Biology a Level F215

* State the biotechnology is the industrial use of living organisms to produce food, drugs of other products * Explain why microorganisms are used in biotechnological processes * Describe the standard growth curve in closed culture * Compare and contrast the processes of batch and continuous culture * Describe the difference between primary and secondary metabolites * Describe the importance of manipulating the growing conditions in a fermentation vessel in order to maximise the yield

* Industrial use of living organisms to make products for human use * Products include: drugs, food and enzymes * Used in agriculture, food science, medicine and animal husbandry * Microbes are mainly used (ie: bacteria, fungi or protoctista)

* +’s of using microbes: * Rapid life cycle * Grow rapidly in optimum conditions * Asexual reproduction is used in many bacteria * ˳˚˳ genetically identical * Secrete proteins out of cells ˳˚˳ easily harvested * Can be genetically modified by genetic engineering * ˳˚˳ can produce human proteins such as insulin * Simple growth requirements can be maintained in a fermenter * Can be grown in any climate inside a fermenter * Product is purer (ie: free from chemical contamination compared to chemical synthesis in labs) * Can be given “waste” food materials (eg: potato peelings) * No/very few ethical problems of industrial microbe use

* Population growth of bacterial culture in a closed system:

* 0-8 = LAG phase: * Initial slow rate of population growth * Slow because initial low number of individuals * ˳˚˳ doubling by mitosis = relatively small increases * Bacteria have to acclimatise to conditions * Eg) synthesise enzymes needed to hydrolyse nutrients in the culture * 8-24= LOG/Exponential Phase: * Very rapid population growth * Rate of reproduction exceeds death rate * Optimum conditions for growth (eg: nutrients available) * 24-25= deceleration phase: * Rate of increase is in decline * 1 0r more factors in becoming limiting * 25-30= Stationary phase: * Population is stable * Rate of production=rate of death * 30-end= Death phase: * Population decreases to ) only in a closed system * Death rate exceeds reproductive rate * Bacteria die because: * Lack of nutrients * ˳˚˳ no aerobic respiration due to no ATP for metabolism * Lack of O2 ˳˚˳ no aerobic respiration * ˳˚˳ lactic acid/ethanol are made from anaeroib respiration = toxic * Build up of wastes eg) CO2 * ˳˚˳ low pH * ˳˚˳ enzymes are denatured * Excess heat is produced from many reactions (respiration) * ˳˚˳ enzymes are denatured * Industrial scale fermenters (see diagram) * Fermenters are open systmes ie) allowing inputs and outputs * These are large structures the size of a 2 storey buildig * Conditions are kept to optimum for mirobes * 2 types: * Continuous Culture: * products made are removed at regular intervals ˳˚˳ micrboes remain in the LOG phase * nutrients are added regularly * used to generate primary metabolites ie) products of metabolism made during the main growth phase (LOG) * any protiens secreted by cells including those made due to genetic engineering * eg) insulin * Batch Culture: * Nutrients added intitally only @ the start * Microbes are allowed to grow and reach to stationary stage * Used to produce secondary metabolites * Ie) products made after the main growth phase * Eg) antibiotics – penicilin: made by microbes to kill competing microbes in natural conditions * Whole fermenter is emptied to harvest the product Continuous | Batch | Allows a higher growth rate | Restricts the growth | Nutrient levels are maintained | Nutrient levels decline | More difficult to set up and maintain | Generally easier to set up and maintain | More efficient because it is used constantly | Less efficient because fermenter has to be emptied, cleaned, sterilized and restarted | Used to make primary metabolites | Used to make secondary metabolites |

* Explain the importance of asepsis in the manipulation of organisms

* Asepsis = free from contamination of unwanted microbes * Achieved using aseptic techniques * Importance in preventing contamination: * Stops competition for nutrients and space * Yield would decrease * Product could be contaminated with toxins from others microbes * Contamination microbes could kill wanted microbes

* Describe how enzymes can be immobilised * Explain why immobilised enzymes are used in large-scale production

* Enzymes are immobilised to be kept separate from the products * 4 ways of immobilising enzymes: (see diagrams) * Entrapment: * Enzymes are trapped within a gel bead eg) alginate * Substrates have to diffuse into the bead to reach enzymes * Carrier Binding: * Enzymes are attached to the surface of an insoluble bead eg)glass/clay particle * Cross Linking: * Enzymes are covalently bonded to a large, inert molecule * Eg) glutaraldehyde * Membrane Separation: * Enzymes are kept in 1 place by an artificial membrane – partially permeable * Enzymes are too big to pass through the membrane * Substrate can pass through * Products can pass through * Any of these methods can be used inside fermenters * +’s * Products are free from enzymes * Cheaper processing * Enzymes are reusable * Continuous processing is possible * Immobilised enzymes are more stable – less easily denatured by high temperatures of pH because 3˚ structure bonds are protected * Therefore reaction can occur at higher temperatures therefore increased rate of reaction

* define the term recombinant * explain that genetic engineering involves the extraction of genes from 1 organism in order to place them in in another organism * Describe how sections of DNA are extracted from donor using restriction enzymes * Explain how isolated DNA can be placed in plasmids with reference to the role of ligase * State other vectors which fragments of DNA may be incorporated * Explain how plasmids may be taken up by bacterial cells in order to produce transgereic microorganism that can express a desired gene product * Describe the + to microorganisms of the capacity to take up plasmid DNA from the environment * Genetic Engineering
Outline genetic markers in plasmids used to identify bacteria that have taken up recombinant plasmid * Outline the process in genetic engineering of bacteria to produce human insulin
EG) production of human insulin for diabetes type 1 patients 1. Isolation of human gene: a. Starting with mRNA: * mRNA coding for human insulin is extracted from cells from the Islets of Langerhans in the pancreas * mRNA is copied to make single stranded cDNA (complementary DNA) using reverse transcriptase and free DNA nucleotides * Single stranded cDNA is turned into a double helix using DNA polymerase – replicating DNA * DNA double helix is added to a restriction endonuclease enzyme – cutting DNA, making a staggered cut and leaving single stranded sticky ends b. Starting with DNA: * This starting point is used when the required gene is not expressed in only 1 cell type * Eg) respiratory enzyme * A gene probe is used to identify the required gene from the rest of the DNA * This is: * Single stranded DNA * Has a complementary base sequence to the gene * Labelled (ie- florescent dye – visible under UV) * Required gene is cut from rest of DNA using a restriction endonuclease enzyme

2. Preparation of Vector: * A vector is a bacterial plasmid (or other means) that can carry a required gene into a bacterial all therefore a gene from 1 species is carried into another species * The plasmid is a circular loop of DNA, separate from the main DNA and is relatively small * TTAA
AATT

Sticky end
Restriction endonuclease enzyme
Marker gene turned into gene for penicillin
The plasmid is cut open with the same restriction endonuclease that was used to cut the human DNA * It will have complementary sticky ends to the human gene

3. Formation of recombinant DNA, identification and cleaning: c. Formation: * The human gene + cut open plasmid are mixed together with DNA ligase * Sticky ends of gene and open plasmid form H+ bonds with each other * DNA ligase joins the sugar-phosphate backbone with the DNA * This forms recombinant DNA DNA from two different species * The recombinant plasmids are introduced into bacterial cells using heat shock and Ca2+ ions. * Increasing permeability of bacterial cell walls and cell surface membrane * Some bacteria do take up the recombinant plasmids but others don’t * Bacteria that have taken up recombinant plasmids are called transformed bacteria d. Identification: * Grow bacteria in presence of penicillin * Only transformed bacteria have the marker gene for penicillin resistance therefore they will survive * Other bacteria will die * Transformed bacteria are cloned (ie) allowed to grow in optimal conditions 4. Manufacture: * The transformed bacteria are grown industrially in fermenters * Optimum conditions are maintained eg) pressure, O2, pH, temperature and nutrients * Fermenters are set up for continuous culture ie) nutrients are continuously added * Insulin is produced during LOG phase because it is a 1˚ metabolite made @ same time as normal bacterial proteins * Insulin is extracted and purified – separated from bacterial wastes and other solutes in the culture

* +’s of bacteria taking up plasmids from the environment: * Bacteria can take up plasmids naturally * Bacteria can take up plasmids from different strains and even different species of bacteria * Allows exchange of genetic mutation therefore produces more variation therefore natural selection and evolution can pass on their genes for antibiotics resistance

* Outline the process involved in genetic engineering of Golden Rice TM * Explain the ethical concerns raised by genetic manipulation of microrganisms * Golden Rice TM has a gene for β Carotene expressed in the food stores of its rice grains * therefore the rice is yellow-orange * When eaten - β Carotene is converted to vitamin A needed for production of rhodopsin for low light vision and healthy epithelial tissues * Lack of vitamin A = blindness because the cornea = dry, cloudy and white * Millions of children becomes blind in areas * eg) India where rice is the main diet and vitamin A is lacking due to less yellow-orange vegetables. * Ethical Concerns: * Reduces the genetic diversity of rice crops if golden rice is widely grown * Biotech company owns golden rice TM therefore makes high profits * Golden Rice TM is engineered to be infertile. * Therefore rice cannot be save and used for next year’s crop * Some Golden Rice TM is grown by the biotech company and are fertile. * Therefore some could cross-pollinate with wild grasses. * Therefore a new gene is added to these. Unknown long-term effects * Golden Rice TM rains are expensive

* Outline how animals can be genetically engineered for Xenotransplantation * Explain the ethical concerns raised by genetic manipulation of animals

* Xenotransplantation = the use of animals organs for transplantation to humans * Unaltered animal organs result in rapid rejection * Because animal cell antigens trigger an immune response * Therefore organ is attached by antibodies, T killer cells + phagocytes * Genetic engineering can either: * Introduce human genes coding for human antigens OR block the animal antigens * Ethical Concerns: * Animal rights * Unknown long-term consequences * Because irreversible * Religious objections * Especially if pigs are used

* Outline how DNA fragments can be separated by size using electrophoresis * Describe how probes are used to identify fragments containing specific sequences * Outline PCR can be used to make multiple copies of DNA fragments * Define gene therapy

* used for: * Mass replication of DNA * Ie) produces many copies of identical DNA or certain genes * For forensic evidence * Eg) hair follicle or white blood cells * Gene therapy * Adding healthy genes to people that have genetic diseases * Making gene probes * Identify genes * Mechanism (see sheet): * H+ bonds between bases are broken (A(2H+) T), (G(3H+) C) * Primer DNA (= a short length of single stranded DNA) allows DNA Polymerase to attach * Heat stable DNA polymerase is not denatured at high temperature * From bacteria in hot springs – adapted * Process can be automated in a machine * Add original DNA, Primer DAN, free DNA nucleotides + heat resistant DNA Polymerase * Electrophorosis – from a sample * EG) hair follicle/White Blood Cells – check epithelial cell from mouth swab * A number of restriction endonucleases cut the DNA into fragments. * Cut @ different random repeat sections * Single stranded garments * heat is applied to break H+ bonds between complementary bases * Electric current applied to gel plate * Phosphate groups of DNA fragments make them negative * Therefore they move towards the + end * Smallest fragments move the furthest to + * A DNA Finger print is produced * Like a bar code * Use of Electrophorosis: * To identify individuals * Suspects, victims, fathers * Identify specific genes * Using radioactive florescent gene probe * Gene probe binds if carrier * No bond if not * Have to have 2 gene probes if recessive * Binds to complementary if it’s separate * To identify genes to be used for genetic engineering

* Outline the steps involved in sequencing the genome of an organism * Outline how gene sequencing allows for genome-wide comparisons between individuals and between species * A Genome = all the genes (+ the DNA base sequence) for a species (or individual * Human Genome Project = identified the base sequence of all DNA in human cells * Genome Sequencing: * DNA with an unknown base sequence is used initially * Free DNA nucleotides are added, some of which are fluorescently tagged terminator bases. * These terminator bases are different colours (one for each base)these stop DNA replication * The unknown DNA strand is replicated many times * DNA chains are formed each as a different length + has a specific coloured terminator base @ the end * DNA lengths are separated by size using gel Electrophorosis * Colours are scanned, this gives base sequence (starting from smallest)