CHAPTER 17: BIOTECHNOLOGY WHERE DOES IT ALL FIT IN? Chapter 17 investigates contemporary uses of biotechnology based on the principles of classical and molecular genetics. This chapter gives the instructors many opportunities to ask students critical thinking questions about applications of genetics knowledge. The topics in this chapter also stir much debate in society and should be addressed as part of the coverage. Chapter 17 is the capstone chapter for gene expression coverage from Chapters 15 and 16 of the book should be revisited when covering genomics information in Chapter 18. SYNOPSIS Science has reached the exciting, but potentially dangerous stage, at which we are learning to manipulate the materials of heredity. The first human genes isolated and inserted into bacteria turned these cells into miniature factories producing interferon. Many bacteria possess restriction endonucleases to protect themselves from invading viruses. Scientists use these enzymes to chop up strands of DNA at specific locations. Such specificity assures that a given enzyme will always break up a specific kind of DNA into exactly the same size and number of fragments. These fragments constitute a library of DNA sequence information. Restriction enzyme specificity also assures that all of the fragments possess identical, short sequences called “sticky ends.” Each strand of a sticky end is complementary to the other strand and can be joined to the other ends when treated with a DNA ligase. DNA Fragments, even those from different organisms, that have been cut with the same restriction enzyme can be joined enabling the insertion of foreign genes into a plant, animal, or bacterial genome. Bacterial plasmids and viruses are the vehicles by which such genes are inserted into the host DNA, the crux of genetic engineering. There are four steps in this process: cleavage, producing recombinant DNA, cloning, and screening. Cleavage is accomplished using the restriction endonuclease that will produce the desired sticky ends. The fragments are then inserted into the desired vehicle. Unfortunately, very few vehicles actually receive DNA fragments and even fewer get the desired piece. At this point, vehicles not carrying fragments are eliminated, generally by prior association with an antibiotic resistance gene. Each colony of cells is cloned and allowed to multiply, thus replicating not only its own genome but the added fragment as well. The clones are then screened to determine which clonal line contains the desired fragment. Polymerase chain reaction is another new molecular technique that amplifies DNA in an in vitro sample. Frequently the DNA in a sample (of blood for example) is so small that it cannot be analyzed directly. With PCR, the DNA is copied using a microprocessor-controlled thermoregulator. The DNA unzips as the temperature is increased. When it is lowered, polymerase enzymes catalyze the replication of DNA from special primers, making a new strand from each original strand. Thus the amount of DNA is doubled at each cycle – 2 strands to 4 strands to 8 strands to 16 strands and so forth. This method is substantially quicker than cloning the DNA strand via plasmids or viruses. DNA is readily identified using a technique called Southern blotting. Differences in DNA sequences are identified by RFLP analysis. Each individual can be identified by the RFLP patterns possessed, what is referred to as a DNA 138 fingerprint. Biotechnology uses genetic engineering techniques to solve practical problems. The biological community is busy sequencing the entire human genome, certainly an enormous task. DNA fingerprinting has been used to identify and convict numerous criminals. Dozens of commercial applications exist to utilize this revolutionary technology. The most obvious application, pharmaceuticals, however, encounters additional problems of separating the desired product from the rest of the cellular material. Attempts are being made to construct piggyback vaccines, placing genes coding for the exterior of a virulent virus within the harmless vaccinia virus. Agricultural uses range from developing resistance to herbicides, viruses, and insects; to inserting genes for nitrogen fixation and improving growth and plant nutritional value. Society must be informed about these biological processes to ensure our safety and economic wellbeing, as well as that of future generations. Lack of sufficient biological knowledge is the source of most of the public’s concern about genetically engineered products. Many assume that BST in milk products may cause human growth problems; they lack the physiological knowledge that this protein is degraded in the stomach like all other proteins. A great many people do not trust governmental safeguards and fear the inadvertent or intentional development of lethal viruses and bacteria. Although there is little scientific need for labeling genetically modified food products, the public has the right to insist upon it. If properly done, labeling should serve to educate consumers as well as inform them. LEARNING OUTCOMES Understand the importance of plasmids and viruses to genetic engineering. Know the natural function of restriction endonucleases and how a normal bacterial cell protects its DNA from their activity. Understand how “sticky ends” are formed and their importance to gene technology. Describe how a chimeric genome is constructed. Explain the four steps of genetic engineering experiments. Distinguish between the techniques of selection and screening of clones. Explain how to screen for clones that contain a desired gene fragment. Understand the value of and the processes involved with the polymerase chain reaction (PCR). Describe techniques used to characterize DNA. Discuss the different applications of gene technology. COMMON STUDENT MISCONCEPTIONS There is ample evidence in the educational literature that student misconceptions of information will inhibit the learning of concepts related to the misinformation. The following concepts covered in Chapter 17 are commonly the subject of student misconceptions. This information on “bioliteracy” was collected from faculty and the science education literature. Students believe that all biotechnology is genetic engineering Students do not fully understand the role of genetics and environment on determining 139 observable variation in organisms Students do not differentiate the genetic differences between prokaryotes and eukaryotes Students believe that genetic modification is more unpredictable than selective breeding in determining an organisms characteristics Students believe that genetically modified organisms are inherently dangerous Students believe that genetically all modified foods are unsafe or cause allergies Students believe that gene transfer introduces many characteristics of one organism into another Students believe the virus vectors used in gene transfer are more dangerous than natural viruses Students believe it is not possible to introduce the genes of animals into plants Students believe it is not possible to introduce the genes of plants into animals Students believe that cloning is an unnatural process Students believe that genetic modification and cloning introduces unpredictable mutations INSTRUCTIONAL STRATEGY PRESENTATION ASSISTANCE Here we have what’s happening in biology today; where the money is to be made! Discuss the mechanisms of gene technology before discussing its implications. Students have a hard time with the nature of the “sticky ends” resulting from treatment with restriction enzymes. To complicate matters, some blunt cutting enzymes have been discovered as well. It might be helpful to present them with several DNA sequences and show how different restriction enzymes would fragment the sequence. You can then show how identical “sticky ends” can be joined together. Genetic engineering would be significantly more difficult without plasmid and viral vectors. Plasmids were presented in the last chapter, viruses were discussed to some extent in the chapter before that. Recall in either case, how the vector is naturally able to insert genetic material into a complete genome, the plasmid into bacteria, the virus into eukaryotes causing some forms of cancer. Science is merely adapting a natural phenomenon to its own benefit. Screening is not only the most difficult part of genetic engineering to do, it is the hardest part to understand. Include the presence of the antibiotic resistance gene at the onset of your discussion. Explain its function at the screening step. The many technical terms associated with gene technology can be confusing; most are associated with genetic engineering in that they are means for identifying the cell with the correct stuff. Probes have been developed for a number of tumor cell lines and Huntington’s disease. The latter is 95% to 98% accurate in determining whether the gene is present. Thus persons with the disease in their family background can be tested long before the onset of the disease itself (most individuals refuse testing or are tested and don’t want to be told the results). Knowledge of test results may impact personal lifestyle and plans for having children as well as insurance and health policies. 140 One merely needs to pick up the science section of the weekly newspaper, or a lay science magazine to see examples of gene technology in action. As a result, it is important to discuss the implications of such research and the necessary scientific and governmental regulations. This is one of the stronger reasons to have some knowledge of biology, to be able to make informed decisions, and to determine if the decisions made by those in power are indeed in the best interest of the populace. Someone will need to make difficult decisions in the not-so-distant future. Just because science can perform certain technological feats doesn’t mean that it should be allowed to do so. Conversely, just because some gene technology is potentially dangerous, doesn’t mean that all related technology should be brought to a halt. It’s your students who will be making the political decisions for the future of the world. HIGHER LEVEL ASSESSMENT Higher level assessment measures a student’s ability to use terms and concepts learned from the lecture and the textbook. A complete understanding of biology content provides students with the tools to synthesize new hypotheses and knowledge using the facts they have learned. The following table provides examples of assessing a student’s ability to apply, analyze, synthesize, and evaluate information from Chapter 17. Application Analysis Synthesis Evaluation Have students design a theoretical vector for introducing a bacterial gene into an animal cell. Have students hypothesize about feasible traits that can be introduced into crops. Ask students to hypothesize how ligase can be used to make new genes.. Have students to compare and contrast traditional selective breeding to genetic engineering as a means of producing new agricultural organisms. Ask students to determine the problems of introducing eukaryotic DNA into prokaryotes using genetic technology. Ask students to explain why it is possible to damage existing genes when new genes are introduced into genomic DNA. Ask students develop a hypothetical expression vector that would prevent genetically modified crops from reproducing with related wild plants. Ask students develop ways of using restriction enzymes as a tool for controlling viral diseases. Ask students come up with a way strategy of using plants to remove hazardous wastes from the soil. Ask students to evaluate the pros and cons of growing genetically modified crops such as bt corn. 141 Ask students to assess the value of cloning in agriculture. Ask student to debate the safety concerns associated with the creation of a new gene. VISUAL RESOURCES Palindromes are words that exhibit two-fold rotational symmetry (bob, kook, deed). The phrase “a toyota” is a palindrome as is “a man, a plan, a canal, panama.” Search the web for thousands of examples, but start here: http://www.cs.rdg.ac.uk/archive/evihcra/ ku.ca.gdr.sc.www//:ptth/. Hopefully you will notice that the URL itself is a palindrome! The scifi film “Gattaca” touches on future (or maybe not so future!) gene technology and the ethical implications of genetic control. Substantial information is available at the movie website http://www.sciflicks.com/gattaca/. IN-CLASS CONCEPTUAL DEMONSTRATIONS A. Virtual Vector Introduction This demonstration provides a tangible model for showing students design elements of an expression vector. Materials Thick permanent markers White clothesline or thick rope Yellow wool or twine Red wool or twine Blue wool or twine Green wool or twine 1 inch thick slivers of duct tape Scissors Procedure & Inquiry 1. Review the use of an expression without describing its components . 2. Tell the students you will be using rope to represent a plasmid to be used as an expression vector a. Take a 2” loop of white clothesline or rope and tape it into a loop b. Tell the class that the loop can be a plasmid or a yeast artificial chromosome (YAK). 3. Then ask the class what they would need to make a eukaryotic expression vector. 142 4. Use the materials in the following manner as the class is making suggestions: a. Use the scissors to represent restriction enzymes for cutting open the plasmid b. Use the duct tape to present ligase bonded regions of the vector c. Use the wool or twine to represent different components of the vector 5. Cut an paste the expression vector based on students comments 6. Then have the class evaluate the accuracy of their vector B. Virtual Biotechnology Concept Map Introduction This fun and fast way to build a concept map engages students in developing a scheme for reviewing all the facts and concepts associated with DNA replication. It helps student select relevant information needed to understand DNA replication. In addition, it helps them incorporate concepts learned in other sections of the book that contribute to an understanding of DNA replication. The simple click and drag animated concept mapping tool should be practiced before using in class. Materials Computer with live access to Internet LCD projector attached to computer Web browser with bookmark to Michigan State University C-Tool: http://ctools.msu.edu/ctools/index.html Procedure & Inquiry 1. Tell students that you would like to do a quick review of the concepts associated with DNA replication. 2. Then go to the Michigan State University C-Tool and add the concept map term “Biotechnology”. Use the “Add” and “Concept Word” feature to place a term on the map background. 3. Solicit a few more terms or concepts and then ask the class how the concepts are connected to each other. Use the “Add” and “Linking Line” feature to build a connecting line. 4. Then ask the students to justify the concept linking lines. Use the “Add” and “Linking Word” feature to place student comments on the map. 5. Continue the activity until you feel the students made a comprehensive map. USEFUL INTERNET RESOURCES 1. Animations are a valuable classroom resource for reinforcing the DNA concepts needed to understand biotechnology. The Cell Biology Animation website provides a well-done animation sequence showing the three-dimensional structure of DNA and its location in the cell. This website can be found at http://www.johnkyrk.com/DNAanatomy.html 2. Gene and vector bank databases are valuable tools used by researchers who desing 143 expression vectors. Students can be shown how these databases are used by looking up different types of vector components available to researches. A website maintained by the Human Genome Project links to gene and vector resources that can be shown on an LCD project. The website can be found at http://www.biologia.uniba.it/rmc/01a_pagina/1-1_PAC.html. 3. The history of biotechnology is an interesting topic discuss with students during a lecture on biotechnology. Excess Excellence has a biotechnology timeline that can be projected on a screen to review with students. The website is available at http://www.dna.gov/. 4. Case studies are a highly effective way to reinforce the learning of issues related to biotechnology. A case study developed by the University of Buffalo called “Frankenfoods? The Debate Over Genetically Modified Crops” encourages students to rationally investigate the scientific and societal issues related to genetically modified crops. The website can be found at http://www.sciencecases.org/gmfoods/gmfoods.asp. LABORATORY IDEAS Biotechnology research requires computational studies using a DNA database before proceeding with the laboratory work needed to produce a genetically modified organism. This activity introduces students to the use a DNA sequence search engine called BLAST. a. Introduce students to the value of knowing gene sequences before designing expression vectors used to produce genetically modified organisms. Tell them that researchers use electronic on-line databases to search for gene sequences for a particular protein they want to place into an organism. b. Provide students with the following resources: a. Computer with Internet access b. Web browser with a bookmark to BLAST Tutorial (http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html) c. Web browser with a bookmark to BLAST Search (http://www.ncbi.nlm.nih.gov/blast/index.shtml) d. A list of amino acids with their single letter designations e. Codon chart c. Tell the students go the BLAST tutorials on Information Page of the BLAST website. d. Then tell the student to type in the amino acid sequence into the Input box on Part 2 of the PSI-BLAST tutorial at http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/psi1.html: a. GLNKSVEEFENELKNKLTEEAKNKMENIKKELEDVGFKVKDIIVVGIPHEE IVKIAEDEG b. Have them press the Search button without making any other changes to the search. c. Then have them discuss with each other the diversity of genes programming that amino acid order in its DNA sequence. e. Now have the students go to the BLAST Search and click on the Protein-protein BLAST (blastp) link. f. Instruct them to type in the same amino acid sequence and analyze the results. g. Then have them analyze the results including any information provided in the links from 144 the completed search page. h. Then instruct the students to calculate the approximate DNA sequence for the gene. i. Have the students go to the Nucleotide search and type in the purported DNA sequence. j. Have the students assess the outcomes of their search. They should be able to explain why the search may or may not have found the appropriate gene for the amino acid. LEARNING THROUGH SERVICE Service learning is a strategy of teaching, learning and reflective assessment that merges the academic curriculum with meaningful community service. As a teaching methodology, it falls under the category of experiential education. It is a way students can carry out volunteer projects in the community for public agencies, nonprofit agencies, civic groups, charitable organizations, and governmental organizations. It encourages critical thinking and reinforces many of the concepts learned in a course. 1. Have students do a presentation on biotechnology to a civic group. 2. Have students design an educational PowerPoint presentation of genetic engineering for high school teachers. 3. Have students tutor high school biology students studying genetics. 4. Have students design a series of educational posters about biotechnology for a local school or library. This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity employer and does not discriminate on the following basis: against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity. . 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