Reading quiz • Identify the term that best represents each description • 1. When a bacteria is not affected by chemicals that interfere with its life processes • 2. A rod shaped bacterial cell • 3. Chemicals that interfere with bacteria’s life processes • 4. Substance discovered by Alexander Fleming that can be used to treat bacterial diseases • 5. Draw a picture of a typical bacterium and label with cell wall, pili, nucleoid region (genetic material)
Identification (“DNA fingerprinting”) What Are Common Uses of Biotechnology? • Diagnosing and treating genetic diseases • Genetically modifying organisms to make medicines or other useful products • - Human Growth Hormone • - Rice with added vitamins and minerals for use in third-world countries - Human tissues grown in animal donors
Your Health • Genetically Modified Organisms • Pesticide producing crops • Human organs in animals for transplants • Testing for and treating genetic diseases • Cancer • SCIDS • Making new or cheaper drugs • Insulin from bacteria • Antibiotics in milk
Genetic Engineering • Selective breeding • Advantages? • Disadvantages? • Increasing variation • Induce mutations
This is the Biotechnology Age You have been born in it and will live during it You must be informed in order to make intelligent decisions And… you deserve to be qualified to work in an exciting field - as scientists, technicians, receptionists, business execs, and artists
Prokaryotes are identified by their shape, chemical nature of cell walls, movement, and how they obtain energy. SHAPE: Rod- bacilli Spiral- spirilla Spherical- cocci CELL WALLS: Scientists use Gram staining to tell cell wall type apart. If the Gram testing is positive, then the bacteria has thick peptidoglycan walls If the Gram testing is negative, the bacteria has thin walls inside an outer lipid layers. MOVEMENT: Flagella.
Metabolic Diversity Identifies prokaryotes by how they obtain energy. Heterotrophs Chemoheterotrophs- consume organic molecules for energy and a supply of carbon. Photoheterotrophs- use photosynthesis; need organic compound like a carbon source. Autotrophs Photoautotrophs- use photosynthesis EX: cyanobacteria- Chemoautotrophs- make organic carbon molecules from CO2. They obtain energy directly from chemical reactions. Releasing energy- by fermentation, cellular respiration, or both Obligate aerobes- constantly need oxygen Obligate anaerobes- live without oxgen Facultative anaerobes- live with/without oxgen EX: E. coli
Life Cycle Bacteria can divide at high speeds, (every 20 min. ) Binary Fission- (asexual). Conjugation- Bacteria exchange genetic info by forming a bridge between two of them. Different genes are transferred to each other to increase genetic diversity in bacteria populations. Transformation-To be discussed later Spore formation
Antibiotics and resistance • Alexander Fleming and Penicillin • Antibiotics • Antibiotic resistance
Bacteria’s impact on life • Pathogenic bacteria • Toxins • Biowarfare • Production of chemicals and foods • Mining, environmental use • Nitrogen fixation • Decomposers
What Are The Basic Tools Of Biotechnology? Micropipets Gel boxes
Used for accurately measuring very small amounts of fluids Used in almost all biotechnology to measure 1 μL = .001 mL = .000001 L = 1 x 10^-6 L 2 mL = μ L = L Micropipets • Pipet types • P-20 (2-20 μ L) P-200 (20-200 μ L) P-1000 (200-1000 μ L)
Reading quiz • Viruses contain a coat made up of ____ and their genetic material is _____ • Lytic and lysogenic are examples of what? • How is HIV different than many other viruses?
The problem… • A disease is causing large pale spots on the leaves of tobacco plants • 1892, Ivanovski identified the cause of the disease to be in the juice • 1897, Beijerinck suggested that tiny particles caused the disease, and named them viruses (Latin-poison)
HIV and AIDS • HIV is the virus that can lead to AIDS • Transmission • Prevention • AVOID any of the forms of transmission!
HIV mutates, evolves very rapidly • Evades defenses of the immune system • Destroys Helper T cells
Is it living? • Contains genetic material? YEP • Evolves? YEP • Interacts with other organisms? YEP (parasitic) • Metabolic activity? IN A HOST CELL • Reproduces? IN A HOST CELL • What do you think?
Reading quiz (get SG 3,4C) • Write the term that best represents each description • 1. Circular piece of DNA that replicates independently of the chromosome(s) • 2. Protein that can “cut” DNA at precise spots • 3. Purposeful manipulation of DNA • 4. Overhanging sequence of bases left after DNA is “cut” • 5. Substance that can be used to identify cells with our gene of interest
1. Circular piece of DNA that replicates independently of the chromosome(s) 2. Protein that can “cut” DNA at precise spots 3. Purposeful manipulation of DNA 4. Overhanging sequence of bases left after DNA is “cut” 5. Substance that can be used to identify cells with our gene of interest Plasmid (vector okay) Restriction enzyme Genetic engineering Sticky end Antibiotic (tetracycline okay) 1 point each
Genetic engineering defined: Recombination of genetic information by transferring genetic information from one organism to another, usually by way of a recombinant “vector” At a minimum from different species -- from one bacterial species to another... Or… as dramatic as DNA from different kingdoms! Bacteria Plant Animal
Uses in basic research Simple: - to produce multiple copies of DNA - to produce multiple copies of a protein More complex: - monitor spread of virus infecting tobacco plants
Transgenic organisms • Organisms contain genes from other organisms • Microorganisms • Animals • Plants
Restriction Enzymes Protein molecules used to cut DNA Ligase Enzymes Protein molecules used to put together DNA Plasmids Small DNA molecules used to put new genes into bacteria Viruses Used to put new genes into other living things Tools to accomplish genetic engineering
How is DNA cut? • DNA is cut using restriction enzymes. • Examples for different restriction enzymes: • Eco RI, Hind III • Restriction enzymes cut DNA at specific four or six-base-pair-sequences called restriction sites. • Examples for different restriction sites: • Eco RI at G AATT C • C TTAA G • Hind III at A AGCT T • T TCGA A
Gene therapy • 1. Identify gene • 2. Clone gene • 3. Replace the faulty or missing gene • 4. Result? The correct protein should eliminate cause of the disorder • Advantages • Disadvantages
Transformation • Griffith experiment • Outside DNA gets incorporated into bacteria • What you have: pGLO plasmid (outside DNA) and E. Coli (bacteria)
How do you get pGLO into E. Coli? How do you grow transformed E. Coli? How do you identify transformed cells? Compentency CaCl2 Heat/ice shock Spread E. Coli on petri dish with agar and incubate Amp resistance Arabinose GFP protein Transformation procedure
A plasmid used as a vector must have Origin of plasmid replication ON/OFF switch a. An origin of replication b. A gene conferring resistance to an antibiotic. araC ori In addition, pGLO contains pGLO c. The Green Fluorescent Protein - the protein of interest GFP bla d. An ON/OFF switch that controls the synthesis of GFP by the presence or absence of the sugar, arabinose. Beta-lactamase to break down ampicillin Green Fluorescent Protein
Tranformation prelab • Read thoroughly through WB 204-206 • Make notes on any procedures you don’t understand. Highlight important information • Use the background information to answer the following • What is the role of the following items in the transformation lab and identify each as a gene, protein, or other: Ampicillin, GFP, Ampicillin resistant gene, arabinose, pGLO plasmid, E. Coli,
Interpreting results • LB + DNA : Prediction, why? • LB + DNA + amp : Prediction, why? • LB + DNA + amp + arabinose : Prediction, why? • Satellite colonies
Lab report • Introduction • Purpose • Deduction • Background info • Materials/procedures • Include amounts • SUMMARIZE procedures (don’t copy from lab) • Results • Data table, picture, bar graph?, written paragraph • Analysis • Support/doesn’t support hypothesis? Interpret results from each petri dish. Difference between your results and class average? How can you improve the lab? Importance of lab?
Reciprocal teaching: “Are viruses alive” • As we’ve done before, take turns reading through the article (rotate after each paragraph). Each person will have a different role • Summarizer: Summarizes EACH paragraph read • Predictor: Predict what will happen next • Connector: Discusses connections with class content/experiences/society • Questioner: Asks under and on the surface questions. Asks if anyone else has questions • Have your assignment sheet out. I will stamp WB 206 (transformation activity sheet) and give you a stamp for your RT read based on your discussion
Reading quiz (get SG and transformation activity stamped, and take out 207) • Write the term the best represents each description • 1. A weakened or killed pathogen • 2. A disease when the body launches an immune response against its own cells • 3. The first one was produced by Jenner using cowpox • 4. The body system HIV attacks
Vaccines • A weakened or killed virus is injected into the individual • Elicits an immune response (antibodies) • Does not harm the individual
Used to separate chemicals like DNA based on size, charge, etc.. Used for identification/DNA fingerprinting, researching genes, etc.. Gel Boxes
Lab B analysis • Voltage vs. current • When was electricity being conducted? Why? • Electricity allows electrolysis to occur • Role of phenol red? • Role of buffer? • Requirements for running DNA through a gel: Current and a stable pH
Note bubbles and swirls of changing pH H2O + NaCl + phenol red + TAE buffer
The Principles of Gel Electrophoresis • Charge of DNA: ____ • If put into an electric field, it will move from the _____ to the ____ pole. • A gel serves as the porous matrix for the movement of the DNA molecule. • DNA is cut into fragments of different size.
libproto RFLP (DNA FINGERPRINTING) Protocol