' refers to presence of unwanted microbes at a given place or time. Contaminants can cause infection, spoilage to food, medicines, cosmetics, materials etc. May lead to illness and cause economic losses.
Microbistatic vs Microbicidal effects on microbes
Antimicrobial agent - kills/inhibits growth of microbes. Antibiotics - used on bacteria. Antifungals - used on fungi. Antimicrobial agent can be: Microbisatic (or microbiostatic). ~static means to inhibit growth and multiplication (only when in contact with the microbes). Microbicidal (or microbiocidal): ~cidal means to kill. Antiseptics and drugs used for humans tend to have microbistatic effects (microbicidal compounds may be too toxic)
Modes of action of Antimicrobial treatments/agents
Disruption of cell wall (inhibit synthesis or initiate breakdown)
Can be achieved by either inhibiting cell wall synthesis (e.g. penicillin) or digesting cell wall, causing it to break down (e.g. lysozyme, ethanol and detergents). Cell is unable to maintain structural integrity. Bacterial cells (not animal cells) will lyse easily due to differences in osmotic pressure.
Disruption of cell membrane
The agent damages lipids and/or proteins that make up the lipid bilayer. Cell membrane cannot retain its selective permeability. This can result in valuable cellular contents leaking into the surroundings and damaging chemicals to enter the cells.
Inhibition of protein synthesis or cause protein denaturation
Target of antimicrobial agent could be to prevent function of bacterial ribosomes or to denature cellular proteins. Ribosomes are involved in protein synthesis (to from enzymes, flagella, pili and other important cell structures). Some antibiotics can prevent ribosome function, thus preventing protein synthesis. Antimicrobial agent like alcohols and acids inactivate protein by destroying their native structure (denaturation); metal ions behave as competitive inhibitors of enzymes. Microbial cell can no longer function normally.
Inhibition of nucleic acid synthesis
Antimicrobial agent could permanently inactivate or prevent synthesis of DNA or RNA. Physical agents like X-rats, UV rays and gamma rays can cause DNA to mutate. Cell would not be able to undergo cell division, transmit correct genetic instructions or synthesize proteins.
Inhibition of cellular metabolic processes
Antimicrobial agents could inhibit important processes (e.g. folic acid synthesis). Folic acid is a vitamin synthesised by bacterial and fungi (but not humans). It is an important precursor for DNA and RNA synthesis. It is also involved in the synthesis of some amino acids (serine and methionine).
Physical methods of microbial control
Heat (moist heat, dry heat, refrigeration, freezing)
Moist heat (steam): pure steam under pressure (autoclaving)(sterilisation). 121 degC, 15 psi, 15-20 minutes (depending on load size). Able to penetrate and kill micro-organisms (including endospores). Item must not be heat or moisture-sensitive.
Moist heat (boiling water): used for household items (cutlery, baby supplies, drinking water etc.). Achieves disinfection. Exposure to 100 degC for 30 minutes kills most vegetative cells and viruses. Some bacterial endospores can withstand 100 degC for 60 minutes.
Dry heat (hot air oven): required higher temperatures and longer exposure periods than for moist heat. Denatures proteins, oxidises macromolecules in cells. Used to sterilise materials that must remain dry; they must be able to withstand high temperatures of 170 degC (for 1 hour) or 180 degC (for half an hour).
Dry heat (incineration by burning): achieves sterilisation because it kills microbes. Examples: flaming a metal wire inoculating look with a Bunsen burner (hottest point = 1870 degC). Furnace - for incinerating medical wastes (800 to 6500 degC)
Refrigeration and freezing: Most microbes do not reproduce at ordinary refrigerator temperature (0-7 degC). Many microbes survive but do not grow in freezers (below 0 degC). It is more microbistatic, rather than microbicidal. This method is used to preserve food, culture media and microbial cultures.
Radiation
Ionizing radiation: e.g. Gamma rays, X-rays.
Shorter wavelength but has more energy (deeper penetration). Damages DNA by causing breakage (DNA mutations)
Effectively penetrates solids and liquids. Can cause DNA breakage (mutation), produces oxidising agents (peroxides), therefore it disrupts H bonds and covalent bonds.
Alternative sterilisation method to materials sensitive to heat/chemicals like drugs, vaccines, meat, fish, fruits, vegetables, sterile disposable needles, swabs, catheter, syringes etc. Microbicidal and sporicidal.
Non-ionizing radiation e.g. Ultraviolet (UV) rays
Longer wavelength but has lesser energy (shallow penetration). Damages DNA by causing abnormal bond formation between adjacent thymine bases (DNA mutations).
Does not penetrate paper, glass and cloth. Can cause DNA mutation through the formation of thymine dimers (which inhibits DNA transcription and translation). Can also generate free radicals that can damage DNA, RNA and proteins
Alternative disinfectant used in germicidal lamps in BSCs, hospitals, food preparation areas. Can kill fungal cells/spores, protozoa, viruses and bacterial vegetative cells and endospores (only after prolonged exposure).
Desiccation
Traditional way of preserving food (e.g. vegetables, fish, meat jerky) together with addition of solutes like salt, vinegar or sugar. Dehydrate cells to remove water content. Microbes are not killed. Rehydration can cause them to restart cellular activities. Decreases cellular metabolism, retards/inhibits cell growth/ reproduction - but does not work for psychrophiles. Alternatively, we can use lyophilization (freeze drying). Lyophilised food products that are vacuum-packed can be stored at room temperature.
Chemical methods of microbial control
Chemical agents are present in the liquid, gaseous, solid state (rare) and serve as disinfectants. Preferred properties: rapid action at [low], good penetration ability, chemical stability (in water or alcohol), non-corrosive/staining, brad spectrum, affordable, low toxicity.
Alcohols
Germicidal ability: intermediate (destroys fungal spores)
Only ethyl alcohol and isopropanol are suitable for microbial control. Fast acting, leaves no residue. Denature proteins, dissolves lipids and damages cell membranes. [Optimum] = 70-90% (minimum 50%). Has limited activity against mycobacteria and are not sporicidal.
Cationic detergents
Germicidal ability: very low
Detergents are soluble chemical compounds that have polar and non-polar groups (amphipathic) and are used as surfactants due to their strong cleansing ability. Cationic detergents have a positive charge at one end of a long hydrocarbon chain. Quaternary ammonium compounds ('quats') are most commonly-used as disinfectants. Increased permeability of cell membranes (disrupts their selective permeability). Applications: consumer products like hand soap, disinfectant spray, toothpastes, mouth rinse etc.
Halogens
Germicidal ability: intermediate (destroys fungal spores)
Chlorine
Common disinfectant, used in the form of hypochlorites (chlorine bleach). Denature proteins by disrupting disulfate bonds, damages DNA, RNA and fatty acids. Mixing clorine gas and water forms hypochlorous acid (HClO), which is uncharged and enters cells easily. HClO partially dissociates to from hypochlorite ions (ClO-). both HClO and ClO- are strong oxidisers and the primary disinfection agent of chlorine solutions. Applications: disinfection of drinking/ swimming water, raw sewage, wastewater, inanimate objects
Iodine
Commonly used in the form of iodophors, which are solutions that contain iodine and a solubilizing agent, when applied, it gradually releases small amounts of iodine. Strong oxidising agents which can interfere with DNA synthesis, bind to and inactivate proteins. Applications: medical and dental (mild disinfectant, antiseptic)
Phenol and its compounds
Germicidal ability: low to intermediate
Phenolics are chemically-altered phenols (e.g. triclosan and chloroxylenol). Disrupts cell membranes and denature proteins. They are bactericidal, fungicidal, virucidal (but not sporicidal). Applications: cosmetics, hand soap, household cleaning products, textiles, kitchen utensils, plastic materials etc.
Heavy metals and their compounds
Germicidal ability: low
[Low] of mercury and silver kills vegetative cells (but no endospores) by inactivating proteins. Bacteriostatic agents. Silver nitrate is used in eye drops for new born infants; silver is also used in medical and hygiene products (e.g. plasters, toothpaste etc.). Use of mercury as an anti-microbial substance has been discontinued due to its toxic effects on humans.
Ethylene oxide
Germicidal ability: high (destroys endospores, mycobacteria, viruses)
Strong alkylating agent, destroys all life forms by chemically-reacting with nucleic acids and proteins. Being a gas, it diffuses easily into paper, rubber and plastics, therefore it is used for sterilisation and disinfection. Application: EtO is the official sterilant for heat-sensitive plastics and delicate instruments in hospitals and industries.
Effectiveness of chemical as antimicrobial agents depends on their: concentration, exposure period, temperature, bacterial population (load, type), presence of organic matter (e.g. blood, oil etc.)
Mechanical methods of microbial control
Filtration is used to physically-remove microbes from air or liquid. Fluid is forced through a filter that retains microbes. Range of pore sizes commonly used in the lab: 0.2 micrometre or 0.45 micrometre. Selection of pore size is done with consideration of the molecules to be filtered - membranes with tiny pore sizes may damage larger proteins (e.g. serum) in liquid culture media.
Liquids: membrane filtration or filter-sterilisation is applied to liquids that contain heat-sensitive ingredients (e.g. serum, drugs, vaccines, IV fluids, enzymes and some culture media>
Air: membrane filtration of air is done using High efficiency Particulate Air (HEPA) filters to remove particles greater than 0.3 micrometre in size. Provides a flow of sterile air to hospitals and sterile rooms. Applications: manufacturing of pharmaceuticals, cosmetics, food products or medial devices etc. - especially when contamination control is essential.
Terms used in microbial control processes
Antisepsis: destruction of microbes or inhibition of their growth on living tissues (e.g. skin or mucous membranes).
Cleaning: physical removal of all visible signs of dirt and organic matter, but does not necessarily destroy microbes.
Decontamination: A chemical and/or physical process to remove and prevent the spread of undesirable microbes (contaminants) so that they no longer pose a hazard.
Disinfection: the process of using a chemical or physical agent to destroy or inactivate vegetative cells of microbes (excluding endospores) on non-living surfaces.
Sanitation: treatment to reduce the microbial load on eating and drinking utensils to achieve safe public health levels.
Sterilisation: the process of destroying all forms of microbial life, including endospores, using physical or chemical means.
Chapter 9: Central dogma in molecular biology
Central dogma of molecular biology
DNA replication
DNA replication is the process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules.
Semi-conservative model of DNA replication: each new double strand consists of one parental strands and one new daughter strand.
Semi-conservative model of DNA replication involves: 1. Uncoiling of parental DNA molecule at a predetermined location (called origin of replication). 2. Unzipping of H bonds between base pairs, resulting in 2 parental strands that act as templates. 3. Synthesis of 2 new DNA strands complementary to each template DNA.
Requires 3 things:
Something to copy: parental DNA molecule (templates).
Something to help with copying: many enzymes
Building blocks to make a copy: deoxyribonucleotide triphosphate (dNTPs) to serve as building blocks for DNA. Always added to the OH at the 3' end of the growing strand. Catalysed by DNA polymerase III.
Enzymes involved in DNA replication
Helicase: Unzipping of the DNA helix
Primase: Synthesizing an RNA primer
DNA polymerase III: Adding bases to the new DNA chain, proofreading the chain for mistakes. Can only add nucleotides to ana existing DNA strand, need primase to initiate. Can only add nucleotides to a DNA chain in the 5' to 3' direction.
DNA polymerase I: Removing RNA primers, replacing gaps between Okazaki fragments with correct nucleotides, repairing mismatched bases.
Ligase: Final binding of nicks in DNA during synthesis and repair.
DNA replication processes
Initiation
Before replication can take place in a prokaryote: at the origin of replication (Ori), helicase (enzyme) unzips parental DNA by breaking hydrogen bonds between complimentary bases. Replication fork is where the 2 parental DNA strands separate. 'Opening' of circular DNA results in 2 replication forks.
Elongation
Leading strand (is synthesized continuously from 5' to 3' diection)
Primase synthesizes a short RNA primer. DNA polymerase III binds at the primer and initiates replication. DNA polymerase III needs a primer to provide a 3' -OH end to which new nucleotides can be added. DNA polymerase III adds complementary nucleotide bases to the strand at the 3' end. When replication is completed, DNA polymerase I replaces the RNA primer with DNA fragments. Ligase then seals the gap in the backbone. Entire leading strand consists of DNA only.
Lagging strand (is synthesized discontinuously in short stretched, each one 5' to 3' direction.
Numerous RNA primers are made by primase. These primers bind at various points along the lagging strand. Multiple DNA polymerase III enzymes bind at the primers and initiate replication, Short strands of DNA (Okazaki fragments) are made, each one in a 5' to 3' direction. DNA polymerase I enzymes removes the old primers and replace it with DNA. Okazaki fragments are finally linked by ligase (forms phosphodiester bond). Lagging strand consists fully of DNA.
Termination
DNA polymerase I removes the primers and replaces them with complementary DNA. Ligase then joins the DNA fragments together to form one continuous length of DNA.
DNA transcription
Transcribed mRNA undergoes translation to from proteins. This involves tRNA and rRNA.
Messenger RNA contains codes for the sequence of amino acids in a protein. It carries the DNA master code (for a protein) to the ribosome for translation.
Transfer RNA: contains codes specifying a given amino acid. It carries amino acids to the ribosome during translation.
Ribosomal RNA: carries codes for several large structural rRNA molecule. It is a major part of a ribosome and is involved in protein synthesis.
Requirements for transcription
Transcription is the copying of genetic information from DNA to synthesize an mRNA strand. Like DNA replication, governed by complementarity. Unlike DNA replication, only one DNA strand is copied. It requires RNA polymerase and it catalyses polymerization in the 5' to 3' direction only. The gene that is being transcribed is called a structural gene. Within the gene, the two DNA strands have different names, Like DNA polymerase in DNA replication, RNA polymerase aslo catalyses in a 5' to 3' direction. But it does not need a RNA primer to initiate, nor does it require helicase. RNA polymerase can bind to DNA, unwind it and synthesize RNA directly.
Transcription unit is the sequence of nucleotides (in DNA) that codes for a single mRNA strand, together with the sequences necessary for its transcription. It contains a promotor - located upstream of the gene, it is a location where RNA polymerase binds. The structural gene - located on the template strand, codes for mRNA strand. A terminator - located downstream of the gene, stops the transcription process.
Initiation
The promoter forms a recognition and binding site for RNA polymerase. It is found upstream of the start site (template strand). It is not transcribed into mRNA.
Elongation
RNA polymerase moves along the DNA template (strand) in a 3' to 5' direction to synthesize the corresponding mRNA. The new mRNA strand (transcript) elongates in a 5' to 3' direction as more nucleotides are added at its 3' end. Transcription bubble: contains RNA polymerase, DNA template and growing RNA transcript. After the transcription bubble passes, the now-transcribed DNA is rewound as it leaves the bubble.
Template strand (3' to 5') is transcribed into mRNA by RNA polymerase.
Coding strand (5' to 3') is not transcribed.
Termination
RNA polymerase continues transcribing until it reaches terminator sequence on DNA. Terminator sequence is located towards the 3' end (downstream) of the coding strand. Provides the stop signal that defines the end of transcription. Length of mRNA strand depends on the protein it encodes.
In prokaryotes, Transcription is coupled to translation. mRNA is translated (to make proteins) before transcription is finished (polyribosomes).
DNA translation
Genetic code is universal, degenerate, non-ambiguous.
Ribosome
Consist of large subunit and a small subunit. Each ribosome has multiple tRNA binding sites: P site - binds the tRNA attached to the growing peptide chain. A site - binds the tRNA carrying the next amino acid. E site - binds the tRNA that carried the last amino acid.
Ribosomes serve 2 functions: decode the mRNA and form peptide bonds.
Initiation
Ribosomal subunits assemble in a way that forms sites to hold mRNA and tRNAs. Small subunit binds at 5' end of the mRNA and moves towards the 3' end. Large subunit holds the tRNA and is also involved in peptide bond formation. Small subunit binds to START codon (AUG) on the mRNA and aligns it with the P site of the large subunit. An initiator tRNA molecule brings the first amino acid, methionine. It bonds with the start codon using its anticodon, UAC. The large subunit then binds to the small subunit.
Elongation
After the ribosome is assembled around the initiator tRNA and mRNA, then elongation begins: an appropriately charged tRNA binds to the codon at the empty A site of the ribosome. A peptide bond forms between the adjacent amino acids, catalysed by peptidyl transferase; the tRNA holding the proteins chain moves from the A site to the P site. The empty tRNA moves to the E site and is then released. The ribosome then shifts to the next codon and the cycle continues. Note: the growing polypeptide lies at the P site and the A site is always open for the binding of the next tRNA molecule.
Termination
Elongation continues until the ribosome encounters a stop codon (UAA, UGA or UAG) at the A site. A release factor binds to the stop codon and causes the polypeptide to be released from the ribosome. The ribosomal subunits separate from the mRNA; and from each other. Polypeptide undergoes folding into secondary and tertiary structures to become a fully-functional protein.
The central dogma of molecular biology. DNA is transcribes to make mRNA, which is translated to make a protein
Gene expression: The process by which information from a gene is used in the synthesis of a functional gene product (usually a protein) by the cell.
Relationship between genotype and phenotype
DNA (genotype) encodes the amino acid sequence for proteins that determine phenotype. Synthesized proteins spontaneously fold into a complex 3D- shape. They may: form important cell/body structures and determine body functions (e.g. enzymes used in glycolysis or Krebs cycle). General rule, gene influences the phenotype (by specifying the kinds of proteins made in the body), which largely determines how the body functions.
Causes and effects of genetic alterations
Genetic alterations can cause permanent, heritable change in the genetic material. It has a major effect if it alter the identity of the amino acid encoded there. This could lead to a new version of the protein where it may fold differently, causing its function to be altered or destroyed. Many diseases are caused by gene mutations (e.g. Down syndrome, Sickle-cell anemia etc.)
Alterations in DNA can alter phenotype
E.g. the recessive disorder, sickle-cell anemia is caused by a malformed haemoglobin (protein) molecule. It can be traced back to a single mutation on chromosome 11, exchanging an amino acid (valine, instead of glutamic acid). This results in the production of defective haemoglobin (protein) and abnormally-shaped RBCs.
Effects of genetic alterations
Pathogenic effects leading to increased susceptibility to diseases.
Provides biological diversity. Genetic variability, phenotypic changes (new variations to a trait), creates organisms evolve and adapt to environmental changes.
Causes of genetic alterations
DNA repair and its importance
The term DNA repair refers toa collection of processes by which a cell identifies and corrects damages done to its molecules. DNA in human cells undergo several thousand to a million damaging events per day, generated by both normal metabolic activities and environmental factors such is exposure to radiation or chemicals. Genomic mutations can also be carried over into next generations of cells if the nutation is not repaired prior to mitosis.
Specific DNA repair
Targets a single kind of lesion (damage to the molecular structure of DNA) and repairs only that damage. For one particular form of damage caused by UV light (thymine dimer formation)
3 steps: 1. Recognition of damaged site. 2. Enzyme (DNA photolyase) absorbs energy from light in visible range. 3. It uses this energy to cleave the bond between thymine dimers.
Photoreactivation
A photolyase enzyme recognises the damage and binds to the thymine dimer. The enzyme absorbs visible light and uses its energy to cleave the bond between the thymine dimer, therefore restoring the DNA.
Regulation of gene expression
Gene expression is the process by which the information encoded in a gene (in DNA) is used to direct the assembly of a protein molecule.
Strategies to regulate gene expression: exert control at DNA (replication), RNA (transcription), protein (translation), protein (post translation).
Gene regulation in prokaryotes ensure that genes are expressed only when their products are required. Prokaryotes utilise operons to perform gene regulation. Operons are sections of DNA that contain: 1 (or several) structural gene(s) involved in the same metabolic pathway, operator (controls transcription) and promoter (for binding).
Inducible operon
Lactose (lac) operon regulates metabolism of lactose in bacteria (e.g. E. coli). In the absence of lactose, genes are "turned off". Presence of lactose, genes are "turned on".
In the absence of lactose, a repressor protein attaches to the operator of the operon. This effectively blocks the transcription of structural genes downstream. Suppression of transcription prevents the unnecessary synthesis of enzymes for processing lactose.
Upon entering the cell, lactose becomes a genetic inducer by attaching to the repressor, which is rendered inactive and falls away. The operator is no longer closed off and its DNA becomes accessible to the RNA polymerase. The RNA polymerase transcribes the structural genes, and the mRNA is translated into enzymes that can act on the lactose substrate.
Repressible operon
Principle of operation is opposite to that of inducible operons (e.g. lac operon). Repressible operons (e.g. Arginine (arg) operon are responsible for the synthesis of anabolic enzymes that are used in the production of amino acids and nitrogenous bases (purines and pyrimidines). Similar features as lac operon: promoter, operator, structural genes, repressor proteins. One key difference: by default, operon is turned on. Repressible operon is turned off only when the product of the pathway is no longer required. Excess product behaves as a co-repressor that slows/stops transcription of genes.
A repressible operon remains on when its nutrients products are in great demand by the cell. The repressor has the wrong shape to bind to the DNA operator without a corepressor. RNA polymerase is free to actively transcribe the genes and translation actively processes.
The operon is repressed when arginine builds up and, serving as a corepressor, activates the repressor. The activated repressor complex affixes to the operator and blocks the RNA polymerase and further transcription of genes for arginine synthesis.
Genetic alterations
Chapter 2: Nutritional and environmental conditions for microbial growth.
Classification of nutrients required for microbial growth
Quantities required by cells for growth.
Macronutrients: needed in large amounts for cell structure and metabolism. E.g. sugars and amino acids that contain C, H, O, N, P, S.
Micronutrients: needed in small amounts for enzyme function and protein structure. E.g. trace elements like Mg, Fe, Mn, Ni, Zn, Cu etc.
Carbon content
Organic nutrients: molecules that contain C in their structure. E.g. macromolecules like carbohydrates, proteins, lipids, vitamins or even simple molecules like methane.
Inorganic molecules: molecules that do not contain C. E.g. water, mineral salts like ferric nitrate, magnesium sulfate, sodium phosphate and gases.
Sources of carbon and energy
To determine an organism's nutritional type, we need to know its source of carbon and source o energy for metabolism. Carbon is an essential element required for cell structure and metabolism. Organisms can be classified as autotrophs or heterotrophs based on how they obtain carbon.
Autotrophs (autotropic nutrition)
Source of carbon: CO2 (inorganic source). Carry out photosynthesis, convert CO2 and water into organic compound (sugar) with the help of sunlight energy captured by chlorophyll.
Heterotrophs (heterotrophic nutrition)
Source of carbon: organic source. Organisms must feed on other organisms to obtain organic nutrients (E.g. macromolecules). Can be further sub-divided into:
Parasitic nutrition: feed off another organism
Saprophytic nutrition: feed off dead organisms/ organic matter
Holozoic nutrition: ingestion and digestion of organic food particles
Organisms can also be classified based on how they obtain energy. Two key sources of energy are sunlight and chemicals.
Phototrophs: organisms that obtain energy from the sun are photosynthetic and are called phototrophs.
Chemotroph: Organisms that gain energy from simple inorganic chemicals are called chemotrophs.
Environmental factors that affect growth
Temperature
Microbes have no internal temperature control. They assume temperature of their surroundings. Each microbial species grows over a range of temperatures. There are three cardinal temperatures: minimum, optimum and maximum. Usually dictated by metabolism (enzymatic activities) (10 degC rule): enzyme activity doubles fro every 10 degC increase in temperature, until enzymes become denatures by high temperature.
Minimum temperature - lowest temperature that permits a microbe's growth and metabolism.
Maximum temperature - highest temperature that permits a microbe's growth and metabolism.
Optimum temperature - promotes the fastest rate of growth and metabolism.
Psychrophiles: minimum temperature, less than 0 degC. Optimum temperature 10-15 degC. Maximum temperature: less than 20 degC. Grows best at relatively low temperature.
Mesophiles: minimum temperature, 10 degC. Optimum temperature, 20-40 degC. Maximum temperature: less than 45 degC. Most bacterial especially those living in association with warm-blooded animals.
Thermophiles: minimum temperature 45 degC. Optimum temperature 45-80 degC. Maximum temperature, more than 100 degC. There's a wide variation among thermophiles in optimum and maximum temperature.
Oxygen availability
Organisms can be classified based on their oxygens requirement and tolerance. Aerobes (aerobic organisms) grow in the presence of oxygen. They require oxygen for cellular respiration. They produce enzymes to deal with toxic forms of oxygen, turning them into harmless substance like water and oxygen.
Obligate aerobes - can only grow in the presence of oxygen (21%). Microaerophiles - can only grow in limited amounts of oxygen (1-15%). Facultative anaerobes - have a preference for oxygen (carry out aerobic respiration) but can also grow in its absence (switch to anaerobic respiration). Obligate anaerobes (anaerobic organisms) - grow in the absence of oxygen. Cannot grow in the presence of oxygen. Do not produce enzymes to deal with toxic forms of oxygen. Aerotolerant anaerobes - do not require oxygen for growth. They utilise fermentation to produce ATP (whether there is oxygen in the environment or not). Produces enzymes to protect them against toxic forms of oxygen.
pH
Microbes vary in their tolerance to pH, but most prefer pH 6-8 as pH extremes can damage cellular proteins (especially enzymes). Most bacteria are neutrophiles - grows best at pH 5.5 - 8.0. Fungi (and some bacteria) are acidophiles - grows best at pH 0.1 - 5.4. Some bacterial are alkalinophiles - grow best at pH 9 and above.
Osmotic pressure
Water is required for cell growth. Water is essential to all living cells as the primary solvent in which chemical activities takes place and as a reactant in metabolic reactions such as hydrolysis.
Microbes obtain water by osmosis. High [solute] = high osmotic pressure (or low water potential). Low [solute] = low osmotic pressure (or high water potential). Water always moves from a region of low osmotic pressure (dilute) to high osmotic pressure (concentrated).
Halophiles are organisms that thrive in conditions with high salt concentrations. Obligate halophiles (e.g. Halobacter sp.), a member of the Archaea, need high salinity to grow (9-25% NaCl). Facultative halophiles (e.g. S. aureus) do not normally reside in saline conditions, but can survive in high salt concentrations (0.1-20%), allowing them to colonise the skin and nasal cavity of mammals.
Slightly halophilic: 2-5% NaCl
Moderately halophilic: 5-20% NaCl
Extremely halophilic: 20-30% NaCl
Monitoring the growth of microbial populations
Total cell count
Counts all cells (whether dead or alive). The cells are counted with a special microscope slide called a hemocytometer.
Viable plate count
Counts only live cells (because they can divide and form colonies). 1 viable cell will form 1 colony (Colony Forming Unit). Optimal for counting (25-250 CFU per plate), therefore appropriate dilutions are made. By multiplying the number of colonies in a sample by the volume of the broth, we can get an estimate of the total cell population at a given point in time. This growth of the cell population over time can be plotted over time (cell growth curve).
Cell growth curve
Broth is inoculated with bacterial, population size of cells increases with time (3-4 days). Cell growth curve (batch culture).
Lag phase: no increase in cell numbers. Cells are metabolically alive but not dividing. Cells preparing to divide by synthesising various components like DNA, enzymes that are necessary for growth and division.
Log (or exponential) phase: cells are growing and dividing at an exponential rate. Period of fastest growth, lowest death. Growth rate is maximal and constant, generation time is minimal. All required nutrients are available in sufficient quantities.
Stationary phase: Growth rate equals death rate so total number of viable cells remain constant. May be due to nutrient depletions and accumulation of wastes, leading to unfavourable physical conditions for cell growth.
Death phase: Growth rate is much less than death rate, resulting in logarithmic decline in viable cell numbers. Conditions become less and less conducive for growth.
CFL/mL = Av. no. of CFU/plate x 1/Dilution factor x 1/mL of aliquot plated
Growth is defined as an increase in quantity of all cellular components and structures. In multicellular organisms, they become bigger in size (their cells divide and differentiates into different kinds of cells). In unicellular organisms, there are more organisms in a population.
As microbes grow, they consume nutrients and release waste (toxic metabolites).
Batch culture: As the cells grow, there is no input of fresh nutrients, nor waste removal.
Continuous culture: As the cells grow, there is constant input of fresh nutrients and removal of waste and old cells.
Obtaining a pure culture (from a mixed culture)
Streak plate Technique
The streak plate method is based on the dilution during the process of mechanical spreading of inoculum over the surface of solidified culture media in order to obtain well-isolated colonies of the original specimen at the terminal streaks.
Chapter 6: Structure, reproduction and symbiotic relationships of fungi
Introduction to Fungi
Eukaryotic organisms (cells with nuclei and many membrane-bound organelles). They do not possess flagella, cilia or chloroplasts. All fungi need water and oxygen (no obligate anaerobes). Unicellular (yeasts) or multicellular (molds).
General structure of fungi
Molds are multicellular fungi that consist of long, threadlike filaments called hyphae. Some hyphae are aseptate - not divided by septa (cross walls). Other hyphae are septate (divided by cell wall). A network of hyphae is called mycelia. Mycelia grows though and digest the substrate.
Molds have 2 types of hyphae: reproductive hyphae and vegetative hyphae
Fungal cell walls contain polysaccharides: chitin + b-glucan (80-80% of cell wall). Chitin is also found in the hard shells (exoskeletons) of insects, prawns etc. Fungal cell membranes contain ergosterol rather than the cholesterol that is present in mammalian membranes.
Fungal nutrition
Fungi are chemoheterotrophs - obtain carbon and energy from organic macromolecules. Fungi are adapted for efficient absorption of nutrients. They obtain nutrients by secreting digestive enzymes into the surroundings (substrate). The substrate is digested by these extracellular enzymes (external digestion). Then the digested organic molecules are absorbed into the fungus.
Most fungi are saprobes as they can obtain their nutrients from dead animals and plants. They decompose their substrates with the help of extracellular enzymes.
Fungal symbioses
Fungal symbioses can be: obligare symbiosis (essential fro fungal survival) or facultative symbiosis (non-essential for survival).
Kinds of symbiotic relationships: mutualism (relationship between 2 organisms that is mutually beneficial) or parasitism (relationship between 2 organisms where 1 partner benefits but the other is harmed).
Mutualism
Lichens are symbiotic associations between a fungus and a photosynthetic partner (e.g. green algae). Fungus: unable to grow normally without their partners; provides shelter to partner (give protection from strong sunlight and drying). Partner: carries out photosynthesis; provides sugar to fungal partner. Lichens can grow in harsh habitats and are resilient to drying. They are good indicators or environmental pollution.
Parasitism (on plants)
Fungi are the cause of many plant diseases. Many are harmful parasites of living plant host. Cause billions of dollars in agricultural losses. Some fungi may secrete substances that make food unpalatable. Some of these chemicals may be poisonous or even carcinogenic. e.g. Aspergillus flavus is a fungus that secretes aflatoxin in corn and peanut. These chemicals can cause severe damage to kidneys and nervous system to immunocompromised patients
Parasitism (on animals)
Human and animal diseases caused by fungi are called mycoses. Range of mycoses: superficial infections to invasive infections (fungus invading parts of the body that are normally free from germs (sterile), usually among immunocompromised patients. Athlete's foot is a mycosis commonly caused by the fungus, Trichophyton rubrum.
Reproductive strategies
Unicellular yeast
Unicellular yeast reproduce through binary fission or budding (more common). Yeasts can ferment carbohydrates (glucose) into ethanol and carbon dioxide; used to make bread, beer and wine.
Budding process in yeast: 1. Small projection (bud) develops from the parent cell and enlarges in size. 2. Nucleus of parent cell divides mitotically and a daughter nucleus passes into the bud. 3. The bud forms a constriction at the base to cut itself off the parent cell. 4. The bud could fall off the parent cell or a new bud forms before separation, resulting in a chain of buds.
Multicellular molds
Except for unicellular yeast, all fungi develop from spores (reproductive cells). Spores allow fungi to spread to new food sources and are resistant to environmental stresses. Molds are capable of both asexual and sexual reproduction.
Asexual reproduction: One parent copies itself to from a genetically-identical offspring. It does not require male and female individuals (hence, no fusion of haploid cells). Spores are formed by mitosis. Fungi undergo asexual reproduction to colonise a habitat.
Sexual reproduction: Fusion of haploid sex cells (n+n) which arise from different mycelia. This involves: 1. plasmogamy: fusion of cytoplasm and temporary coexistence of 2 nuclei in the fused cell. 2. Followed by karyogamy ('nuclear marriage): haploid nuclei fuse to from a diploid nucleus.3. After meiosis, spores are produced and scattered into the environment. Fungi undergo sexual reproduction to introduce genetic variation.
Chapter 10: Molecular cloning and genetic engineering
Introduction to molecular engineering
Molecular cloning is a process where a gene (target gene of interest, GOI) is placed in a cloning vector (e.g. plasmid). This results in recombinant DNA, which is then placed into a suitable cloning host (e.g. bacteria cells) where it can be cloned (multiplied)
A cloning vector is a DNA molecule that can carry foreign DNA into a host cell. It has the ability to self-replicate and integrate into the host cell.
Recombinant DNA is a single DNA molecule made from 2 different species (that will be inserted into a host organism to produce new traits that are of value to man).
Tools and techniques of DNA technology
Restriction enzymes
Function is to recognise and cleave (cut) specific DNA sequences, about 4-12 base pairs long. Each RE has its own restriction (or recognition) site. Restriction sites of REs are usually palindromic sequences.
There are 3 types of termini that can be generated after DNA has been cut by restriction enzymes: 5' overhang, 3' overhang, and no overhang (blunt ends).
Restriction enzymes cleave the DNA in the 3' carbon and 5' phosphate of the phosphodiester bond. Fragments produced by RE has 5' phosphates and 3' hydroxyls. The gene of interest (GOI) and vector must be cleaved by the same restriction enzyme to ensure that their ends are complementary. This makes it easier for DNA ligase to join them later.
Agarose gel electrophoresis
Standard lab procedure to separate DNA by size (length in base pairs; bp). Electrophoresis uses an electrical field to move the negatively-charged DNA through an agarose gel matrix towards a positive electrode. Shorter DNA fragments migrate through the gel more quickly than longer ones. A DNA ladder (a collection of DNA fragments of known lengths) is used to gauge the approximate lengths of DNA fragments. Both run alongside the agarose gel.
An agarose and buffer solution is poured into a plastic tray. A comb is placed into the tray on one end. 2. The agarose polymerises into a gel as it cools. The comb is removed from the gel to from wells for sample. 3. DNA samples coloured with a tracking dye are pipetted into the wells. 4. The tray is placed into a chamber that generates electric current through the gel. The negative electrode is placed on the side nearest to the samples. The positive electrode is placed on the other side. 5. DNA has a negative charge and will be drawn to the positive electrode. Smaller DNA molecules will be able to travel faster through the gel. 6. One well, called DNA ladder, will contain DNA fragments of known sizes This ladder is used to determine the sizes of other samples.
Polymerase Chain Reaction (PCR)
Sometimes called 'molecular photocopying'. PCR is a fast inexpensive technique used to 'amplify' (copy) small fragments of DNA. Important applications in molecular and genetic analysis as both require large amounts of DNA as starting material: DNA fingerprinting, detection of bacteria or viruses (e.g. COVID-19 testing), diagnosis of genetic disorders. Amplified PCR products can be sent for sequencing, visualised by gel electrophoresis, or cloned into a plasmid for further experiments
PCR process: 1. Sample is first heated to denature DNA (separate into 2 pieces of single-stranded DNA). 2. Taq polymerase enzyme synthesizes 2 new strands of DNA, using the original stands as templates (like DNA replication). This gives 2 DNA molecules, each one containing an old strand and a new strand of DNA. 3. Then each of these strands is used to create 2 more new copies, and so on. 4. The cycle pf denaturing and synthesizing new DNA is repeated as many as 30-40x, which yields more than 1 billion exact copies of the original DNA segments.
Key ingredients: Taq polymerase, primers, template DNA and nucleotides (DNA building blocks)
3 basic steps of PCR:
Denaturation (94-96 degC): heat the reaction strongly to denature (or separate) the DNA strands. This provides single-stranded template for the next step.
2, Annealing (55-65 degC): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA..
Elongation (72 degC): Raise the reaction temperatures, allowing Taq polymerase to extend the primers, synthesizing new strands of DNA.
Vectors (cloning and expression)
Vehicles that carry foreign DNA into a cell.
Cloning vectors
They are small pieces of DNA that can be stably maintained in a host cell by replicating autonomously. It is used to introduce genes into cells. It is used for reproducing gene of interest (GOI, transgene) inside the cell (cloning host)
Has: origin of replication (Ori), unique restriction site (known as multiple cloning site - MCS; or polylinker), selectable marker
3 key features of plasmid cloning vectors: they are small, circular, double stranded DNA molecules with:
Multiple cloning site (MCS), also called polylinker. It contains unique sites for several restriction enzymes (i.e. cloning sites for the insertion of target DNA)
Origin of replication (Ori). It is used to maintain the vector DNA in host cells.
Marker gene: 1. Selectable marker (e.g. antibiotic resistance gene). It selects host cells that have successfully taken up the vector DNA (transformants) using media + antibiotics. 2. Screenable marker (also called reporter gene). It causes genotypic change (e.g. colour change) in transformants (e.g. GFP)
Usually a plasmid, but can also be a phage, artificial chromosomes, cosmids etc.
Expression vectors
It is a vector that is used for expressing the GOI contained within the cloning DNA by reproducing large amounts of mRNA, and by extension, proteins of interest. It is typically used to express the GOI inside the cell (expression host).
Has features of a cloning vector and a promoter sequence, ribosomal binding site, sequences to initiate/terminate transcription, sequences to initiate/terminate translation, and a protein purification tag sequence.
4 feature of plasmid expression vectors:
They have the same 3 features as cloning vectors, plus a few more: They have an enhancer (increases rate of transcription) and a strong promoter (initiates transcription). They have a ribosome binding site (where ribosome binds and initiates translation). A start codon (ATG) (to initiate translation) and a stop codon (TAA, TAG, TGA) to terminate translation). They also have a terminator (terminates transcription). They have tag sequences which enables purification of the protein produced; or allow us to identify the location of the protein in the cell, e.g. Histidine tag (6x His-tag).
Usually a plasmid.
Process of DNA cloning
4 key steps in DNA cloning.
Isolation of target DNA
Carry out restriction digestion. A process where DNA is cleaved (cut, digested) with the chosen restriction enzyme(s). Performed on vectors (cloning or expression) and target DNA using the same RE(s). The cutting generates compatible termini to facilitate joining of the insert DNA and vector DNA. or the chosen vector, cleavage is made at the multiple cloning site (MCS).
To ensure success in the next step, ligation, we must prevent self-ligation (of the vector) and ensure that the insert is added into the vector in the correct orientation. Thus, 2 different restriction enzymes are used: one enzyme for the 5' end and one enzyme for the 3' end.
Ligation of target DNA into cloning vector (e.g. plasmids)
During ligation, two linear DNA molecules are joined by covalent bonds, More specifically, a phosphodiester bond is formed between the 3' OH of one nucleotide and the 5' phosphate of another nucleotide. Facilitated by DNA ligase enzyme. Recombinant DNA molecule consist of vector and target DNA.
Introduction of recombinant plasmids into suitable host cells (e.g. bacteria)
Process is known as transformation. Only 'competent' cells can take up exogenous DNA. Chemical transformation or electroporation. Host cell serves as a 'copying machine' and is able to replicate the recombinant DNA molecule. As each host cell reproduces, the recombinant DNA molecules are passed on to all the progeny (daughter cells), giving rise to a population of cells (colony) carrying the cloned DNA insert.
Screening/selection of successful clones
Transformed cells are host cells that have taken up the recombinant DNA molecule. Successfully-transformed cells must be identified and isolated from those that are not.
Method used depends on the type of marker in the vector. Selectable marker (antibiotic resistance e.g. resistance to ampicillin), Screenable marker - reporter gene (e.g. green fluorescent protein).
The word 'clones' refer to there resulting cell population (after a cell has multiplied many times). The recombinant plasmids which have also replicated many times within the cells.
Key components of pGLO
Bla gene: Beta-lactamase, confers resistance against B-lactam antibiotics like ampicillin.
GFP gene: reporter gene; green fluorescent protein
araC gene: Arabinose C protein that regulates expression of the PBAD promoter.
PBAD gene: promoter for the transcript of GFP gene
MCS: multiple cloning site which contains 'cut sites' fro restriction enzymes (hindIII, EcoR1 etc.)
Application of genetic engineering
Genetic engineering is the artificial manipulation, modification, and recombination of DNA or other nucleic acid molecules in order to modify an organism or population of organisms
Uses of genetic engineering: Production of therapeutic proteins and drugs, plants with improved qualities (pest resistance, better quality and longer-lasting crops), animal (fish, cows etc.) that grows faster, 'meat-free' meat, bacterial for degrading/clean up oil spills.
Genetic mutation: alteration to the nucleotide sequence of a gene.
Point mutations: A mutation where a single base is altered.
Silent mutation
Alteration of a single base does not change the amino acid encoded.
Missense mutation
Alteration of a single base changes the sequence of the amino acid encoded.
E.g. sickle cell anemia
Nonsense mutation
Alteration of a single base results in the production of a stop codon
Frameshift mutation: Insertion/deletion of 1, 2 or 4 nucleotides within an open reading frame (coding sequences). Produces more serious consequences than point mutation.
Two effects of frameshift mutations: changes the whole sequence of amino acids downstream of the mutation; and/ or promote premature protein chain termination. Might get a new protein (beginning portion identical to the original protein, but the portion after the frameshift is different).
UV-induced thymine dimer formation (frameshift mutation)
Effects of UV radiation on DNA: Formation of abnormal chemical bonds between adjacent pyrimidine molecules (mostly between adjacent thymines, forming thymine dimers). Disrupts the normal pairing of T bases with corresponding A bases on the opposite strand. Affects transcription and replication, UV light exposure is the primary cause of melanoma (skin cancer).
Triplet repeat (expansion) mutation: insertion of triplet sequences of DNA (trinucleotide). The cause of many genetic diseases. Depending on location in the chromosome, the unstable trinucleotide repeat can cause: defects in the protein encoded by the gene, change in the regulation of gene expression, Chromosome instability.
Chapter 8: The genetics of inheritance
Introductions to terms used in genetics
Genetics is the study of heredity. Heredity is the passing of traits from one generation to the next. The term traits refers to specific characteristics that are unique. Traits affect: the way we look (e.g. hair - straight/curly, eye colour, handedness etc.) Traits are specified by out genes (found on chromosomes and contain DNA). A gene is a specific location on a chromosome that determines a certain trait.
Alleles are different versions of a gene. And codes for different protein.
Phenotype is a term for the physical appearance of an organism. Genotype is the genetic makeup of an organism.
Locus is a specific physical location of a particular gene on a chromosome.
Mendelian genetics #1
Phenotypic ratio of dominant to recessive is 3:1. Genotypic ratio is 1:2:1.
Principle of segregation
Two alleles fro a gene segregate from each other during the process that give rise to gametes; and are rejoined at random, one from each parent during fertilisation.
Monohybrid cross
Genetic diagrams must include: Parental phenotype, Parental genotype, Possible gametes, Offspring genotype, Offspring phenotype, and ratio of each phenotype
Test cross (for monohybrid cross)
Purpose: to determine the unknown genotype of an individual with dominant phenotype. Cross the individual with unknown genotype with a homozygous recessive.
Mendelian genetics #2
Principle of Independent assortment
The law states that during gamete formation, different pairs of alleles assort independently of each other. This is the case with a dihybrid cross.
In a dihybrid cross, if 2 heterozygous parents for both genes are crossed, you will always get 9:3:3:1. Unless there is autosomal linkage or crossing over at meiosis.
Non-mendelian genetics
Mendel's model of inheritance has 3 assumptions: each trait is controlled by a single gene, Each gene has only 2 allele. There is a clear dominant-recessive relationship between the alleles. Mendel had studies inheritance of traits that were controlled by unlinked genes.
Some genes do not meet the criteria of Mendel's model of inheritance.
Linked genes (alleles located close by on a chromosome are inherited together)
Autosomal linkage of genes that are not on the sex chromosomes. Assortment of genes happens most frequently if the genes are located on different chromosomes or if they are far apart on the same chromosome. But sometimes, assortment of genes is not independent. e.g. linked genes occur on the same chromosome, therefore they tend to be inherited together (i.e. they do not segregate independently).
Incomplete dominance
A mixture of alleles in the genotype is seen in the phenotype.
A case in which one allele is not completely dominant over another is called incomplete dominance.
Codominance
Both alleles in the genotype are seen in the phenotype. Both dominant alleles are expressed.
Codominance occurs when 2 alleles contribute towards a phenotype. Unlike incomplete dominance, aspects of both phenotypes are seen in the heterozygote offspring.
E.g. Human ABO blood group system
Human ABO blood type is determined by the I gene. The I gene has 3 alleles: I^A, I^B and i. I^A and I^B are codominant to each other; but both I^A and I^B are dominant over i. The I gene encodes an enzymes that adds different sugar molecules to membrane proteins (glycoproteins = antigens) on the cell membranes of red blood cells (RBCs) - they serve as cell recognition markers.
Chapter 3: Culture media for microbial growth
Introduction to culture media
Culture medium contains nutrients that meet the nutritional needs of microbes (bacteria and fungi), enabling their cell division and metabolism. Viruses are obligate parasites and can only replicate within living cells. Culture media can be classifies in 3 ways:
Physical state
Liquid
Semi-solid
Solid
Chemical composition
General purpose
Selective
Selective and differential
Enrichment
Function of the medium
Chemically defined
Complex
Classification of culture media
By physical state
Culture media can occur in 3 physical states depending on the amount of agar it contains: solid (1.0-1.5% agar), semi-solid (0.2-0.5% agar), liquid (no agar). Agar (polysaccharide from seaweed) is used to solidify culture media, liquefies when heated (100 degC), but it forms a firm gel upon cooling (~37 degC).
Solid medium
Advantages: colonies have a specific morphology that is useful in identification. Allows formation of discrete colonies for counting. Can tell when there is culture contamination as colony morphology will be different.
Disadvantage: Poor diffusion of nutrients and waste products. Lower colony count.
Liquid medium
Advantages: Better diffusion of nutrients and waste products. Higher cell count.
Disadvantage: Growth - no special characteristic colony appearance (identification). No discrete colonies for isolation (or counting). With turbid cultures, cannot tell if the culture is contaminated.
Semi-solid
Test for motility of bacterial cells.
Chemical composition
Two types:
Chemically defined (synthetic) medium: Full chemical composition is known.
Complex medium: contains one (or more) components with unknown chemical composition. Obtained from animal/plant tissues. Possible additives: Blood (sheep, horse etc.), meat extract, yeast extract, soybean extract, casein, peptone etc.
Function
Routine cultivation of microbes that have no special growth requirements.
General purpose media
Basal media to support growth of a large variety of microbes. Considered to be complex media as they often contain peptone, blood serum, yeast extract, blood etc. to promote cell growth. E.g. nutrient agar and LB (Luria-Bertani) agar.
Isolation and/or identification of microbes
Selective
Contains additives that suppress the growth of microbes, but encourage the growth of others. Useful for isolating a particular type of microbe from a mixture. E.g. Sabouraud dextrose agar. Useful fro the cultivation and identification of fungi. Has a low pH of 5.6; fungi outcompete bacteria at low pH. Addition of 5% NaCl; favours halophiles. Chloramphenicol is added to further inhibit bacterial growth.
Selective and differential
Considered selective as they contain additives that allow the growth of some, but suppresses the growth of others (i.e. isolates one microbe from a mixture). Also considered differential as they contain additives that change visibly by interacting with the microbes as they grow. Examples include Eosin methylene blue (EMB) agar, Baird-Parker (BP) agar.
Eosin-methylene blue (EMB) Agar
Selective: contains bile salts, eosin and methylene blue dyes (inhibit Gram positive bacteria). Selective for Gram negative bacteria; Gram positive bacteria cannot grow.
Differential: Contains sucrose and lactose (carbohydrate sources). Differentiates between lactose fermenters (black colonies) and non-lactose fermenters (pale pink or colourless colonies)
Escherichia coli: Gram negative, lactose fermenter. Forms dark purple (black) colonies with a metallic green sheen.
Pseudomonas aeruginosa: Gram negative, non-lactose fermenter. Forms colourless or pale pink colonies
Baird-Parker Agar
Selective: contain glycine and pyruvate )promotes growth of staphylococci); lithium chloride and tellurite (inhibits growth if other microbes). Selects for Gram positive staphylococci bacteria; Gram negative bacteria and other bacteria cannot grow.
Differential: contains egg yolk emulsion (enables lipolytic activity of staphylococci to be detected). Differentiates between those with lipolytic activity (clear rings) and those without (no ring).
Staphylococcus epidermidis: Gram positive, no lipolytic activity, Black colonies (no clear ring)
Staphylococcus aureus: Gram positive, lipolytic activity, Black colonies (with clear ring)
Enrichment
Non-selective liquid culture medium that contains additives that favour the growth of a particular microbe. Aims to increase cell numbers to enable isolation and identification. Often used for testing of food, soil or fecal samples.
E.g. Alkaline peptone water (broth). Promotes growth of bacteria that can multiply under alkaline conditions (e.g. Vibrio cholerae). Contains meat peptone and 2% NaCl. Inhibits the growth of contaminating flora (other intestinal microbes) that cannot multiply at an elevated pH of 8.6 +/- 0.2
Preservation of culture
Constant sub-culturing is time-consuming and laborious. For longer term storage, colder temperatures are used - slow down/ stop the rate of chemical reactions in the cells, yet maintain cell viability.
4 degC: storage up to a week.
-20 to -40 degC: storage up to a year.
-80 degC: storage for several years.
Measures taken to prevent/ minimise dehydration of agar and prevent culture contamination
Topic 4: Structure and replication of viruses
Introduction to viruses
Viruses are non-cellular, non-growing, non-metabolic entities, therefore not in any domain. They can only replicate within living host cells (plants, animals, fungi, bacteria). They are not called dead or alive but inactive or active. Virion is an infective virus particle outside of a cell.
Structure of viruses (capsid, envelope, nucleic acid)
Each virus contains 2 key structures which are needed to invade and control host cells: covering (capsid) and the central core (nucleic acid). Nucleic acid (genome) can be DNA or RNA. Circular or linear, single- or double-stranded. Specialized enzymes (some viruses), reverse transcriptase.
Capsid
Capsid surrounds the nucleic acid core. It is a protein coat, sometimes called nucleocapsid. It protects the nucleic acid (genome). Many animal viruses have an envelope derived from host cell membranes with viral proteins added and it surrounds the capsid.
Capsid determined the shape of the virus. Made of repeating protein subunits called capsomers. Usually, viral capsids consist of 1 type of capsomer; some viral capsids may contains several types of capsomers. Protect nucleic acids from degradation and sometimes facilitates penetration of virion into a host cell. Two main viral shapes depending on geometry of capsids: helical viruses, icosahedral viruses. Complex viruses (e.g. T-even bacteriophage have an icosahedral head and helical axis tail).
Envelope
Members of the 13 of the 20 families of animal viruses possess a lipid envelope. It is an additional covering the surrounds the capsid. Usually a modified lipid bilayer, takes when they bud outwards from host cell membrane/ nuclear membrane or even ER. Viral proteins called spikes are inserted into the lipid envelope. They are essential for host cell recognition and viral attachment to host cells.
Nucleic acid
Viruses vary greatly in type of nucleic acid (genome) and number of strands. They carry either DNA or RNA. Their genes encode proteins that allow viruses to invade host cells and control the cells to make new viruses. Most DNA viruses have double-stranded DNA (ds DNA), and are replicated in the nucleus of eukaryotic host cell. Most RNA virus have single-stranded RNA (ss RNA), and are replicated in the host cell's cytoplasm. Replication in the cytosol is error prone, which leads to high rates of mutation and will become difficult targets for immune system and vaccines/drugs.
Viral replication
Animal virus
Viruses lack ribosomes and enzymes for protein and nucleic acid synthesis. They hijack the host cells transcription and translation machineries to express their genes (and make viral nucleic acids and proteins). Host cell assembles all the newly-formed parts into viruses before they are released into the environment. In animal viruses, viral replication occurs in 5 stages:
Attachment: Virus become attached to a target cell.
Entry: the cell engulfs the virus by endocytosis.
Uncoating: viral contents are released. Viral RNA enters the nucleus where it is replicated by the viral RNA polymerase.
Synthesis: viral mRNA is used to make viral proteins.
Assembly and release: New viral particles are made and released into the extracellular fluid. The cell, which is not killed in the process, continues to make new viruses.
Bacteriophage
Called 'phage' for short, they are viruses that infect bacteria and archaea. Diverse structure, but most phages contain double-stranded DNA. Phages that infect E. coli are best studied (e.g.T4 phage and lambda phage). They have an icosahedral head and helical tail, with tail fibres for attachment to host cell.
Phages reproduce by the lytic or lysogenic cycle, only few viruses are capable of both.
Lysogenic cycle e.g. Lambda phage
Host cell does not lyse immediately; only after it switches over to lytic cycle. There is integration of phage DNA with host bacterial DNA and is called the prophage. Integration allows viral DNA to be replicated along with the host cell's DNA as the host divides. Lambda phage is also known as lysogenic (or temperate) phage.
Events following phage infection depends on host cell conditions - determines which cycle the virus undergoes: lytic or lysogenic. Lysogenic cycle - phage DNA integrates with host DNA (prophage) and is multiplied as cell divides multiple times (host cell does not lyse, new phages not formed yet). Process of induction (occurs when host cell undergoes stress (e.g. physical factors like exposure to UV or X-rays; or chemical factors such as exposure to antibiotics) Prophage is excised and lytic cycle is initiated. Requires turning on the gene expression necessary for the lytic cycle.
Lysogenic cycle continues until host cell undergoes stress. This leads to induction whereby the prophage is excised from the bacterial chromosome and viral genes are expressed. This marks the start of the lytic cycle. In the lytic cycle, viral DNA directs the production of new viral particles by the host cell. The virus kills the cell by lysis.
Lytic cycle: e.g. T4 phage
Host cell lyses after new phages are formed to allow new viruses to escape to the environment. There is no integration of phage DNA into E. coli chromosome. T4 phage is also known as lytic (or virulent) phage.
Adsorption (attachment) of virion to host cell: precise attachment of tail fibres to bacterial cell wall.
Penetration or injection of phage DNA into host cell: injection of DNA through cell wall (phage does not enter).\
Synthesis of viral components using host cell enzymes and ribosomes: in host cytoplasm, phage may immediately take over the cell's replication and protein synthesis enzymes to synthesize phage parts.
Assembly inside the cytoplasm: Phage parts spontaneously assemble into bacteriophages.
Release of viruses from host cell: mature virions are released when host cell lyses (aided by viral enzymes)
Spike proteins are important fro animal viruses to recognise host cells. Each type of virus has a limited number of hosts due to specific binding of viral spike protein to host surface receptors. In a multicellular host, viruses may infect only certain tissues, Some viruses can remain dormant or latent for years. (e.g. chicken pox can re-emerge as shingles).
Chromosomal aberration: alteration of the structure or number of chromosome
Causes of structural abnormalities
Deletion: loss of a (large) portion of the chromosome. Can be fatal to an individual.
Duplication: Duplication of a region of the chromosome. May or may not have fatal consequences. e.g. if the duplication occurs outside of a gene, then it has no effects. If it happens next to the original region, it is called tandem duplication.
Inversion: occurs when a chromosome is broken into 2 places, reversed and put back together. If this occurs outside of a gene, then there would be no phenotypic effects.
An abnormality in chromosomes which can be grouped into 2 broad classes: numerical abnormalities (having extra chromosomes or missing chromosomes), structural abnormalities (extensive changes to chromosomal structures)
Chapter 5: The genetics of prokaryotes
Introduction to bacterial genetics
Prokaryotic organisms have a single, circular chromosome located in the nucleoid. They may also possess additional circular DNA molecules called plasmids.
Plasmids: contain only a few genes and are capable of self-replication. Are able to autonomously synthesize proteins. Are present in prokaryotic cells and some eukaryotic cells (e.g. yeast). May encode advantageous information (that are not required for normal function).
In eukaryotes, genetic variation is achieved during sexual reproduction by the process of genetic recombination. Although prokaryotes do not reproduce sexually, they have a more primitive means for sharing of DNA with each other. Horizontal gene transfer: movement of genes between cells that does not involve transmission from parent to offspring. Can result in a new bacterial strain (cells are called recombinant cells as they are different dorm donor cell and recipient cell)
Having genetic diversity is important for a bacterial population as it provides the genes for the cells to adapt to changing environments. Natural mutations do occur and this increases the chances of the survival of at least some of the organisms in a population. Recombinant is more beneficial to a microbe than mutations. New combination of genes might allow bacteria to carry out new functions, e.g. acquire resistance to antibiotics.
Prokaryotes display 3 types of horizontal gene transfer:
Conjugation
Requires cell-to-cell contact for gene transfer
Transduction
Uses bacteriophages for gene transfer
Transformation
Free DNA is captured from the environment
Horizontal gene transfer methods
Bacterial conjugation
Requires physical joining of 2 living bacterial cells. Typically, DNA transfer only goes one way, with the donor cell using a sex pilus or conjugation bridge. To produce pili, donor cells must have a plasmids called to F factor (fertility factor plasmid). Cells with F factor plasmid are called F+. Cells without the plasmid are termed F-. The F+ condition is heritable. If an F+ cell undergoes cell division, both the resulting cells will be F+. This condition is also 'contagious'.
Process of DNA transfer:
F+ cells produces F pilus that connects it to F- cell. 2. Transfer of F plasmid occurs through conjugation bridge (pilus). 3. A single DNA strand from the F plasmid of the F+ cell is copied in the F- cell using a process called rolling circle replication. This results in a newly-synthesized second DNA strand that is complementary to the donor DNA strand. 4. The F plasmids circularize in both cells. The F- cell is now renamed to F+. The end result is two F+ cells. Conjugation bridge dissociates.
Bacterial transduction
Bacterial transduction is mediated by bacteriophages. Unlike conjugation it does not require physical contact between the donor cell and the recipient cell. Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into the genome of a host cell. Bacteriophages can carry out the: lytic cycle (host cell is destroyed); or lysogenic cycle (viral DNA integrates into the host genome to become a latent prophage; then enters the lytic cycle upon induction)
Phage serves as a carrier of DNA between donor cell and recipient cell. Two types of transduction:
Generalised transduction: virtually any gene can be transferred. Occurs during the lytic cycle. Happens when the viruses accidentally package host bacterial DNA (instead of their genome) in their capsids. Bacterial DNA is then transferred to a different bacterial cell when the phage initiates a new infection.
Accidents occurs during the lytic cycle. During viral assembly, several possibilities: bacterial host DNA is randomly packaged into the head, then viral DNA; or only bacterial DNA is packaged (no viral DNA). If this transduced phage goes on to infect another bacterium, it could inject bacterial DNA instead of its own.
A bacteriophage attaches to a susceptible bacterium. 2. The phage genome is injected into the bacterium where its enzymes degrade the bacterial chromosome. it also directs the cell's metabolic machinery to synthesize viral components and enzymes. 3. During phage assembly, occasionally the capsid is mistakenly packed with either a fragment of the the donor bacterium's chromosome or plasmid, instead of the phage genome. 4. Host cell lysis causes phages to be released. Each newly-made phage carries a fragment of bacterial genome instead of phage genome. 5. The phage (carrying the donor bacterium's DNA) then attaches to another recipient bacterium. 6. The phage inserts the donor bacterium DNA into the new recipient bacterium. 7. Homologous recombination occurs and the donor bacterium's DNA is exchanged for some of the recipient's DNA.
Specialised transduction: Only a few host genes can be transferred. Occurs via accidents in the lysogenic cycle. Happens because of imprecise excision of prophage DNA during induction. Phages contain viral DNA and flanked by portions of host DNA. Bacterial DNA is then transferred to a different cell when the phage initiates a new infection.
Accidents occur during the lysogenic cycle of some temperate phages (e.g. lambda phage). First, the phage DNA has integrated into bacterial DNA (prophage) at certain locations and becomes dormant. Upon induction, the lytic cycle is initiated as the prophage is excised, sometimes imprecisely because it includes extra (adjacent portions of) bacterial host DNA. Defective viral DNA is multiplied, then packaged into new phages. This DNA can be transferred to other bacteria that the phage infects subsequently,
Viral attachment and penetration. The phage infects a cell. 2. Integration. The phage DNA becomes incorporated into the host genome. 3. Excision. The phage is excised from the bacterial chromosome along with a short piece of bacterial DNA. The DNA is then packaged into newly formed capsids. 4. Infection. Phage containing both viral and bacterial DNA infects a new host cell. 5. Recombination. The phage DNA, along with the attached bacterial DNA are incorporated into the new cell.
Bacterial transformation
Transformation is the genetic alteration of a cell resulting from the direct uptake, incorporation and expression of exogenous genetic material (DNA) from its surroundings. Does not need sex pili for DNA transfer,
For cells to take up exogenous DNA, they must be in a state of competence. Only competent cells are able to take up foreign DNA (either naked or in the form of plasmids) from the environment. Non-competent cells cannot.
Some bacterial species (e.g. E. coli) do not naturally undergo transformation in the lab. We need to induce artificial competence so that cells are passively permeable to DNA. 2 common ways:
Competent cell preparation at 4 degC
For heat-shock transformation, treat cells with cold CaCl2.
For electroporation, wash cells with ice-cold D.I. water several times to remove salts etc.
Heat-shock transformation
Induce competence by incubating bacterial cells in a solution containing plasmids and divalent cations (e.g. CaCl2) under cold conditions. Cells are then exposed to a pulse of heat shock (e.g. 42 degC, 30 sec) that causes DNA to enter cells. Plasmids enter the cells through pores in the cell membrane. Cells are allowed to 'recover' in warm, liquid culture medium (pores close). Cells are transformed.
Errors occurring during DNA replication: leading to erroneous DNA sequences replicated. Evade the proofreading function of DNA polymerases at the replication fork leads to spontaneous mutation,
Exposure to mutagens: induced mutation - exposure to mutagens that could increase frequency of mutations. Mutagens: radiation (e.g. UV rays and X-rays); and chemicals (e.g. nitrous acid, ethidium bromide etc.). Mutagens react with parent DNA causing DNA breakage or structural change (affects base-pairing ability of altered nucleotide).