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Today’ s Agenda: Journal Questions: (1) Describe what you know about DNA. (2) What is genetic engineering (Biotechnology)? *1. Lecture: Genetic Engineering (Biotechnology) & Recombinant DNA Technology -slide …. 80 5/23/2017 1 Genetically Modified (GM) Crops around the World 5/23/2017 2 5/23/2017 3 5/23/2017 4 Genetic Engineering Dr. Rick Woodward 5/23/2017 5 Genetic Engineering 5/23/2017 6 Genetic Engineering 5/23/2017 7 Genetically Engineered Boneless Chicken Ranch 5/23/2017 8 DNA, the Law, and Many Other Applications – The Technology of DNA Fingerprinting A DNA fingerprint used in a murder case. The defendant stated that the blood on his clothing was not his. What are we looking at? How was it produced? 5/23/2017 9 DNA Fingerprinting Basics A. Different individuals carry different alleles. B. Most alleles useful for DNA fingerprinting differ on the basis of the number of repetitive DNA sequences they contain. 5/23/2017 10 DNA Fingerprinting Basics A DNA fingerprint is made by analyzing the sizes of DNA fragments produced from a number of different sites in the genome that vary in length. 5/23/2017 11 The DNA Fragments Are Separated on the Basis of Size The technique is gel electrophoresis. The pattern of DNA bands is compared between each sample loaded on the gel. 5/23/2017 12 Gel Electophoresis A. Technique used to separate nucleic acids or proteins by size and charge. 5/23/2017 13 California Biology Content Standards: California Content Standards: 5 b. Students know how to apply base-pairing rules to explain precise copying of DNA. 5 c. Students know how genetic engineering (biotechnology) is used to produce novel biomedical and agricultural products. 5 e. Students know how exogenous DNA can be inserted into bacterial cells to alter their genetic makeup and support expression of new protein products. 5/23/2017 14 DNA Review: A. Structure: Double Helix B. Location: Nucleus of Cell C. Function: “Blue print of Life” – Creation of Proteins/Amino Acids – Transcription. D. Nitrogen Base: ATCG E. Nucleotide: Phosphate, Sugar (Deoxyribose), Nitrogen Base (ATCG). F. Base Pairing Rules: A-T C-G 5/23/2017 15 Genetically Modified (GM) Food Genetically Modified Cotton (contains a bacterial gene for pest resistance) 5/23/2017 Standard Cotton 16 Genetically Modified (GM) Food 5/23/2017 17 What is Genetic Engineering? “Genetic engineering is the technology for modifying the genetic information in a plant, animal or human in order to produce some desired trait or characteristic” 5/23/2017 18 Genetic Engineering Vocabulary A. Restriction Enzymes “molecular scissors” are enzymes that cut DNA only at particular sequences. B. Plasmids are small circles of DNA found in bacteria. C. Plasmids are used to replicate a recombinant DNA. D. Vector - A vector is a small piece of DNA used to carry a gene of interest. 5/23/2017 19 Genetic Engineering: Recombinant DNA Technology 5/23/2017 20 Restriction Enzymes are Enzymes that Cut DNA Only at Particular Sequences The enzyme EcoRI cutting DNA at its recognition sequence Different restriction enzymes have different recognition sequences. 5/23/2017 21 DNA Cut by a Restriction Enzymes Can be Joined Together in New Ways These are recombinant DNAs and they often are made of DNAs from different organisms. 5/23/2017 22 Plasmids are Used to Replicate a Recombinant DNA A. Plasmids are small circles of DNA found in bacteria. B. Plasmids replicate independently of the bacterial chromosome. 5/23/2017 23 Human Insulin Production by Bacteria 1. 2. 3. 4. 5. 6. Isolate human cells and grow them in tissue culture. Isolate DNA from the human cells. Isolate plasmid DNA from a bacterium. Use the same restriction enzyme to cut the plasmid DNA. Mix the recombinant plasmid with bacteria. Allow the new bacteria to incorporate the recombinant plasmid into the bacterial cell. 7. Grow trillions of new insulin producing bacteria (this is when cloning takes place). 8. A fermentor is used to grow recombinant bacteria. 9. Collect the bacteria, break open the cells and purify the insulin protein. 5/23/2017 24 Harnessing the Power of Recombinant DNA Technology – Human Insulin Production by Bacteria 5/23/2017 25 Human Insulin Production by Bacteria and cut with a restriction enzyme 6) join the plasmid and human fragment 5/23/2017 26 Human Insulin Production by Bacteria Mix the recombinant plasmid with bacteria. Screening bacterial cells to learn which contain the human insulin gene is the hard part. 5/23/2017 27 Route to the Production by Bacteria of Human Insulin One cell with the recombinant plasmid A fermentor used to grow recombinant bacteria. This is the step when gene cloning takes place. The single recombinant plasmid replicates within a cell. 28 Route to the Production by Bacteria of Human Insulin The final steps are to collect the bacteria, break open the cells, and purify the insulin protein expressed from the recombinant human insulin gene. 29 Overview of gene cloning. Route to the Production by Bacteria of Human Insulin. 5/23/2017 30 Genetic Engineering: Insulin Production Overview 5/23/2017 31 Monday (March 12, 2012) Genetic Engineering Journal Question: What is a plasmid? *1. Lecture II: Genetic Engineering 2. Comprehensive Exam next Monday. 5/23/2017 32 Reviewing Genetic Engineering 5/23/2017 33 Banking Genes A. The massive Svalbard Global Seed Vault is built into the permafrost deep in a mountain on a remote arctic island in Norway 5/23/2017 34 Banking on Genes B. Built in 2008 After receiving its first deposits, a “doomsday” seed vault on an Arctic island has amassed half a million seed samples, making it the world’s most diverse repository of crop seeds. 5/23/2017 35 Most Widely Used Genetically Modified Crops are… 1. Cotton plants with a built-in resistance to insects. 5/23/2017 36 Most Widely Used Genetically Modified Crops are… 2. Corn and Soybeans resistant to the herbicide Roundup. a. Allowing Farmers to employ no-till techniques to farming. 5/23/2017 37 Which country is the leader in plant biotechnology? Answer: China A. They have recently sequenced the rice genome. 5/23/2017 38 Problems with Genetic Engineering Technology 1. Environmental Problems 2. Food Safety 3. Access to the New Techniques 5/23/2017 39 Environmental Problems A. Pest –resistant properties of transgenic crops. B. If pests have a broad exposure to the toxin or some other resistance incorporated into the plant, it is possible that they will develop resistance to the toxin and thus render it ineffective as an independent pesticide. 5/23/2017 40 Food Safety A. Food safety issues arise because transgenic crops contain proteins from different organisms and could trigger an unexpected allergic response to people who consume the food. 5/23/2017 41 Access to the New Techniques A. Relates to the developing world. B. Farmers in the developing countries are unable to afford the higher cost of the new genetically altered seeds. 5/23/2017 42 Other Types of Genetic Engineering: 1. Transgenic Engineering a. Putting genetic information from one type of plant or animal into another. 2. Cloning a. Making exact genetic copies of an existing plant or animal. 5/23/2017 43 Transgenic Organisms A. An organism is called “transgenic” if it has genetic information added to it from a different type of organism. B. Viruses do something of this sort when they infect plants, animals or humans. C. Humans have begun to do this with plants and animals. 5/23/2017 44 Transgenic Organisms D. This is the work that is furthest along: (1) Corn with its own insecticide. (2) Soybeans & cotton resistant to herbicides. (3) Papayas resistant to viruses 5/23/2017 45 Transgenic Organisms F. Human genes have been inserted into: (1) Bacteria (Prokaryotes) (2) Mice G. To produce various human proteins for treating diseases. 5/23/2017 46 Making Transgenic Mice 5/23/2017 47 Advantages of Transgenic Organisms A. Plants: (1) More disease-resistant. (2) Larger yields. (3) More transportable. B. Animals: (1) Make proteins for medicinal purposes. (2) Make organs for transplant to humans. 5/23/2017 48 Cloning: “Exact Copies” A. A “clone” is an exact copy. B. In genetics, a clone is a genetic copy of another organism. C. Clones occur naturally: Asexual breeding in plants & lower animals Identical twins (triplets) in higher animals D. For centuries it has been known that simple animals – worms & starfish – can be cloned by cutting them in half. E. This doesn’t work for higher animals! F. Part of the problem is cell specialization: Nerve, Bone, Muscle, etc. 5/23/2017 49 Cloning in the th 20 Century A. We now realize that each specialized cell has all the genetic information, but much of it is turned off. B. Problem – how to reset the “program” so this information is usable? C. Cloning of frogs successful in 1950s D. Cloning of livestock from fetal cells in 1970s. 5/23/2017 50 Cloning in the 20th Century E. The human genome (an organism’s genetic material) consists of 3 billion base pairs of DNA and about 30,000 genes. (1) 97% of our DNA does not code for protein product. -mostly consisting of repetitive sequences that never get transcribed. 5/23/2017 51 Cloning in the 20th Century: Hello Dolly F. Clone from an adult sheep cell by Scots researchers under Ian Wilmut. G. Had only one success in 300 attempts. H. Dolly grew to maturity, and successfully had a lamb by natural means in 1998. I. But Dolly seems to be prematurely old. 5/23/2017 52 Cloning 5/23/2017 53 Cloning 5/23/2017 54 Genetic Engineering Genetic engineering (also known as genetic manipulation or GM) is not the same as cloning. -Though cloning techniques are used in genetic engineering, the two processes should not be confused. 5/23/2017 55 Genetic Engineering versus Cloning A. Cloning: 1. Produces exact copies 2. Genes replicated within the same species. B. Genetic Engineering: 1. Produces a totally unique set of genes. 2. Genes can be swapped across species. 5/23/2017 56 Selective Breeding versus Genetic Engineering A. In the past, humans have brought about change in the genetic make-up of organisms by means of selective breeding (artificial selection) i.e. Purebreds B. Genetic engineering brings about such change by scientifically altering an organism's genetic code. 5/23/2017 57 Genetic Engineering Overview 1. In genetic engineering enzymes are used to cut up and join together parts of the DNA of one organism, and insert them into the DNA of another organism. 2. In the resulting new organism the inserted genes will code for one or more new characteristics - for example producing a new substance, or performing a new function. The organism has been genetically reengineered 5/23/2017 58 Other names for Genetic Engineering: A. This technique is also known as gene splicing or recombinant DNA technology (because the DNA is recombined in the vector molecule. B. Vector - A vector is a small piece of DNA used to carry a gene of interest. Besides the gene being studied, a vector may contain elements which are used to help the gene integrate into a genome. 5/23/2017 59 Why does genetic engineering work? A. Genetic engineering works because there is only one code for life. The set of instructions for which a gene is responsible work whichever organism the gene is in, and whatever instructions that gene gives are carried out within the cells of the recipient. B. Theoretically the possibilities are limitless, although this sort of manipulation gives rise to strong feelings for and against. 5/23/2017 60 Applications of Genetic Engineering A. One field in which genetic engineering has had a huge impact is the mass production of insulin to help diabetics. Scientists have isolated the gene responsible for making human proteins, including the insulin hormone. This gene is inserted into the bacterial DNA, and the microbes then clone themselves rapidly, making identical copies of themselves, all with the new gene and all capable of making human insulin. B. This is a cheap way of producing sufficient quantities of exactly the right hormone, for everyone who needs it. 5/23/2017 61 Other Applications of Genetic Engineering 5/23/2017 C. Producing interferon, a human protein which stops viruses multiplying inside the body. D. Producing human growth hormone to treat growth abnormalities E. Blood clotting factor to treat hemophiliacs. F. Used in industry to produce enzymes for use in biological washing powder. G. Producing pest resistant crop varieties. H. Producing tomatoes and other produce that stay fresh much 62 longer. Introduction to Genetic Engineering 1. With genetic engineering scientists directly manipulate genes. a. It frequently involves the use of recombinant DNA, which is composed of DNA segments from at least two different organisms. 5/23/2017 63 Commercial Applications 1. An example is the use of recombinant DNA technology to make interferon, a virusdestroying protein naturally produced by the human body. 5/23/2017 64 Production of Synthetic Interferon Involves: 1. Isolating the human gene that codes for the interferon production. 2. Splicing this gene into a strand of bacterial DNA. 3. Inserting recombinant DNA into a bacterium. 4. Cloning the bacterium and collecting the product: Interferon. 5/23/2017 65 Isolation of a Gene A. The first step in the process is isolating the human interferon gene. B. Genetic engineers use restriction enzymes, proteins that cut a DNA molecule into pieces. 5/23/2017 66 Isolation of a Gene C. The restriction enzyme EcoRI cuts DNA wherever the sequence C-T-T-A-A-G occurs. D. Other restriction enzymes cut DNA at different nucleotide sequences. E. By using the proper restriction enzymes scientists can cut the human interferon gene out of its chromosome. 5/23/2017 67 Gene Splicing 5/23/2017 68 Isolation of a Gene F. Once the gene for interferon is removed, it is separated from the rest of the DNA and then inserted into a strand of bacterial DNA. 5/23/2017 69 Gene Splicing A. Gene splicing is the process by which a gene from one organism is placed into the DNA of another organism. B. The human interferon gene is placed into the DNA of E. coli, the common bacterium of the human intestine. 5/23/2017 70 Gene Splicing C. In addition to a single, circular chromosome, E. coli contains a single, small ring of DNA called a plasmid. (Plasmid = a single ring of DNA in bacteria) D. Human DNA is inserted into this plasmid. 5/23/2017 71 Gene Splicing E. The plasmid ring is removed from the bacterium and the opened with a restriction enzyme. F. The human interferon gene and the bacterial plasmid have “sticky ends” –unpaired bases at each end of the DNA segment, where they were cleaved by restriction enzymes. 5/23/2017 72 Gene Splicing G. As the human DNA is spliced into the plasmid DNA, the unpaired bases of each bond readily. H. Consequently, a newly formed plasmid contains both human and bacterial DNA. 5/23/2017 73 Insertion, Cloning, and Collecting A. Once a DNA fragment is incorporated into a plasmid, the plasmid is inserted into another bacterium, which is then placed in a culture medium, where it divides and replicates. 5/23/2017 74 Insertion, Cloning, and Collecting B. Each time a bacterium divides a new copy of the plasmid DNA, which includes the human DNA gene, is created. C. This process by which the human gene is replicated is called gene cloning 5/23/2017 75 Insertion, Cloning, and Collecting D. Because E. coli can divide every 20 minutes, gene cloning is an efficient way to produce many copies of a specific genetic sequence. 5/23/2017 76 Insertion, Cloning, and Collecting E. The gene for human interferon is thus expressed in bacterial cultures and the resulting interferon protein is collected and eventually used by physicians. 5/23/2017 77 5/23/2017 78 5/23/2017 79 Genetically Modified (GM) Crops 5/23/2017 The ear of genetically engineered corn at top contains a toxin that kills worms. 80