GENETIC · 2016-01-05 · • Genetic make-up is received from both parents, and is expressed as traits which can be predicted (pp. 128-130). • The genetic code instructs the production

GENETIC ENGINEERING

This unit was developed by

California Foundation for Agriculture in the Classroom2300 River Plaza Drive

Sacramento, California 95833Telephone: (800) 700-AITC

[email protected]

Mailing address:Post Office Box 15949

Sacramento, California 95853

All or part of this educational unit may be reproduced for teacher and student classroom use.Permission for reproductions for other purposes must be obtained from the

California Foundation for Agriculture in the Classroom.

1st Printing

October 1997

California Foundation for Agriculture in the Classroom 1997 1

TABLE OF CONTENTS

ACKNOWLEDGMENTS ......................................................................................2

INTRODUCTION ..................................................................................................4

GETTING STARTED

UNIT OVERVIEW ......................................................................................5

BEFORE YOU BEGIN ..............................................................................8

BACKGROUND INFORMATION ON GENETIC ENGINEERING .............9

LESSONS

LESSON 1: WHAT CAN WE DO?..........................................................13

LESSON 2: THE MEETING ...................................................................21

LESSON 3: THE PLASMID ...................................................................35

LESSON 4: THE RESEARCH STARTS: TRANSFORMING DNA .......51

LESSON 5: HOW DO GENES GET INTO PLANTS? ............................67

LESSON 6: THE PRESENTATION .......................................................76

TEACHER RESOURCES

WHERE DO GENES COME FROM? ......................................................81

“CAREERS IN BIOTECHNOLOGY” STUDENT HANDOUT ..................82

TEACHER RESOURCES AND REFERENCES ......................................83

GLOSSARY ............................................................................................86

California Foundation for Agriculture in the Classroom 19972

ACKNOWLEDGMENTS

This unit was made possible through grants and support from Calgene, Inc., Monsanto Company,Bio-Rad Laboratories, the California Farm Bureau Federation and the California Foundation forAgriculture in the Classroom.

Calgene, Inc. is an agricultural biotechnology company which develops improved varieties of plantsand plant products for the fresh tomato, cottonseed and industrial and edible plant oils markets.

Monsanto Company is a global agricultural company which focuses on agricultural biotechnology andthe development and marketing of value-added food and fiber crops, as well as crop protectionproducts, performance materials, food ingredients and pharmaceuticals.

Bio-Rad Laboratories is a multinational manufacturer and supplier of products for life scienceresearch and education.

The California Farm Bureau Federation actively represents, protects and advances the social andeconomic interests of farm families and California communities by organizing productive agricultureto provide group benefits and manage issues which affect its membership.

The California Foundation for Agriculture in the Classroom is dedicated to fostering a greater publicknowledge of the agricultural industry and seeks to enlighten students, educators and leaders in thepublic and private sector about agriculture's vital, yet sometimes forgotten, role in American societyand the effect all citizens have on agriculture's well being.

We would like to thank the following people who helped create, write, revise and edit this unit. Theircomments and recommendations contributed significantly to the development of this unit. However,their participation does not necessarily imply endorsement of all statements in the document.

Writers

Jean KennedyScience TeacherArmijo High SchoolFairfield-Suisun Unified SchoolDistrictFairfield, CA

Suzanne WeiskerScience TeacherWill C. Wood High SchoolVacaville Unified School DistrictVacaville, CA

Editor

Pamela EmeryCurriculum SpecialistCalifornia Foundation forAgriculture in the ClassroomSacramento, CA

Field Testers

Dorothy ReardonScience TeacherDel Campo High SchoolSan Juan Unified School DistrictFair Oaks, CA

Francesca LoweScience TeacherConcord High SchoolMount Diablo UnifiedSchool DistrictConcord, CA

Kathleen McCarthyScience TeacherConcord High SchoolMount Diablo UnifiedSchool DistrictConcord, CA

Illustrators

Patricia Houk

Maceo Montoya

Layout, Typing and Design

Margaret Anderson

Karin Bakotich

Sherri Hughes

Tami Gutschall

Rae Lehutsky

Cinamon Vann


Curriculum Advisory and Review CommitteeDavid Anderson, Ph.D.Director of Cotton BiotechnologyJ.G. Boswell CompanyCorcoran, CA

Carol BastianiResearch AssistantDepartment of Medical Biological ChemistryUniversity of CaliforniaDavis, CA

Beth BrookhartFreelance JournalistBakersfield, CA

Lucas Calpouzos, Ph.D.Former Dean of AgricultureCalifornia State UniversityChico, CA

Steve ClarkScience TeacherMonterey High SchoolMonterey Peninsula Unified School DistrictMonterey, CA

Norma ClawsonScience TeacherCajon High SchoolSan Bernardino Unified School DistrictBlue Jay, CA

Constance ComanBiology TeacherWinters High SchoolWinters Joint Unified School DistrictWinters, CA

Lane ConnChairmanStanford Human Genome CenterStanford UniversityPalo Alto, CA

Jenny CuccinelloScience TeacherFlorin High SchoolElk Grove Unified School DistrictSacramento, CA

Judy CulbertsonManager of Programs and ServicesCalifornia Foundation forAgriculture in the ClassroomSacramento, CA

Jerry DelsolAgriscience TeacherWoodland High SchoolWoodland Joint Unified School DistrictWoodland, CA

Jim ElamAnimal NutritionistAgricultural Technology, IncorporatedSolvang, CA

Richard EngelProject CoordinatorCalifornia Foundation for Agriculturein the ClassroomSacramento, CA

John FedorsScience Education ConsultantSan Diego, CA

Mary Jo Feeney, MS, RD, FADADirector of EducationCalifornia Beef CouncilPleasanton, CA

David HammondEducational ConsultantSacramento, CA

Carolyn HayworthManager, Investor and Public RelationsCalgene, Inc.Davis, CA

Catherine Houck, Ph.D.Vice President, Product Developmentand Variety DevelopmentCalgene, Inc.Davis, CA

Lauren HubbardGraduate Student ResearcherPlant Molecular GeneticsUniversity of CaliforniaBerkeley, CA

Gay JividenSenior Director of ResearchCotton, Inc.Raleigh, NC

Andy KennedyField Representative/BuyerColusa County Canning CompanyWilliams, CA

Jean LandeenAgricultural Education ConsultantCalifornia Department of EducationSacramento, CA

Jeanne LaytonResearch BiologistMonsanto CompanyChesterfield, MO

Mark LinderPresidentCalifornia Foundation forAgriculture in the ClassroomSacramento, CA

Ron MardigianProduct ManagerBio-Rad LaboratoriesHercules, CA

Martina McGloughlinAssociate Director of BiotechnologyBiotechnology DivisionUniversity of CaliforniaDavis, CA

Craig McNamaraFarmerSierra OrchardsWinters, CA

Donna MittenGenetic Engineering ConsultantWoodland, CA

Jeffrey O’NealExtension CoordinatorBiotechnology ProgramUniversity of CaliforniaDavis, CA

Janette OaksResearch ScientistCalgene, Inc.Davis, CA

Maria OsbornBiology TeacherFoothill High SchoolGrant Joint Union High School DistrictSacramento, CA

Frank PlesciaManager, Government AffairsMonsanto CompanyRoseville, CA

Pam SchallockFourth Grade TeacherSandrini Elementary SchoolPanama-Buena Vista Union School DistrictBakersfield, CA

Claudia SellersGraduate Student ResearcherDepartment of Plant and Microbial BiologyUniversity of CaliforniaBerkeley, CA

Wynette SillsFarmerPleasant Grove FarmPleasant Grove, CA

Roger SitkinFarmerOld Dog RanchLinden, CA

Barbara SootsAssistant Education DirectorCenter for Engineering Plant ResistanceAgainst PathogensUniversity of CaliforniaDavis, CA

Nancy StevensBiology TeacherSan Rafael High SchoolSan Rafael City High School DistrictSan Rafael, CA

KarenBeth TraigerScience Resource TeacherGraystone Elementary SchoolSan Jose Unified School DistrictSan Jose, CA

Denise Van HornFourth Grade TeacherMcSwain Elementary SchoolMcSwain Union Elementary School DistrictAtwater, CA

John VogtMiddle School Science TeacherCreekside Middle SchoolCotati-Rohnert Park Unified School DistrictRohnert Park, CA

Thea Wilkins, Ph.D.Associate ProfessorDepartment of Agronomy & Range ScienceUniversity of CaliforniaDavis, CA

Mary YaleMiddle School Science TeacherGrange Middle SchoolFairfield-Suisun Unified School DistrictFairfield, CA

4 California Foundation for Agriculture in the Classroom 1997

INTRODUCTION

The Science Framework for California Public Schools emphasizes the need to make scienceeducation more meaningful to students so they can apply what they learn in the classroom to theirdaily lives. Since all students eat food and wear clothing, one natural connection between scienceand the real world is agriculture. Advances in agricultural technology, especially in geneticengineering, are continually making headlines and are an excellent way for educators to connectcurrent trends and issues in science to the lives of every student.

Agriculture is an enormous industry in the United States, especially in California. As more ruralareas become urbanized, more challenges exist to maintain and improve the quality of life on theplanet and feed the people of the world. It is extremely important to educate students about theirenvironment, agriculture and the current technologies and research that continue to make the Earth aviable planet. Genetic engineering is a relatively new science that has blossomed over the last 40years. Most genetic engineering research relates to agriculture and medicine. This unit will focus onplant genetic engineering.

Genetic Engineering in Agriculture is a thematic high school unit that can be used at the end of agenetics unit or an advanced unit on the cell. Throughout the unit, students are provided with thescientific principles of genetic engineering and are encouraged to use their knowledge to thinkcritically, creatively and freely about the viability and ethics associated with genetic engineering andagriculture. The introductory lessons present biotechnology terms as the students are presentedwith a hypothetical scenario associated with two major environmental concerns—food and fiberquality and the efficient use of resources. Students prepare interactive reports and perform DNAtransformation laboratory activities that teach genetic engineering concepts, such as how geneticinformation is transferred from one organism into another through the use of plasmids and tissueculture. The students then use their acquired knowledge to prepare a presentation to a hypotheticalresearch funding board to report their findings on one issue associated with the cotton industry—gossypol quantities in cottonseed.

Genetic Engineering in Agriculture is one of many lesson plans provided by the California Foundationfor Agriculture in the Classroom. The Foundation appreciates the support from Calgene, Inc.,Monsanto Company and Bio-Rad Laboratories for assisting in the funding and development of thisunit. Please contact the Foundation for assistance with furthering the integration of agriculture intoyour curriculum. Comments on this unit and on other Agriculture in the Classroom resources arealways welcome and appreciated.


UNIT OVERVIEW

BRIEF DESCRIPTION

This interdisciplinary thematic unit consists of six lessons on genetic engineering in agricultural cropsalong with the background knowledge needed to complete the lessons. This unit is designed forcollege preparatory biology or physiology students who have a basic understanding of genetics andadvanced knowledge of cell structures and functions. The lessons are designed to introducestudents to biotechnology and genetic engineering in a format that supports critical thinking andproblem-solving on current issues associated with these sciences.

As indicated in the first lesson, one agricultural challenge is presented to the students—the presenceof a chemical called gossypol in cottonseed. This chemical acts as a defense mechanism for theplants, but can be potentially harmful to animals, including humans, if consumed in large quantities.Through background information gathered from hypothetical, yet realistic, memos, meeting minutesand newsletters, the students are asked to address the gossypol challenge. The lessons guide thestudents to investigate gossypol using a genetic engineering approach and to test their procedures inthe laboratory, where they perform a hands-on transformation activity. As part of a research team,the students are asked to present their findings to a research board requesting further funding of thisproject.

PRIOR STUDENT KNOWLEDGE REQUIRED

Before beginning this unit, your students should understand:

• The basic components of cells.

• The structures and functions of bacterial cell components, especially plasmids.

• The structure of DNA and the role of DNA in coding for traits.

• Cell transcription and translation.

• How the order of DNA base pairs determines amino acid selection and that the sequence ofamino acids determines specific protein structure.

• Basic genetic principles.

• Sterile bacterial plate streaking techniques, including hands-on experience with this practice.

UNIT LENGTH

In its entirety, this unit will require approximately three weeks of class time plus several evenings ofstudent homework. It is possible, however, to perform only selected lessons from this six-lessonunit. Long-range planning is required by the instructor so appropriate materials can be ordered andprepared.


SCIENCE CONCEPTS

According to the 1990 Science Framework for California Public Schools, the students shouldunderstand that:

• All living things have genetic material in the forms of RNA and/or DNA (pp. 128-130).

• Genetics is the study of heredity, the passing of traits from one generation to the next(pp. 128-130).

• Genetic make-up is received from both parents, and is expressed as traits which can bepredicted (pp. 128-130).

• The genetic code instructs the production of enzymes and other proteins in cells (p. 130).

• Genetic engineering is a biotechnological process where material from one organism isinserted into the genetic code of another organism (p. 136).

• Genetic changes can be achieved through genetic engineering (p. 130).

• Practical and ethical issues are important to consider in the field of genetic engineering(pp. 135-136).

• Manipulative models can be used to enhance the understanding of complex ideas andtechnologies (pp. 153-155).

LANGUAGE ARTS CONCEPTS

The 1988 Language Arts Framework states that students will:

• Read significantly meaningful literature that introduce new vocabulary and concepts (p. 29). • Formulate and share ideas with others in small group work and discussion (p. 12). • Participate in an oral language program that encourages a variety of writing, reading,

speaking and listening activities (p. 4).

SOCIAL STUDIES CONCEPTS

The 1988 California History-Social Studies Framework states that students will:

• Develop an appreciation of the many people who work to supply their daily needs(pp. 37-38).

• Acquire information by listening, observing and using community resources (pp. 40-42).

• Develop group interaction skills, such as a willingness to listen to the differing views ofothers, decision-making, compromising, resolving conflicts and leadership skills (p. 21).


OBJECTIVES

The students should:

• Understand that all genes are composed of the same basic compounds.

• Understand that genetic engineers can bring bits of DNA together from many differentsources.

• Realize the importance of sterile and safety techniques in a laboratory.

• Discover that bacteria and viruses exist in nature and naturally inject pieces of DNA into otherorganisms.

• Understand how bacterial DNA can be transformed, having different genotypes than theyonce had.

• Analyze how genetic engineering has influenced and continues to influence agriculture.

• Realize the complexity of advancing science technologies, including genetic engineering.

• Understand the importance of having a strong science knowledge base.

KEY VOCABULARY

It is assumed that students should be able to define and understand the following words which arediscussed in this unit. Definitions for many of these words are incorporated into the lessons. Apartial list of definitions is also provided in the glossary on pages 86-87.

• Agrobacterium

• Amino Acids

• Antisense Sequence

• Base Pairing

• Biotechnology

• Chimeric Gene

• Chromosomes

• Cleavage Site

• Cotyledon

• Crown Gall

• DNA

• Enzyme

• Gene Splicing

• Gene Therapy

• Genes

• Genetic Engineering

• Kanamycin

• Ligating Enzyme

• mRNA

• Mutation

• Plasmid

• Polygalacturonase

• Promoter

• Protein

• Recombinant DNA

• Restriction Enzyme

• RNA

• Selectable Marker

• Terminator

• Tissue Culture

• Transcription

• Transformation

• Transgenic

• Translation

• Vector


BEFORE YOU BEGIN

1. Review the lessons to gain understanding of the unit. Determine which lessons you will use andwhen you will teach them.

2. Order the appropriate supplies needed for the DNA transformation activities your students willperform. Lesson Four: The Research Starts requires the Bacterial Transformation Kit#166-0003-EDU from Bio-Rad Laboratories, plus other specialized equipment such as ultra-violet lights and incubators. Refer to the lesson on pages 51-54 and the resources on page 83for specific ordering information. Allow four to six weeks for the delivery of materials.

3. Obtain various references on general plant microbiology and genetic engineering. Several aredescribed in the Teacher Resources and References section on pages 83-85.

4. Due to the complexity of the subject matter, many assumptions and generalizations have beenmade so your students can complete the activity without becoming overwhelmed. Several of thefacts and generalizations are discussed below. Share this information with your students as youfeel appropriate.

• The gossypol content in cottonseed is an actual issue in agriculture today. Many attempts havebeen made to resolve this issue—from producing glandless cotton plants that do not containgossypol to detoxifying the gossypol in the cottonseed. Unfortunately, to date, none of themethods have been successful at a commercial level.

• In this unit, references are made to a gossypol “gene.” In actuality, there are numerous genesinvolved in gossypol production. Gossypol production is not a single gene trait.

• Gossypol is actually a secondary metabolite (sesquiterpene), a chemical compound that deterschewing insects from eating the cotton plant. Gossypol is not a protein.

• “Gossypol synthase” is the name of the gene which encodes for gossypol synthase enzymeproduction. This enzyme causes gossypol formation.

Enzyme

Precursor Gossypol (Gossypol Synthase)

• The main objectives of this hypothetical research scenario are described in the flow chart below.

Understand the agricultural issues regarding gossypol.

Brainstorm ways to resolve the gossypol challenge.

Decide on a genetic engineering research approach.

Perform numerous activities to insert the hypothetical gossypol gene into a bacterial plasmid.

Understand how the gossypol gene can be inserted into a cotton plant using bacteria and tissue culture.

Present a continued research proposal to find a regulator gene that will allow gossypol to exist in thevegetable parts of the plant, but not in the cottonseed.


TEACHER BACKGROUND INFORMATION ONGENETIC ENGINEERING

While the sciences of genetics, genetic engineering and biotechnology are complicated, there aremany components that can easily be incorporated into the classroom. The following information canhelp you better understand the subject matter and relay this information to your students.

What is biotechnology?

Biotechnology is a number of technologies which use biological organisms to produce usefulproducts, processes and services. Production can be carried out by intact organisms, such as yeastor bacteria, or by natural substances, such as enzymes, from organisms. The use of yeast in bread-making is a form of biotechnology. The use of bacteria and molds in cheese-making is anotherexample of simple biotechnology. In the 1970s, a new type of biotechnology began—geneticengineering or recombinant DNA technology.

What is genetic engineering?

Genetic engineering is a process where genetic material (DNA) is taken from one organism andinserted into the cells of another organism. Genetic engineering also can be the rearrangement ofgene location or the removal of genes. The “altered” organism then makes new substances orperforms new functions based on its new DNA. For example, the protein insulin, used in thetreatment of diabetes, now can be produced in large quantities by genetically engineered bacteriaand yeasts. Insulin was formerly extracted from pigs or cows. Some say the genetic engineering ofplants can make food more nutritious and plentiful, helping to feed the ever-rising world population.

What can genetic engineering do?

It can improve the ability of an organism to do something it already does. For example, anadjustment in the amino acid balance in a particular corn variety improves the corn’s ability to bestored.

It can suppress, or stop, an organism from doing something it already does. For example, the genethat codes for the softening of tomatoes is “turned off” in a genetically engineered tomato variety sothe tomatoes do not soften as quickly.

It can make an organism do something new. For example, particular bacteria and yeasts have beengenetically engineered to produce chymosin, an enzyme used in cheese production.

What is a gene?

Genes are sequences of DNA which serve as blueprints for the production of proteins in all livingthings. DNA is found in all cells, usually in the nuclei. In bacteria and viruses, which do not havenuclei, the DNA floats within the cell. DNA is composed of six molecules: sugars, phosphates andfour bases. A gene produces a specific protein or has an assigned function.


What is a protein?

Proteins are chains of amino acids that perform the necessary functions of living organisms. Whena gene is “expressed,” that means it is transcribed into mRNA which is used as a template fortranslation into a protein. Some of these proteins perform specific functions themselves (such asbecoming insulin or muscle); others participate in the production of cell components (such asbecoming enzyme proteins that assist in making carbohydrates and fats); still others are regulatoryand modify gene expression.

What are some examples of genetically engineered products?

• Human growth hormone, normally produced in the human pituitary gland, can be made inbacteria to give to people who lack this hormone.

• Rabies vaccine can protect against the rabies virus.

• Oil-eating bacteria can clean up oil and gasoline spills efficiently.

• Healthier edible oils can be produced by genetically altered canola plants.

• Tomato plants can be altered to delay the onset of softening and rotting of fruit.

• Herbicide-resistant cotton can withstand the effects of sprays so weeds can be eradicated withoutharming the crop.

• Viral-resistant fruits and vegetables can resist viruses.

• Cheeses can be made using bacterial-produced rennet (an enzyme formerly taken from calves’stomachs).

• Insecticidal proteins produced internally by plants can reduce the need for chemical pesticides.

How do we know that genetically engineered plant foods are safe?

Advanced technology, as well as standards and regulations set by food producers and governmentalagencies, have allowed the United States to maintain its safe food record. In fact, the United Stateshas the safest food supply in the world. The following information will help you better understand thegenetic engineering food safety guidelines.

Before any plant food developed through biotechnology is made available to the public, it undergoesa safety evaluation. In 1992, the Food and Drug Administration (FDA) issued testing guidelines forgenetically engineered foods. The specific policies are under the title “Foods Derived From NewPlant Varieties.” There are other policies for products other than plants. The genetically engineeredplant food product guidelines are summarized as follows:

• Genetically modified plant foods shall be regulated exactly as traditionally produced foods.

• The food products will be judged on their individual safety, allergenicity, toxicity, etc., rather thanon the methods used to produce them.

• Any new food additive produced via biotechnology will be evaluated for safety employing thesame guidelines used for a traditional food additive (such as food coloring).


• Any food product that is found to contain material that could render it unsafe will not be allowed toenter commerce.

• If the introduced product contains an allergen, or if the production of the food has altered itsnutritional value, the FDA may require informational labels.

As in the case for any food product, any genetically engineered plant food found to containsubstances not in keeping with the safety guidelines may be removed from the marketplace by theFDA. The United Nation’s World Health Organization continues to debate the policies revolvingaround genetically engineered food products.

How do we know if genetically-engineered plants are safe for the environment?

To insure that genetically engineered crop plants are safe, the United States Department ofAgriculture (USDA) oversees all field testing of genetically engineered products. Before a new cropcan move into commercial production, the USDA reviews the field-testing results. Field-testingresults and studies must demonstrate that plants altered using biotechnology react with ecosystemsin the same ways as do their traditionally produced plant counterparts.

What are some risks associated with genetic engineering?

As with any new technology, risks must be considered. Some criticism of genetic engineeringpractices include the possibility that modifications in the genetic make-up of the plant could result insome type of unknown toxin. The odds of that occurring in normal plant breeding and selection arefar greater than that occurring in genetic engineering. Genetic engineering involves only themovement of specific genes with specific functions. In traditional plant breeding, crosses betweendifferent varieties and wild relatives result in the transfer of many genes. The science of geneticengineering is carefully monitored and the risks associated with any products and processes, suchas allergens and ecological impacts, are constantly addressed.

How can genetic engineering affect agriculture?

With increasing food needs around the world and the loss of farmland to urbanization, farmers mustconstantly find ways to increase yields and lower production costs. As farmers continue to look forrenewable resources and safe ways to control pests and fertilize plants, genetic engineers continuetheir research to assist agriculture.

• Pest-resistant plants are being developed through genetic engineering. For example,mungbeans, a staple in Asia, can now be commercially grown without the use of pesticides.Strawberries have also been genetically engineered to be resistant to root pests.

• Herbicide-tolerant cotton has been developed through genetic engineering. The herbicide

bromoxynil is broken down by the cotton plant. This allows the cotton field to be sprayed withbromoxynil to kill weeds without affecting the cotton plant itself. This method of weed controlgreatly reduces the amount of herbicide used on cotton while increasing the yield of cotton peracre.

• Genetic engineering is helping farmers diversify their crops. For example, ethanol produced from

starches genetically added to potatoes can be used as a fuel, and genetically engineered plantoils in canola and soybean plants can be used to produce biodegradable plastics.


What are the basic procedures for producing a genetically engineered plant product?

The actual procedures for producing a genetically engineered product are very complex. However,most genetically engineered plant products are produced using the basic steps described below:

a) TRAIT IDENTIFICATION: Traits of organisms are identified.

b) GENE DISCOVERY: Genes for the desired traits are identified.

c) GENE CLONING: The desired gene is inserted into a bacterial cell and, as bacteria reproduce,the desired gene is also reproduced.

d) GENE VERIFICATION: Researchers study the copies of the gene using molecular techniques toverify that the replicated gene is precisely what is wanted.

e) GENE IMPLANTATION: Using a bacterium or other procedure, the desired DNA (gene) istransferred into the chromosomes of the host plant cells.

f) CELL REGENERATION: Researchers select the plant cells that contain the new gene andregenerate whole plants from the selected plant cells.

g) THE NEW PLANT TESTING: Laboratory and field testing occur to verify the function and safetyof the new plants.

h) SEED PRODUCTION: Seeds with the desired traits are produced using standards set forspecific crop production.


LESSON 1: WHAT CAN WE DO?(An Introduction to Biotechnology)

PURPOSE

The purpose of this activity is for students to realize that biotechnology, specifically geneticengineering, is a tool that can be used to meet challenges that exist in agriculture.

CONCEPTS

• Biotechnology is the development of products using a biological process.

• Biotechnology is a science that affects every individual’s life.

• Many career opportunities exist in the field of biotechnology.

• One specialized application of biotechnology is genetic engineering.

• Many current challenges exist in the agricultural industry.

• Practical and ethical issues are important to consider in the field of genetic engineering.

• Agriculturalists, educators and industry representatives can work together to meet many of thechallenges that exist in our society today.

MATERIALS

For the class:

• Reference books on cotton, biotechnology and other pertinent topics (see pp. 83-85)

• Masking tape

For each group of two to four students:

• Here’s An Idea task list (p. 19)

• Butcher paper

• Markers

For each student:

• What Do You Think? memo (pp. 17-18)


TIME

Teacher preparation ..................................................... 10 minutesClass activity ................................................................ One or two 50-minute sessionsHomework .................................................................... Two nights

BACKGROUND INFORMATION

BIOTECHNOLOGY: If you split up the word, it is easy to understand. “Bio” stands for biology, livingthings. “Tech” stands for technology, the tools and techniques used to study things. “Ology” meansthe study of. Biotechnology, in simple terms, means the study of living things using various tools andtechniques. Here, “biotechnology” is the study of how technology is used to impact the function ofliving things. Or rather, biotechnology is the development of products using technology in biologicalprocesses.

This lesson presents facts about cotton and current agricultural challenges associated with it. In thishypothetical yet realistic scenario, students learn about one real challenge that faces the cottonindustry—the high gossypol content in cottonseed. Using this example, the students work asscientists to confront this challenge through research and development.

Gossypol is a naturally occurring chemical found in the seeds and plants of cotton. Located in thepigment glands of cotton plants and cottonseed, it deters chewing insects and other animals fromeating the plants. The presence of gossypol in cultivated cotton has been an issue in the cottonindustry for over 40 years.

Cottonseed is used as a food source for both cattle and other animals. Mature cattle can eat smallamounts of cottonseed as part of their diet and be unaffected by the presence of gossypol. Theproblem with the presence of gossypol in cottonseed, however, is that it is toxic to animals in highquantities. For this reason, only limited quantities of cottonseed are permitted in cattle and chickenfeed and it is not a widespread ingredient in human food. The processing methods used to detoxifythe gossypol in the seed meal have been unacceptable because they decrease the nutritional valueof the meal and increase the cost of production.

One of the objectives of this unit is for students to realize the potential benefits and challenges ofproducing cotton plants that contain gossypol in the leaves and stems, but not in their seeds.

The issue of gossypol content in various parts of the cultivated cotton plants is only one challenge ofthousands that are being researched today. This topic of study was chosen for many reasons, someof which include its interesting history and current research possibilities. The issues addressed inthis unit are being addressed in the agriculture research community. To make this unitunderstandable by high school students, however, many details have been left out of the researchprocess.


PROCEDURE

1. Discuss the idea of this unit—to examine issues associated with agriculture and to investigate andpropose resolutions to one or more agricultural challenges.

2. Divide the students into groups of two, three or four.

3. Distribute the What Do You Think? memo (pp. 17-18) written to Caitlin Noonan from Tom Davis.

4. Have the students read and discuss the memo and then complete the Here’s An Idea task list(p. 19), including an oral presentation of their ideas. Some possible ideas for the brainstorm listare:

• Find wild strains of cotton that have gossypol in the vegetative parts of the plant, but not in theseed. Selectively breed those plants.

• Find an economical way to detoxify the gossypol in the cottonseed through some type ofprocessing.

• Find a way to make gossypol-free cotton plants resist chewing pests.

• Genetically engineer a cotton plant to have gossypol in the plant, but not in the seeds.

• Investigate the effects of heat on gossypol stability. Use this information in the processing ofcottonseed to produce human food and silage.

5. As homework, have the students complete a response letter to Mr. Davis. In small groups, havethe student share their response letters with one another.

CONCLUSION

There are many issues associated with agriculture that affect people and the environment. Thereare also endless possibilities on how major challenges can be resolved through research andinvestigation. The issue of gossypol content in cottonseed is one such issue.

EXTENSIONS

• Have the students find out about other genetic engineering research projects currently under wayby contacting biotechnology companies such as Calgene, Inc., Cotton, Inc., Genentech,Monsanto Company and the University of California Cooperative Extension.

• Throughout this unit, have the students learn more about the agricultural production of cotton.

• Invite a cotton grower or cotton ginning provider to your classroom to discuss the growing,harvesting and ginning of cotton.

• Have your students use the Internet to learn more about the agriculture of cotton and/or issuesassociated with genetic engineering.


M E M OTO: Caitlin Noonan

Research DirectorAgri-Gene, Inc.

FROM: Tom DavisSenior Research InvestigatorCotton Research Associates

WHAT DO YOU THINK?I recently attended the 20th Annual United States Department of Agriculture Symposium at theUniversity of California at Berkeley. It featured a lecture on specific issues in Californiaagriculture. One of the topics discussed was the gossypol content in cottonseed and cottonplants and the issues associated with this naturally occurring chemical.

Cotton is an important California commodity and is currently the leading commodity in Fresnoand Kings counties. It is believed that the first cultivation of cotton was in India. The AmericanIndians grew cotton in the early 1500s with true American cultivation beginning in 1621 whenthe English settlers were provided cottonseed from the West Indies. The history of cottoncultivation and processing techniques is fascinating and I will be happy to talk with you aboutthis further, if you so desire.

The purpose of this memo, however, is to discuss a possible research partnership in the areaof gossypol reduction in cottonseed. The presence of gossypol in cultivated cotton has beenan issue in the industry for more than 40 years. Here are some facts we already know aboutgossypol:

• Gossypol is a naturally occurring chemical found in the seeds and vegetative materialof cotton. This chemical exists in the pigment glands of cotton plants and in thecottonseed. It deters chewing insects, such as caterpillars and other animals, fromeating the plants.

• Gossypol is a secondary metabolite produced from interactions between certain

precursors and an enzyme called gossypol synthase. • Cottonseed is a food source for cattle and chickens. They can eat limited amounts of

cottonseed as part of their diet and be unaffected by the presence of gossypol in thefeed.

• Humans most often consume cottonseed in the form of oil. Many manufactured goods,

including cookies and crackers, list cottonseed oil as an ingredient.

• Cottonseed can be used as a filler in many foods. Since cottonseed is relativelytasteless, flavorings can be added to the seeds to produce items such as chocolateflavored chips and imitation nuts.


• It is the industry’s goal to increase the saleability of cottonseed so this by-product fromcotton fiber production can be utilized more efficiently and effectively; therefore, it canbenefit the cotton growers financially and potentially be a currently untapped foodsource for millions of people each year.

• The problem with the presence of gossypol in cottonseed is that it is toxic in higher

quantities. For this reason, only limited amounts of cottonseed are included in humanfoods and in cattle and chicken feed. Extensive studies have not been done ontolerance levels of gossypol; however, evidence shows that humans and cattleexposed to high levels of gossypol from cottonseed can sustain red blood cell scarring,liver damage and experience other medical problems.

• Gossypol has some positive effects in animals. It is an anti-viral chemical, which

means it reduces the propagation of some viruses, even in humans. Researchindicates that it may also suppress the spread of cancer cells, such as those whichresult in leukemia.

• As stated previously, gossypol is a natural pest deterrent. Plants containing high

quantities of gossypol tend to resist chewing insect pests. Those with low levels ofgossypol are not as successful in the field.

Here is our challenge. Cotton Research Associates would like to develop a cultivated cottonplant that has high amounts of gossypol in its vegetative material, but little or no gossypol in itsseeds. To date, we have not been successful in achieving this goal, but are interested inproviding the funding for research to improve cotton production and the stability of thecottonseed market.

I have been asked by your Board of Directors to investigate the feasibility of this task. I needto gather data to present at its next meeting. At that time, the board will determine if fundingwill be provided for such a project. I request your input on how to reduce or eliminate gossypolin cottonseed. Please include your ideas on the following:

—a definition of the work that you think should/could be done.

—a brief explanation of how this work could be accomplished.

—a list of pros and cons concerning each method you suggest.

I appreciate the time you are giving to this potential joint venture. I respect your opinions andyour continued support of scientific research in agricultural advances and look forward tohearing from you soon.


HERE’S AN IDEA Name(s)

TASK LIST

1) Brainstorm at least four potential ways gossypol levels incottonseed can be reduced or gossypol could become less toxic tolivestock and humans. During this brainstorming, you may useyour textbook or other references to help you obtain ideas.

Note: Think of scientific procedures and technologies used today that have resultedin healthier, more flavorful or more desirable foods. Examples include technologiesused to develop leaner beef, low-fat cheeses, sweeter corn, tastier tomatoes andseedless grapes.

2) Decide on two methods you think might be worth pursuing in regardto the gossypol issue. For each method, provide a brief descriptionon how this could be achieved and the pros and cons for eachmethod.

3) On a large piece of butcher paper, prepare a visual aid that willhelp express your ideas. Prepare a three-minute explanation thatdescribes your two favorite ideas.

4) Present your ideas to the class.

5) Listen to ideas presented by other groups.

6) HOMEWORK—Assuming the role of Caitlin Noonan fromAgri-Gene, Inc., write a reply to Mr. Davis discussing the variousideas presented by your class (research team). Be sure to discusswhich method you suggest Cotton Research Associates pursue.The potential pros and cons of your recommendation should alsobe addressed.


COTTON PLANT


LESSON 2: THE MEETING(The Decision for a Genetic Engineering Approach is Made)

PURPOSE

The purpose of this activity is for students to learn how some research decisions are made and togain background information and begin preparatory work regarding the genetic engineering activitythey will perform.

CONCEPTS

• Genetic engineering is one method used to alterthe genetic make-up of an organism.

• The genotype and the phenotype of an organismcan be altered through genetic engineering.

MATERIALS

For each group of four to six students:

• The Meeting Task Sheet (pp. 32-33)

• Access to a computer for word processing and newsletter layout

• Sample public relations newsletters from a variety of companies

For each student:

• Letter from Tom Davis (p. 25)

• The Gossypol Meeting Transcription (pp. 26-31)

PRIOR KNOWLEDGE REQUIRED BY THE STUDENTS

Since there are quite a few details mentioned in the meeting notes used in this activity, it is stronglysuggested that your students have an understanding of the following concepts and vocabulary priorto this lesson:

• the terms genotype and phenotype, and how the genotype of an organism affects its phenotype;

• how DNA codes for protein;


• transcription and translation, and how they are associated with cell replication and proteinsynthesis;

• the structures of large chromosomes and plasmids in prokaryotes; and

• the term gossypol and why it is a challenge in the cotton industry.

TIME

Teacher preparation .........................................20 minutesStudent activity .................................................Three to four 50-minute class periods, plus homework


The meeting notes are incorporated into this lesson for two reasons. One reason is to continue thescenario with the students so the upcoming lessons have more meaning. Secondly, the meetingminutes inform students, in a unique way, much of the background information needed to completethe upcoming bacterial transformation activity. Based on the prior knowledge of your students, youmay need to explain some of the terminology and concepts. Provide the necessary supportingactivities so your students have comprehended the listed terms by the conclusion of this lesson.

• amino acid

• antisense

• chimera

• complimentarybase pairs

• gene

• genome

• mRNA

• plasmid DNA

• promoter sequence

• protein

• selectable marker

• sense

• terminationsequence

• transcription

• translation

• vector

PROCEDURE

1. Discuss the previous activity, if necessary. Then, have each student individually read the letterfrom Tom Davis (p. 25) and the Gossypol Meeting Transcription (pp. 26-31).

2. Have a brief class discussion about the letter and transcription clarifying any facts that areunclear. If appropriate, make an overhead transparency of page 28 for your discussion.

3. Distribute The Meeting Task Sheet (pp. 32-33) to each student group of four to six students.Review the lesson, which requires the students to do research and develop a company publicrelations newsletter.

4. Allow sufficient time in class for students to develop draft ideas of the newsletter. Also, allowenough time for the students to proof each other’s work before turning in the final newsletter.

5. Upon completion, have the students share their newsletters with fellow classmates.


6. Individually, have the students write a one-page summary describing the most interestingconcepts or facts they have learned. Clarify any misconceptions that have surfaced or planappropriate questions or activities in upcoming lessons that will challenge the misconceptions.

CONCLUSION

Genetic engineering has been used to alter the genetic make-up of many agricultural products.There are many challenges associated with the actual processes of genetic engineering as well asethical issues that must be considered when developing genetically engineered products.

VARIATIONS

• Coordinate time at your school computer lab to create the newsletter on the computer.

• Rather than a newsletter, have the students develop an editorial for a television show thatdiscusses the information. Use video equipment, if available.

• Assign particular students the roles of the people in the meeting minutes. Have them orallypresent the information while the rest of the students listen.

• Turn the meeting transcription into a skit with appropriate characters and props.

EXTENSIONS

• Invite a research scientist to your classroom to discuss how research ideas are formed and howfunding sources for those ideas are obtained.

• Working with the English, social studies, journalism, mathematics and computer teachers at yourschool, expand this lesson into an extensive newsletter which may include editorials, polls,classified ads for appropriate jobs and statistical graphs.

• After completing this lesson, have the students discuss the profit or loss potential of such aproject.


TOM DAVISSenior Research Investigator

COTTON RESEARCH ASSOCIATES123 Leaf Way

Cottonseed, Missouri 54321

September 1, 1999

Caitlin NoonanResearch DirectorAgri-Gene, Inc.Cottontown, California 12345

Dear Caitlin,

Thank you for your suggestion to have an informational meeting about how toreduce the gossypol content in cottonseed. I have included the minutes from onemeeting we had on this topic. The group of people attending the meeting were veryinformative and enlightened me as to the processes our company will be involved in aswe pursue this new venture.

As you will see from the transcription, the Board of Directors decided that agenetic engineering approach to the gossypol issue may best serve our purposes.Thank you again, Caitlin, for your support on our project. I will be in touch with you asthings progress.

Sincerely,

Tom DavisSenior Research Investigator

Enclosures

cc: Alexandra HoeppnerGordon Spicer


GOSSYPOL MEETING TRANSCRIPTION8/20/99

Sacramento, California

Meeting Attendees:

Tom Davis, Senior Research Investigator, Cotton Research AssociatesAlexandra Hoeppner, Research Geneticist, Calgene, Inc.Gordon Spicer, Agronomist, University of California, DavisAssistants to Tom Davis, Cotton Research Associates

Tom: Good morning. Thank you for attending our meeting. Our task today is toinvestigate the feasibility of genetically engineering cotton plants tocontain cottonseed with little or no gossypol. There are several questionswe would like to have answered during this meeting.

• We are aware that Calgene, Inc. created the Flavr Savr® tomato seeds.Why and how was this tomato developed? Can a similar protocol beused for our project?

• I have heard the term “chimeric” used in relation to genetic engineering.What does this mean and will we be using a chimeric gene?

• I understand there is a direct relationship between the DNA in a gene andprotein production. Please explain this relationship to me in regards togossypol and cotton.

I would like to turn this forum over to Alexandra Hoeppner, who willdiscuss how the work at Calgene, Inc. on the Flavr-Savr® tomato seedswas accomplished. The company performed research associated withthis tomato for ten years prior to its development. The geneticallyengineered tomatoes are presently sold in selected stores throughout thenation under the name MacGregor’s® tomatoes.

Alexandra: Calgene developed the MacGregor’s® tomato in response to the tastelesstomatoes usually available in the grocery stores during the winter months.Normally, tomatoes consumed in the winter and spring months are pickedgreen so they can be stored and shipped without becoming too soft. Thetomatoes redden during shipping or in warehouses after being exposed toethylene gas, a gas naturally released by ripening fruit. The softening ofa tomato is associated with a single sense gene that causes thedevelopment of a protein called “polygalacturonase” (PG). At Calgene,we figured out how to place the backwards version of the PG gene, calledthe “antisense PG” gene, into the tomato genome. The backwardsversion is combined with the “sense PG” gene so replication andexpression of the softening PG gene cannot occur. Thus, the level of thePG enzyme is reduced and the softening of the tomato is slowed. As a


result, the tomatoes can be shipped vine ripened and have a moreflavorful taste.

Tom: How can you get the PG antisense strand to be expressed?

Alexandra: In any organism, whether it is prokaryotic or eukaryotic, transcription ofDNA to form mRNA must occur. In order for transcription to occur, anenzyme RNA polymerase must bind to a specific recognition site on thegene. This recognition site is called the promoter region. To reduce PGenzyme production, the PG gene is removed using restriction enzymesthat cut DNA; then, the inverted gene, ASPG, is inserted next to thepromoter. As a result, the ASPG gene instead of the PG gene istranscribed and translated. Thus, the polygalacturonase needed for fruitsoftening is not synthesized.

Tom: How do you get the PG gene out of the genome and the ASPG gene intothe genome? It seems difficult and complex.

Alexandra: Developing the process was complex, but the process itself incorporatesthe natural actions of plants. To accomplish this task, we made use of acommon reaction that occurs when a plant is injured. Frequently, when aplant gets a cut or another type of wound, it may get infected by a soil-borne bacterium called Agrobacterium. When the wound is infected bythis bacterium, some of the bacterium’s DNA is transferred into the plantand a tumor is formed. This particular tumor is called a Crown GallTumor. We used this knowledge to our benefit.

We also made use of a natural feature of many bacteria. Many bacteriacontain genetic material called plasmids. Agrobacterium, like otherbacteria such as E. coli, contain two types of genetic material, one largerchromosome and numerous smaller circular plasmids. Plasmids arerelatively easy to remove from bacterial cells. Using specializedenzymes, called restriction enzymes, the PG gene is removed from thetomato genome, flipped backwards and then placed into a plasmid. Theinsertion of a gene is done with ligating enzymes. The backwards versionof this gene is called the antisense PG gene or the ASPG gene. Theplasmids containing the ASPG gene are put into living Agrobacteria.Then, when a wound is purposely made on a tomato leaf, theAgrobacteria containing the ASPG gene goes into the wound site andcreates a callus—a scar—and transfers the ASPG gene into the plant.From this callus, tissue culture techniques are used to grow tomato plantsfrom a single cell of the callus. Hence, the tomato plants grown from thecallus have the new feature of the reduction of the non-softening PGenzyme because it binds with the ASPG gene that entered the cellthrough the plasmid. It’s a complex process, but is relatively easy nowthat we have the method down. (As Alexandra speaks, she is writing thisschematic diagram on the board.)



Tom: It seems that this new tomato plant genome is composed of DNA fromseveral different sources—the original plant and Agrobacterium.

Alexandra: Yes, it is. This is called a “chimera.” Chimera means “from severalsources.” In this case, the genome is from the original tomato plant aswell as the Agrobacterium, which contains the antisense PG gene clonedfrom a different tomato plant.

Tom: I heard that you grew the Agrobacterium in a solution that contained theantibiotic Kanamycin. Why was this done and what effects will thisantibiotic have on the person who eats the tomato? Can they becomeresistant to this antibiotic by eating your tomatoes?

Alexandra: It is common practice to use what is called a selectable marker whentransferring genetic material. Let me explain. . . When the plasmid ismade, it contains the promoter sequence, the ASPG gene, the selectablemarker and a terminating sequence—a sequence on the DNA which stopsthe formation of mRNA. When the mRNA is made during transcription,both the ASPG gene and the selectable marker gene are transcribed atthe same time. The selectable marker for our tomatoes is the resistanceto the antibiotic Kanamycin. This antibiotic resistance serves severalpurposes. It serves as a control so only the bacteria we want will survive.You see, after the plasmid is placed back into the bacterium, thebacterium is grown on a special medium plate which contains theantibiotic. It will kill all of the bacteria except those that have beentransformed—those that have the softening gene removed and theKanamycin resistance gene. Secondly, it is easy to see antibioticresistance in bacteria—the bacteria either grow or don’t. It is much moretime-consuming to see if the softening occurs or doesn’t occur.Therefore, when a new gene is inserted, a gene for antibiotic resistance isalso included. Sometimes two selectable markers are used.

CHIMERIC GENE CONSTRUCT DIAGRAM

And . . . to answer your questions about humans becoming resistant tothe antibiotic during the process—this is, frankly, not possible. Whenpeople eat the tomatoes, they digest the DNA as well. The DNA is notbeing inserted into the human cells. The antibiotic resistance is part ofthe plant genome and is only recognized by plants.

Tom: What about the question of feasibility concerning the use of thisprocedure for reducing the gossypol content in cottonseed? Gordon, canyou address this?

Gordon: I believe that it is theoretically possible to reduce the gossypol incottonseed without reducing the amount of gossypol in the adult plant.

PromoterSequence

TerminatorSequence

SelectableMarker

Geneof Interest


This question intrigued me and I asked one of my graduate students tobegin researching this question. She found out that it is possible toproduce a gossypol-free plant. This has been done by breeding cottonplants that are homozygous recessive for glandless cotton. These plantswill produce only plants that do not make gossypol. They are not growncommercially at present because they do not resist chewing insects;therefore, they are not as productive and may require more use ofchemical pesticides. These homozygous recessive plants, however, willsuit our research purposes since they produce no gossypol in theirgenomes. I would suggest that we start by determining if a glandlessplant can make gossypol when a genetically engineered plasmidcontaining the gossypol gene is inserted.

If this works, this is half our battle. We still need to somehow transformthe plant so that gossypol is made in the adult plant only, not in the seed.During our research, we found some reference to wild strains of cottonthat have a gene that regulates the production of gossypol synthase sothat gossypol is made only in an adult plant, but not the embryonic part orseed. If we can isolate this gene, we can create glandless plants that canbe infected with a transformed bacterium that contains both a gossypolgene and a regulator gene. This way gossypol will be made only in theadult plant. This is where the real research and funding will be needed.(Gordon draws this schematic on the board as he speaks.)

Alexandra: Do you foresee using the “antisense” method of reducing the gossypolcontent or will you try to just control gossypol production?

Produce glandless gossypol plantsfrom wild strains

Isolate gossypol gene andgossypol regulator genes from

cotton plants

Insert gossypol and gossypolregulator genes into

glandless cotton

Use tissue culture techniques toproduce cotton plants whichhave gossypol in the plants,

but not in the seeds.


Gordon: Good question . . . At this point, I think it would be best to insert thegossypol gene and then regulate it with a promoter sequence rather thanincorporate the antisense–mirror image of the gene. Another thing, reallyquick if I may . . . we are calling this gene the “gossypol gene” forsimplicity. In actuality, it is a group of genes that produce an enzymecalled gossypol synthase. This gossypol synthase is the protein thatcauses gossypol production. We’re after the genes that create thisenzyme. But for ease, let’s continue to call the group of genes neededthe “gossypol gene.”

Tom: Do you have suggestions about how we can do some short transformationexperiments to see what might happen?

Gordon: Well, in fact, I do! Not only do I have a graduate student who has isolatedthis gossypol gene, we think we have a selectable marker that can beused as well. Last summer, a high school student, Jorge Villalobos, tooka five-week summer research internship with us. Jorge not only mappedthe gene adjacent to the gossypol gene, but in a serendipitous event,determined that this adjacent gene becomes luminescent when grown in amedium that contains a special sugar called “arabinose.” It would makean easy second marker gene for you. You could immediately know iftransformation has occurred correctly just by looking at the bacteria grownin the media containing arabinose. Since it glows beautifully under a UVlight, we named this plasmid the PGLO™ plasmid.

The research that you are proposing will be quite involved. I propose thatwe try to attach the gossypol gene to the glow marker, put these genesinto a plasmid and then insert the plasmid into E. coli or Agrobacterium. Ifthis is possible, we should be able to use tissue culture techniques togrow a glandless cotton plant that produces gossypol. This is step one.Step two will be to find a regulator gene that controls gossypol production.Step three will be to insert the regulator gene into this plasmid that we justcreated. This will take quite a bit of research and will require a majorfunding source.

Tom: That sounds great! It gives us a great place to start. I would like topublish an account of all of this information in our PR newsletter. This willkeep the farmers abreast of what is happening in research and will informour staff and stockholders of potential products. Hmmm. . . I wonder whatenvironmental impact studies we will have to do as this progresses! Let’skeep this in mind.

Thank you everyone, for participating in this meeting. There has been anextensive amount of information presented here. My committee and I willassimilate it and ask further questions as they arise.


THE MEETING TASK SHEET Names

Assume the role of the communications department for Cotton Research Associates.You and your colleagues, under Tom Davis’ supervision, have been asked to createone of the monthly public relations newsletters for company members and the public.The goal of this newsletter is to highlight one aspect of research Cotton ResearchAssociates is involved in and to be forthright in discussing issues and challenges thatthis particular research project addresses.

Your newsletter should have a minimum of six articles, plus any other information youwould like to add (pictures, upcoming events open to the public, etc.). Determine howthe newsletter tasks will be divided among your group members. Each member of yourgroup is responsible for the following:

• gathering background information for all articles;

• writing one article;

• proofing all articles for technical accuracy, grammar and spelling;

• participating in the layout, design and typing or illustrating of the newsletter.

POSSIBLE NEWS ARTICLE TOPICS

• What is going to be Cotton Research Associates’ new genetic engineeringventure in relation to the gossypol content in cottonseed?

• Why is the gossypol content in cottonseed an issue in agriculture?

• What is a transformed bacterial cell?

• What is MacGregor’s® tomato and how was it developed? How is thisknowledge going to be applied to the cotton industry?

• What considerations need to be taken into account when placing a gene into aplasmid?

• What are some ethical issues that surround genetic engineering and howshould the public learn about and address these issues?

• Discuss the profit or loss potential of producing genetically engineered cotton.

• What environmental impacts must Cotton Research Associates consider?


The following vocabulary should be included in your articles: Agrobacterium, aminoacid, biotechnology, chimera, chromosome, enzyme, restriction enzyme, gene, geneticengineering, mRNA, plasmid, protein, promoter sequence, selectable marker,terminating sequence, transcription, translation and vector.

_____ 1) Review the meeting minutes and discuss with your colleagues any areasthat confuse you. Assist your colleagues in understanding the key points.

_____ 2) Skim over newsletter samples you have at home and those provided byyour teacher. As a team, discuss format and content ideas you particularlylike or dislike.

_____ 3) Create an overall format for your newsletter, including a title.

_____ 4) Gather the information needed to write the articles and discuss as a groupthe contents of each article.

_____ 5) Write the draft articles individually.

_____ 6) Review the work of your colleagues for technical accuracy, grammar andspelling.

_____ 7) Review the articles to make sure all required vocabulary words areincluded.

_____ 8) Prepare the final version of the newsletter, making sure you follow anyrules and guidelines provided by your instructor regarding size, content andstyle.

_____ 9) Share your newsletter with your classmates.

CREATING THENEWSLETTER


LESSON 3: THE PLASMID(How to Insert a Gene into a Plasmid)

PURPOSE

The purpose of this activity is for students to gain a greater understanding of how DNA from oneorganism can be inserted into DNA of another organism. The focus is on the insertion of thegossypol gene and the PGLO™ and antibiotic resistance selectable markers into a plasmid.

CONCEPTS • A chimeric gene is created by bringing bits of DNA

together from various sources.

• Each chimeric gene has certain elements, including apromoter sequence, a gene of interest, a selectablemarker and a terminator sequence.

• DNA codes for proteins.

• Restriction and ligating enzymes are used to “cut” and“glue” DNA material, respectively.

MATERIALS

For each student:

• Letter to Tom Davis (p. 39)

• Inserting DNA Into a Plasmid news article (p. 40)

For the teacher:

• Inserting DNA Into a Plasmid news article (p. 40)

• DNA Insertion Into a Plasmid answer key (pp. 49-50)

For each partnership:

• Gossypol DNA handout duplicated onto white paper (p. 41)

• Plasmid DNA handout duplicated onto colored paper (p. 42)

• Restriction Enzyme Reference Page (p. 43)

• DNA Insertion Into a Plasmid activity sheet (pp. 44-47)


TIME

Teacher preparation ..................................................... 15 minutesStudent activity ...............................................................One 50-minute session, plus a 15-minute

summary and review


Students should have a basic understanding of the functions of DNA and RNA including theprocesses of transcription and translation. Transcription is the process in which RNA polymerasesynthesizes a messenger RNA chain by reading the code of a DNA sense strand. Translation is theprocess in which the messenger RNA (mRNA) is decoded to produce amino acids. The amino acidslink together in specific sequences directed by the mRNA to form specialized proteins. Theseproteins are then used somewhere in the cell.

A chimeric gene construct generally consists of a promoter sequence, the gene of interest, aselectable marker and a terminator sequence. The promoter sequence indicates the place in whichDNA transcription for protein synthesis should begin. The promoter can also dictate where andwhen the gene of interest is expressed. The gene of interest is the gene that one wishes to transferto another organism. Examples include virus resistance, color or added nutrition. Since it often isdifficult to determine whether a specific gene, such as increased nutrition, is transferred, a selectablemarker is used. A selectable marker is an easily identifiable gene, such as antibiotic resistance orphosphorescense, that is attached to the desired gene so that it is obvious whether or not the genetictransformation has occurred. The terminator sequence is a section of DNA that stops the mRNAtranslation for protein synthesis.

CHIMERIC GENE CONSTRUCT DIAGRAM

Chimeric gene constructs are created by bringing bits of DNA together from various sources. Thesesources may include DNA from bacteria, viruses, plants or animals. A list of genetically engineeredcrops and the source of the gene inserted is located on page 81. Genetic engineers isolate desiredgenes using restriction enzymes. Restriction enzymes identify specific sequences of DNA basesand make a cut at these specific sites on a DNA molecule. Ligating enzymes “glue” bits of DNAstrands together.

By cutting the DNA molecule into small pieces using restriction enzymes, scientists are able todiscover and study genes. There are four restriction enzymes commonly used in geneticengineering. Each type differs in the type of cut it makes. Over 175 different restriction enzymes areknown and are characterized with respect to their cleavage sites and the “sticky ends” that areavailable for attachment. Sticky ends are the single strands of DNA left after a restriction enzymeseparates the base pairs. Some examples are described below. The Bam HI and Sac I restrictionenzymes are used to cut the polygalaturonase genes from tomato genomes. This method is used byCalgene, Inc. to produce the MacGregor’s® tomato—a tomato that resists softening.

PromoterSequence

TerminatorSequence

SelectableMarker

Geneof Interest


Restriction Enzyme Recognition Site Cleavage SiteEcoR I GAATTC G / AATTCHind III AAGCTT A / AGCTTBam HI GGATCC G / GATCCSac I GTCGAC G / TCGAC

/ = cleavage site

This activity shows the students how restriction andligating enzymes work. It is important for the studentsto realize that where a gene inserts itself into anothergenome is dependent on biochemical andenvironmental factors as well as the availability ofsticky ends. Where a chimeric gene constructchooses to attach itself can affect other processes ofthe organism.

A typical gene insertion challenge is that scientistswant a gene to insert itself in a location that will notaffect other plant genes. One such example hashappened in the cotton plant. Researchers were ableto isolate a gene that could increase fiber strength.The problem was that it always inserted itself in themiddle of a gene sequence that caused cotton bollformation. The cotton boll is where the fiber grows.Thus, when the gene for fiber strength was insertedthe mechanism for cotton boll formation wasinterrupted and no cotton bolls would form. Inconclusion, the fiber strength gene could not beexpressed. Since this study, other fiber strengthgenes have been identified and have beensuccessfully inserted into certain cotton varieties.

PROCEDURE

1. Read aloud the memo to Tom Davis from Alexandra Hoeppner (p. 39). Discuss howcommunication and cooperation are very important components of scientific research.

2. Distribute the Inserting DNA Into a Plasmid article (p. 40) to the students. Have them read itindividually and discuss, in small groups, five key points of the article.

3. Have the students individually create a sequencing map which shows the steps that must occurfor DNA insertion into a plasmid. Have them save their ideas and amend them at the conclusionof this activity.

4. Have the students pair up and complete the DNA Insertion Into a Plasmid Paper Model activity(pp. 44-47).

5. Discuss the results and challenges of this activity. Include a discussion of student challenges aswell as actual research challenges.


6. Have the students refer to their pre-activity sequencing maps. With their newly gainedknowledge from this activity, have them amend or rewrite the sequence of events necessary toinsert DNA into a plasmid.

CONCLUSION

Restriction and ligating enzymes are used to cut and paste pieces of DNA into genomes.Restriction and ligating enzymes are specific and affect DNA molecules in specific ways.

VARIATION • Assign the article on page 40 as a homework reading assignment.

EXTENSIONS • In preparation for upcoming activities, have the students practice pouring agar plates, plating

bacteria and using sterile techniques.

• Have student groups research various aspects of cotton production—its life cycle, the ginningprocess, the varieties of cotton, the economic impact of cotton on society, etc.


M E M O

TO: Tom Davis

FROM: Alexandra Hoeppner

RE: Gossypol Gene Insertion into an E. Coli Plasmid

Thank you for including me as a researcher in your project. Enclosed is a readingselection that discusses how DNA is added to a plasmid. I think it explains what we willbe doing. Remember, we will cut out the gossypol gene from a cotton genome andthen put it into E. Coli plasmids. This plasmid example depicts most of the preliminarywork that we will be testing.

The gossypol gene has already been mapped. We now need to determine whichrestriction enzyme we can use to insert the gene into the plasmid genome. We areworking it out on paper prior to trying it in the lab. I hope you can join us in thispreliminary work. By participating in placing the plasmid into the bacterial cell, you willgain understanding of the steps in this process which will help you prepare for yourpresentation to your board.

I have attached an idea that I have used in the past. It shows how DNA is inserted intoa plasmid on the molecular level. I have used this idea during presentations, and itmay be something you can use with your Board of Directors. I hope it helps to clarifyour process.

Attachments

INSERTING DNA INTO A PLASMID(A Genetic Engineering Technique Using Plants and Bacteria)

by Tanisha Bradley

Prokaryotic cells contain one large chromosome.This chromosome contains most of the DNA.Sometimes prokaryotic cells also have several smallcircular pieces of DNA called plasmids. Theseplasmids contain genes which code for proteins thatare beneficial to the survival of the cell.

The first plasmids discovered contained genes forantibiotic resistance. Geneticists believe that plasmidscontain these genes because they neutralize the actionof an antibiotic on the bacterial cell. To counteract theeffects of antibiotics which kill the bacteria, largequantities of the enzyme are required. More copies ofthe antibiotic-resistant gene that produces the enzyme


can be carried on several plasmids than can beincorporated into one large chromosome.

Geneticists take advantage of prokaryotic plasmidsby incorporating the DNA of a desired gene into thegenome of the plasmid. When the plasmid replicates,the desired gene is also replicated. This way theinformation in the gene is passed from one generationto the next as the bacterial cell divides. Moreimportantly, as the DNA in the plasmid is transcribedinto mRNA, the desired DNA from the implanted genealso gets transcribed into mRNA so that translationcan occur. Translation is when the mRNA is decodedto produce amino acids which attach together to makethe desired proteins.

Plasmids have been used in the process of genesplicing in a variety of study areas, includingmedicine and agriculture. To perform the necessary procedure that will place

a piece of DNA into a plasmid, researchers use arestriction enzyme. A restriction enzyme is aspecialized enzyme that cuts the DNA at a site wherethe base pairs are arranged in a specific order. Forexample, the restriction enzyme Bam HI cuts DNAbetween the two Gs in the sequence GGATCC.

G G A T C C C C T A G G You may notice that the DNA is palindromic,

which means the base pairs read the same each way,backward and forward. Since the structure of DNA isthe same in all organisms, the same enzymes can beused in both prokaryotic and eukaryotic cells. When geneticists want to insert a gene into another

organism, they cut out the desired DNA from anorganism using restriction enzymes. Using the samerestriction enzyme, plasmids from a bacterial cell arecut in one spot to open it up. When DNA is cut, orspliced, this leaves the two open ends chemicallyactive. These chemically active ends are called“sticky ends.” Because of DNA’s complimentarybase-pairing rules, a sticky end will readilyrecombine with another piece of DNA withcomplimentary bases in order to chemically bond andonce again become stable. When the new DNA isplaced in with the cut plasmid, ligating enzymes areused to seal the new connection. It is possible forplasmids to recombine with themselves or with othercompatible sticky ends to get a genome arrangementother than the one desired. After the plasmid has been inserted into abacterium, the scientist grows the bacterium on anagar plate to create the colony of bacteria with thenew genotype. A selectable marker is used to identifythe cells that have been transformed in the desiredway.


GOSSYPOL DNA

1 2 3 4

A G G GGGGG C A T G �� CG C A GGGGG T A T G �� CA T T GGGGG A A T G �� CA T C GGGGG G T A G �� CA T G GGGGG C T A G �� CG C C GGGGG G T �� A A �� TG C G GGGGG C T �� A A �� TG C G GGGGG C A �� T A �� TC G G GGGGG C A �� T T �� AA T G GGGGG C A �� T T �� AA T A GGGGG T C �� G T AA T A GGGGG T C �� G T AT A A GGGGG T C �� G C GT A A GGGGG T C �� G G CT A A GGGGG T T �� A C GA T T GGGGG A T �� A C GG C T GGGGG A A �� T A TG C T GGGGG A G �� C A TC G C GGGGG G G �� C A TC G C GGGGG G G �� C G CC G C GGGGG G G �� C G CA GGGGG T T GGGGG A G �� C C GA GGGGG T A GGGGG T A �� T C GA GGGGG T G GGGGG C A �� T T AA GGGGG T G C A �� T T AA GGGGG T G C T �� A T A

Glow Gene Gossypol GeneGGG ��


PLASMID DNA

1 2 3 4

A T A T C ]]]] G A TC G A ]]]] T C ]]]] G A TG C T ]]]] A G ]]]] C A TG C T ]]]] A A ]]]] T C GG C T ]]]] A G C C GT «««« A C ]]]] G A T T AC «««« G C ]]]] G A T G CA «««« T G ]]]] C T A G CA «««« T C ]]]] G C G A TC «««« G A ]]]] T G zzzz C G CG «««« C G ]]]] C G zzzz C G CG C C ]]]] G A zzzz T G CG C T ]]]] A T zzzz A C GG C C ]]]] G A zzzz T T AC G G ]]]] C T zzzz A T AA T A ]]]] T A T A TA T G ]]]] C G C G CG C A ]]]] T C G T AG C T ]]]] A C G T AC G C ]]]] G A T A TC G C ]]]] G A T A TA T C ]]]] G A T A TA T G ]]]] C C G C GG C T ]]]] A C G C GG C A ]]]] T G C G CG C T ]]]] A A T C G


Promoter Sequence Ampicillin GeneTerminator Sequence

RESTRICTION ENZYME REFERENCE PAGE

CCTGG

GGACC

Ava II

CTCGAG

GAGCTC

Sac II

TTCGAA

AAGCTT

Hind III

GGATCC

CCTAGG

Bam HI

GGCC

CCGG

Hpa II

GAATTC

CTTAAG

EcoR I

««« ]]] zzz


DNA INSERTION INTO A PLASMID(Paper Model Student Activity)

I. INTRODUCTION

The purpose of this activity is for you to build a model that will help youunderstand the workings and complexities of restriction and ligatingenzymes as they pertain to inserting a piece of DNA into plasmid DNA.

At the conclusion of this activity, you should be quite familiar with restrictionand ligating enzymes, sticky ends, DNA structure and chimeric genecomponents.

II. MATERIALS


• Plasmid DNA handout (colored paper)

• Gossypol DNA handout (white paper)

• Restriction Enzyme Reference Page

• Tape

• Scissors

III. PROCEDURE

1. Carefully observe the gossypol DNA and the plasmid DNA. Using thekeys, find the areas needed to produce the desired chimeric gene andthen answer Question #1.

2. Observe the available restriction enzymes on the attached page.Remember . . . restriction enzymes cut the DNA. Answer Question #2.


3. Using scissors, cut the plasmid DNA and attach each strip to form onelong strand of DNA. Attach in the order that the lanes are numbered1-2-3-4.

4. Attach the loose end of #4 to the loose end of #1 to form a circularplasmid.

5. Cut and attach the plant DNA in a similar manner as you did theplasmid DNA, but do not attach to form a circle. This represents thepiece of the eukaryotic gossypol DNA that will be placed into theplasmid.

6. From the restriction enzyme page, locate the area on the plasmid whereeach restriction enzyme will cut. Pencil in where the cut would be.Repeat for the plant DNA. Complete the chart under Question #3 andanswer Question #4.

7. Using scissors, cut the plasmid in the same manner that the chosenrestriction enzyme would cut it so that it opens up into a straight line.

8. Cut the plant DNA at the site of the chosen restriction enzyme.

9. Piece together the sticky ends of the plasmid DNA to attach to thesticky end of the plant DNA. All of the pieces should form a circle.

10. Using tape (DNA ligase), attach the plant DNA to the plasmid DNA atthe sticky end sites. You should end up with a larger circular plasmidthan found in #4.

11. Double check to make sure that all of the needed parts of the plasmidDNA are present for the desired protein to be made. Answer Questions#5 through #8 and complete the Conclusion Statement as homework.


IV. QUESTIONS (Write your answers on a separate sheet of paper)

1. Explain what areas of the plasmid DNA and the plant DNA will beneeded to create the desired chimeric gene.

2. List the sticky ends formed by:

a) Hpa II

b) Ava II

c) EcoR I

3. Copy and complete the chart below:

RestrictionEnzyme

Shoulduse?

(State yes or no)

Why? Give your reasons why theenzyme should or should not be used.

Ava II

Sac II

Hpa II

Bam HI

EcoR I

Hind III

4. Which restriction enzyme is the best to use and why?

5. Why was the plasmid DNA taped in a circle while the plant DNA wasnot?

6. If an analogy can be made about the scissors representing therestriction enzyme, what would the tape represent?

7. Does the plasmid you just made contain all of the components neededto make protein synthesis occur? Explain your answer.

8. Many diabetics use human insulin that is made from the bacterial cellE. coli. How can a eukaryotic gene be placed in a prokaryotic cell?


V. CONCLUSION STATEMENT

Write a summary statement for Mr. Davis that he can use as a handout forhis presentation to the Board of Directors.

Special thanks to John Fedors of San Diego, California, for his contributions tothis lesson.


DNA INSERTION INTO A PLASMID(Answer Key)

1. Explain what areas of the plasmid DNA and the plant DNA will be needed to create thedesired chimeric gene.

The glow gene and gossypol gene need to be cut from the plant DNA strand (requiringtwo cuts). The plasmid gene will need to open up after the promoter and before theterminator sequence (requiring one cut). The ampicillin gene on the plasmid DNAmust not be cut.

2. List the sticky ends formed by:

a) Hpa II There will be a two G-C sticky ends.

b) Ava II There will be a one G-T-C sticky end and one C-A-G sticky end.

c) EcoR I There will be a one A-A-T-T sticky end and one T-T-A-A sticky end.

3. Copy and complete the chart below:

RestrictionEnzyme

Should use?(State yes or no)

Why? Give your reasons why the enzyme should orshould not be used.

Ava II No Cuts plasmid DNA, but not plant DNA.

Sac II No Cuts plasmid DNA, but not plant DNA. Cuts in themiddle of the ampicillin gene.

Hpa II Yes Cuts out the glow and gossypol genes in theplant. Opens the plasmid in one spot and doesnot interfere with the promoter sequence,ampicillin gene or terminator sequence.

Bam HI No Cuts plant DNA, but not plasmid DNA. Willinterfere with the glow gene.

EcoR I No Does not cut either plant or plasmid DNA.

Hind III No Does not cut either plant or plasmid DNA.

4. Which restriction enzyme is the best to use and why?

Hpa II. This restriction enzyme cuts the plasmid after the promoter sequence, butbefore the terminator sequence without affecting the ampicillin gene. It also cuts theglow and gossypol genes from the plant DNA so they can be removed as a unit to beplaced into the plasmid.



5. Why was the plasmid DNA taped in a circle while the plant DNA was not?

A plasmid is a circular piece of DNA. The plant DNA is a paper model sequence froma very large model of a cotton chromosome. After the plant DNA has been placed intothe plasmid DNA, the entire sequence will be circular.

6. If an analogy can be made about the scissors representing the restriction enzyme, whatwould the tape represent?

The tape would be the “glue” that fuses the sticky ends of the DNA together after theyfind complimentary base-pair matches. The tape represents the enzyme DNA ligase.

7. Does the plasmid you just made contain all of the components needed to make proteinsynthesis occur? Explain your answer.

Yes. It contains the promoter sequence, the gene of choice, the selectable markersand the terminator sequence. For protein synthesis to begin, the promoter sequencemust be present to start the process. The terminator sequence must be present toend the process.

8. Many diabetics use human insulin that is made from the bacterial cell E. coli. How can aeukaryotic gene be placed in a prokaryotic cell?

After the desired gene has been identified and mapped, the correct restriction enzymeneeds to be identified so that the placement of this sequence can be cut from theeukaryotic cell and placed into the plasmid. Because prokaryotic and eukaryotic DNAare the same chemically, the same restriction enzyme is used to cut both strands ofDNA. The gene of choice needs to be cut in two places so that it can be removed fromthe DNA strand. The plasmid needs to be cut in one spot so insertion can occur. Itshould be cut after the promoter sequence and before the terminator sequence sothat the placement of the gene of choice allows for successful protein synthesis.


LESSON 4: THE RESEARCH STARTS:TRANSFORMING DNA

(A Bacterial Transformation Activity)

PURPOSE

The purpose of this laboratory activity is for students to have firsthand experience transferring DNAfrom a plasmid into a bacterial cell. Specialized laboratory techniques will be taught and practiced.

CONCEPTS • The genome of an organism can be changed by adding or removing DNA.

• Genetic transformation involves the insertion of a gene into an organism in order to change theorganism’s traits.

• Genetic transformation is used in many areas of biotechnology, including agriculture andmedicine.

• Selectable markers are used so successful DNA transformation can easily be identified.

• Examples of selectable markers include antibiotic resistance and bioluminescence.

• Specialized laboratory techniques, such as heat shock, sterile techniques, bacterial platestreaking and quantitative measuring, are important components of biotechnology.

MATERIALS

For the teacher and class:

• Biotechnology Explorer-Bacterial Transformation Kit #166-0003-EDU from Bio-Rad Laboratories(see page 83 for ordering information)

• Microwave oven

• 37° C incubator

• Temperature-controlled water bath

• Celsius thermometer

• 1-liter flask

• 250 ml flask

• 500 ml graduated cylinder

• Distilled water

• Crushed ice


• 10% bleach solution in squirt bottles

• Mini centrifuge

For each team:

Supplied With Kit Not Included in Kit

• 2 microtubes: one for (-DNA) and one for (+DNA)

• Stock bacteria in a Petri dish

• Plasmid

• 4 Petri dishes:1 with LB agar only; 2 with LB agar and ampicillin(amp); 1 with LB agar, ampicillin (amp) andarabinose (ara)

• Sterile transformation buffer

• LB broth

• Pipettes, sterile

• 3 inoculation loops

• Microtube racks

• Recombinant Memo by AlexandraHoeppner (p. 55)

• The Research Starts activity sheet(pp. 56-63)

• Permanent marker

• UV lamp

• Digital watch with second-hand

• 42° C water bath


• Distilled water

• Styrofoam cup with crushed ice

• 10% bleach solution in squirt bottle

• Paper towels

• Waste container with 10% bleach solution

• Safety goggles, gloves and lab apron

• Celsius thermometer

TIME

Teacher preparation ....................................................... 3 hours (see page 7 of the BiotechnologyExplorer, Bacterial Transformation manual)

Student activity ............................................................... Four 50-minute sessions


Your students will perform a genetic transformation laboratory activity. Genetic transformation isused in many areas of biotechnology, including agriculture. Some examples include the insertion ofgenes into plants to make them more frost or pest resistant, drought tolerant or more flavorful.

This activity was adapted with permission from Bio-Rad’s Biotechnology Explorer, BacterialTransformation Kit (#166-0003-EDU). The kit includes background information and lab preparation


instructions as well as a student manual. For your thorough understanding, it is highlyrecommended that you review and follow the instructions provided with the kit. You may alsochoose to provide supplemental information and use student questions that are provided in the kititself.

In this activity, the gossypol scenario will continue as students act as researchers to insert thegossypol gene into glandless cotton plants. This activity will not teach laboratory techniques only, butalso will allow your students to get a flavor of what true research entails. The selectable markers ofantibiotic resistance and bioluminescence will help easily identify the transformed bacterial cells.The antibiotic resistance gene is required so nontransformed cells do not survive—thebioluminescence gene is added for a colorful experience.

TEACHER PREPARATION

Approximately three hours of preparation time will be needed for a well-equipped laboratory.Review and follow the teacher preparation guidelines provided in the Biotechnology Explorer-Bacterial Transformation Kit (#166-0003-EDU) from Bio-Rad Laboratories.

PROCEDURE

DAY 1: INTRODUCTION

1. Attach The Research Starts: Transforming DNA activity (pp. 56-63) to the Recombinant Memo(p. 55) and distribute the packets to the students. Review the memo and discuss what thestudents will be doing.

2. Discuss the purpose of the lab, laboratory protocol and safety instructions.

3. Have the students complete the pre-lab activity questions (p. 57).

DAY 2: EXPERIMENT

1. This is the actual day of the experiment. All supplies should be prepared, organized andavailable to the students.

2. Have the students complete the lab and activity sheet designated Day 2.

3. Assign appropriate questions for homework.


DAY 3: GATHERING DATA

Have the students complete the activities designated for Day 3 on their activity sheets, includingmaking appropriate data tables and answering the questions. Assign the conclusion statementwriting assignment as homework.

DAY 4: CONCLUSIONS

Discuss the laboratory activity, clarifying any confusing points and appropriate applications to thegossypol scenario.

CONCLUSION

• Genetic transformation activities, using sterile techniques, allow scientists to transfer geneticmaterial from one organism to another. Bacteria are often used for transformations because oftheir size and fast replication rates.

VARIATIONS

• Adapt this lesson to other transformation kits available to educators.

• Perform other laboratory activities, such as bacterial plate streaking, preparing agar plates andDNA fingerprinting.

EXTENSIONS

• Complete the Transformation Efficiency* Extension Activity provided on pages 64-65.

• Using the Internet and other sources, have the students research how and why bacterialtransformation activities are used in agricultural, medical and pharmaceutical research.


* This procedure was adapted, with permission, from Bio-Rad’s Biotechnology Explorer, Bacterial Transformation laboratory activity(#166-0003-EDU). For further inquiries contact Bio-Rad at 1-800-4BIORAD or www.bio-rad.com.


M E M O

TO: Tom Davis


RE: The Recombinant

Thank you for the check that your association sent us for preliminary work. Wefollowed up on the suggestion from Ms. Noonan and have found and created a chimeracontaining the gossypol gene, the glow gene and the antibiotic resistance gene. Thissequence has already been inserted into a plasmid. The new sequence is shown inthe diagram below.

We will attempt to insert this chimera into an E. coli bacterial cell next week asscheduled. I am pleased that you will assist us in the laboratory. By participating, youwill have a better understanding of the protocols used in this research. Once we aresuccessful at inserting this chimera into the plasmid, we will need to insert the plasmidinto the cotton plant and determine growth rate success.

If the procedure is successful, we will work on the gossypol regulator gene. This iswhere the sponsorship from your group will become essential. I appreciate ourpartnership as we work closely together to obtain funding for this important endeavor.

I have enclosed the protocol that we will be using next week. We are looking forwardto seeing you again.

Promoter Glow GeneGossypol

GeneAntibiotic

Resistance Gene Terminator


THE RESEARCH STARTS Name TRANSFORMING DNA

Introduction

You are about to complete a genetic transformation activity. Genetictransformation means that one or more genes are inserted into anorganism’s DNA to change the organism’s traits. In this experiment, we aresupposing that you are attempting to insert the gossypol gene into an E. colibacteria. You will know if this transformation is successful because theselectable markers of ampicillin resistance and bioluminescent glow areattached to this gossypol gene. You can detect the transformed bacteria bylocating the bacteria that survive (have ampicillin resistance) and glow (havethe glow gene that causes glowing when the sugar arabinose is present)when plated on an agar plate containing ampicillin and arabinose. Thosethat survive also, hypothetically, carry the desired trait of the gossypol gene.

Special Note

In actuality, the identification of genes and the creation of the chimeric geneconstructs in the plasmids require extensive laboratory work and research.For the sake of time and complexity, we are assuming this preliminary workhas already been done by other researchers.


DAY 1: PRE-LAB QUESTIONS

1. What genes are being placed into the transformed plasmid?

2. What organism is to receive this plasmid?

3. List at least three reasons why a bacterium was chosen for this experiment.

4. What are the two selectable markers used in this procedure? Why were thesemarkers selected?

5. What safety concerns must you use when doing this lab and why?

6. Copy and complete the chart below to predict what you expect to find after yourexperiment is complete. Also, write your hypothesis in an “if . . . then” format.

PREDICTED BACTERIAL GROWTH ON AGAR PLATES

LB (-DNA) LB/AMP (-DNA) LB/AMP (+DNA) LB/AMP/ARA (+DNA)

Will plate showtransformed cells?(+ for yes, - for no)

Estimated numberof colonies

7. What do the labels (+DNA) and (-DNA) represent?

8. Explain what LB, AMP and ARA represent.

9. On which of the four plates listed in #7 do you expect to see growth?

10. Which of the four plates are considered to be control plates and why?

11. What does it mean if growth appears on a plate?

12. Step #7 of the laboratory procedure is only completed for the (+DNA) tube. Why?


DAY 2: THE EXPERIMENT

Materials needed for each team: Other materials needed:

• Microtubes: one for (-DNA) and one for (+DNA)

• Stock bacteria in a Petri dish

• Plasmid

• 4 Petri dishes:1 with LB agar only; 2 with LB agar andampicillin (amp); 1 with LB agar, ampicillin(amp) and Arabinose (ara)

• Sterile transformation buffer

• LB broth

• Pipettes, sterile

• 3 inoculation loops

• Microtube racks

• Second-hand watch or timer


• Distilled water

• Styrofoam cup with crushed ice

• Paper towels

• UV lamp

• Microwave oven

• 37° C incubator

• 42° C water bath with thermometer

• Waste container with 10% bleach solution

• Safety goggles, rubber gloves and lab apron

• Fischer or Bunsen burner

• 10% bleach solution in spray bottle

PROCEDURE

1. Put on safety clothing as instructed. Wearing rubber gloves and goggles, sterilizeyour counter top using paper towels and the 10% bleach solution.

2. Pick up all materials required for your team. Note where community items arelocated.

3. Using the permanent marker, label the topand side of one microtube (+DNA) and thesecond (-DNA). On both tubes, write your labgroup number and the date. Note: One labgroup in your team will work with the (-DNA)tube and one will work with the (+DNA) tube.Be sure to label the top of the vial as the inkmay rub off the side when placed in the waterbath. Place the microtubes in the microtuberack.

4. Using one sterile pipette and sterile techniques, transfer 250 µl of the calciumchloride (CaCl2) solution into each of the microtubes from #3. Store on ice to chill


the calcium chloride. Dispose of the pipette in the designated waste container. Asketch of the pipette with the volume marking is shown.

5. Observe your stock plate by holding it under the UV light. Copy and complete thedata chart below. Using a sterile loop, collect a single average-sized colony ofstock bacteria and transfer it to the microtube labeled (+DNA). Immerse the loopinto the CaCl2 and swirl the loop so that all of the bacteria are transferred into themicrotube. Stir until the bacteria are evenly mixed with no clumps observed.Dispose of the loop in the waste container. Place the tube into the ice bath.

DATA CHART #1

Observe and describe the colonies on the starter plate using the following chart.

Number ofcolonies

Size ofcolonies (estimatein mm using a ruler onthe outside of the Petri

dish)

Largest ___

Smallest ___

Majority ___

Color ofcolonies

Distribution ofcolonies on

the plate

Visibleappearancewhen viewedwith UV light


6. Using another sterile loop, repeat #5 using the (-DNA) tube. Dispose of the loopin the designated waste container.

7. Immerse a third sterile loop into the stock plasmid solution. Pick up some of theplasmid solution in the loop. It should look like a film in the ring. Gently stir theplasmids in the loop into the tube labeled (+DNA). Cap the tube and replace inthe ice. DO NOT ADD PLASMID TO THE (-DNA) TUBE. Close the cap of the (-DNA) tube and place it in the ice. Make sure both tubes are capped and in theice.

8. Check the time and incubate on ice for 10 minutes.

9. While the tubes are incubating, label the bottom of your four Petri dishes asfollows:


10. After your ten minutes are complete, heat shock the bacterial cells. To do this,place the rack of tubes into the 42°C water bath for exactly 50 seconds. Make surethat the bottoms of the tubes sit in the warm water bath. When the 50 seconds areexpired, carry the ice container to the warm water bath, remove the rack of tubesand immediately place them back on the ice, making sure that the bottom of thetubes are in the ice. This timing is critical for successful transformation. Beaccurate with your time! Incubate on the ice for two minutes.

11. After the two-minute iceincubation, remove the rackand tubes from the ice andplace them on the bench top.Open the (+DNA) tube and,using a sterile pipette, add 250µl of LB broth. Close the tube.Dispose of the pipette in thedesignated waste container.Incubate at room temperaturefor 10 minutes.

12. Repeat #11 using the (-DNA) tube. Incubate at room temperature for 10 minutes.

13. After 10 minutes, tap both tubes to mix the bacteria with the LB broth. Use asterile pipette and transfer 100 µl from the (+DNA) tube onto the LB/amp plate.Discard the pipette.

14. Using a sterile loop, gently spread the liquid from #13 evenly around the agar inthe Petri dish. Be careful that you do not gouge the agar. DO NOT LIFT OFFTHE LID TO THE PETRI DISH — JUST TILT UP!

15. Repeat #13 and #14 using a new pipette and loop for each of the other plates. Besure you transfer the (+DNA) to the (+DNA) plate and the (-DNA) to the (-DNA)plates. Discard the used pipettes and loops in the designated waste containerimmediately after each transfer.


16. Allow the liquid to dry for a short time. Turn the Petridishes upside down and stack them. Tape the Petridishes together and incubate at 37°C for 24 hours.If necessary, the Petri dishes can be incubated atroom temperature for three days.

DAY 3: OBSERVATIONS AND CONCLUSIONS

PROCEDURE

1. Carefully remove the tape from the Petri dishes and observe the bacterial growthby placing each Petri dish under the UV light. DO NOT OPEN THE PETRI DISHOR TURN IT. KEEP IT UPSIDE DOWN AND OBSERVE THROUGH THE LIDAND AGAR. Record your observations.

2. Count all of the colonies on each Petri dish. A colony will appear as a small circleof bacteria on the agar. Complete the chart named Data Chart #2.

DATA CHART #2

Observe and record the data from the four plates on Day 2:

Plate SketchQualitative

Observations*Quantitative Observations*

(count the number of colonies)

(+DNA)LB / amp

(+DNA)LB / amp / ara

(-DNA)LB

(-DNA)LB / amp


*Note: Qualitative observations are characteristics such as color, shape and general appearance.Quantitative observations are observations you can measure such as amount and size.

3. After discussing your results with your team, complete the questions andconclusions of this activity.

QUESTIONS

1. What was the purpose of using four types of agar plate(s)?

2. Which plate(s) were considered to be control plate(s)? What purpose did eachserve?

3 Which plate showed the most growth and why? Which showed the least growthand why?

4. By simply looking at your Petri dish, how do you know if the gossypol gene wasplaced into the E. coli?

5. Compare your observations of the starter plate and the transformed plate. Howwas the phenotype of the transformed bacteria different from the phenotype of theoriginal bacteria?

6. Why was an antibiotic gene used in this transformation, since it has nothing to dowith creating a gossypol-producing cotton plant from a gossypol-deficient cottonplant?

7. For the transformed bacteria to glow, what two factors must be available in theenvironment? What would happen to a bacterium with this gene, without thecorrect environment? Explain your answer.

8. Explain how this laboratory activity shows that the following sequence does ordoes not hold true.

DNA RNA Protein Trait

9. Does this laboratory project complete the project for Cotton Research Associates?If so, explain. If not, what must be done after this transformation has occurred?

CONCLUSION STATEMENT

Write a one page explanation of the experiment that you just completed. Be sure toexplain the variables and controls. Explain what the results were and how you knowwhether or not a transformation occurred.


* This procedure was adapted, with permission, from Bio-Rad’s Biotechnology Explorer, Bacterial Transformation laboratory activity(#166-0003-EDU). For further inquiries contact Bio-Rad at 1-800-4BIORAD or www.bio-rad.com.


TRANSFORMATION EFFICIENCY*EXTENSION ACTIVITY

It is important for genetic engineering businesses to know the number of cells that havebeen successfully transformed. Knowing the number of transformed cells is critical indetermining the success of this type of procedure.

To calculate the transformation efficiency, you will need to use the following formula:

Transformation Efficiency = Number of cells growing on the LB/amp/ara plateAmount of DNA spread on the agar plate

To calculate this, you will need to complete the following information below:

1. Check your data chart for the number of glowing colonies on your LB/amp/ara plate.It can be assumed that each of these colonies originated from a single cell. Bycounting the glowing colonies, you know how many cells were transformed.

Total Number of cells =

2. To calculate the amount of DNA spread on the agar plates, you will need todetermine the amount of plasmid that was placed into the microtube. Use theformula below to calculate this information.

DNA (µg) = concentration of DNA (µg/µl) X volume of DNA (µl)

The concentration of the DNA was 0.03 µg/µl. The loop held 10 µl of plasmid.

Total amount of DNA (µg) used in this experiment =

3. You now need to determine the ratio of DNA used. The liquid in your microtubeafter all the ingredients were added consisted of CaCl2, LB broth and bacteriacontaining the plasmid. You now need to calculate the ratio of the bacteria that wasspread to that in the microtube. To do this, determine from your procedure theamount of DNA spread on the LB/amp/ara plate as well as the total amount of fluidin the microtube. (Hint: This will include the CaCl2, LB broth and the amount ofplasmid.) Once that has been determined, use the formula to calculate the ratio ofDNA used.

Ratio of DNA used = Volume spread on the LB/amp/ara plateTotal sample volume in test tube


Ratio of DNA used =

4. The amount of DNA spread on the LB/amp/ara plate now needs to be calculated todetermine the number of micrograms of DNA spread. To do this, use the followingformula:

Plasmid DNA spread (µg) = Total amount of DNA used (µg) x ratio of DNA used

(Hint: The total amount of DNA used can be found in #2, while the ratio of DNA can be found in #3.)

Plasmid DNA spread (µg) =

5. Transformation efficiency can now be calculated. To do this, use the followingformula:

Transformation Efficiency = Total number of cells growing on the agar plate Amount of plasmid DNA spread on the agar plate

(Hint: The numbers for this equation can be found from #3 and #4 above.)

Transformation efficiency = (transformation/µg)

Note: This should be a large number. Scientists usually convert numbers intoscientific notation. To do this, write the first numeral to appear between 1 and10. Count the number of places you moved the decimal to do this. The numberof places you needed to move the decimal will be the number of places to theupper right of the ten. For example, the number 5400 will be expressed as 5.4 x103. Express your transformation efficiency in scientific notation.

Transformation efficiency = (transformation/µg)

6. Most researchers find the transformation efficiency to be between 5 x 103 and5 x 104. How does your transformation efficiency compare to this figure?


* This procedure was adapted, with permission, from Bio-Rad’s Biotechnology Explorer, Bacterial Transformation laboratory activity(#166-0003-EDU). For further inquiries, contact Bio-Rad at 1-800-4BIORAD or www.bio-rad.com.


LESSON 5: HOW DO GENES GET INTO PLANTS?(Using Bacteria to Insert Chimeric Genes into Plant Genomes)

PURPOSE

The purpose of this activity is to sequentially show students how chimeric genes enter plants usingnatural bacteria. Students will discover that naturally occurring bacteria can be used to producegenetically transformed plants.

CONCEPTS • Agrobacterium and E. coli are bacteria that naturally exist in the environment.

• Tissue culture is a method of asexual reproduction that allows genetically identical organisms tobe propagated from one cell.

• Co-cultivation is a process by which two known organisms grow together—one example isAgrobacterium and plant cells.

• Genetic material can be transferred from one organism into another through the use ofspecialized bacteria.

MATERIALS


• Follow-Up Research Memo (p. 70)

• Agrobacterium Summary Sheet (p. 71)

• Tissue Culture Sequencing Activity (pp. 72-73)

• Glue stick

• Scissors

• Ruler

• 12” x 18” paper

• Markers

• Tissue Culture In Plants reading assignment—optional (p. 69)

• Photographs of tissue culture methods in the laboratory (available in many textbooks)

TIME

Teacher preparation ......................................... 10 minutes


Student activity ................................................. One 40-minute session


After this lesson, the students should have a basic understanding of how to produce geneticallyidentical plants using natural processes and tissue culture. Although the processes are quitecomplex, the students will understand that once the system for replication of genetically engineeredplants is refined for a particular situation, laboratory technicians are able to complete the work withadequate training. It is appropriate to talk with students about the various career opportunities in thearea of biotechnology. In fact, encouraging students to participate in laboratory internships could bebeneficial to both the students and the biotechnology industry.

PROCEDURE

1. Explain the concept of tissue culture and its use as a biotechnological tool. If appropriate, havethe students read and complete the Tissue Culture in Plants lesson (p. 69) at home prior to thesequencing activity they do in class.

2. Distribute the Follow-Up Research memo (p. 70) and Agrobacterium Summary Sheet (p. 71) tothe students. Have the students read and discuss the material. Be sure to discuss therelationship between the transformation activity and tissue culture—transformation precedestissue culture.

3. Distribute the Tissue Culture Sequencing Activity Sheet (pp. 72-73) to pairs of students. Havethe students complete the pre-activity questions, the sequencing activity and the follow-upquestions. Discuss the results. A Tissue Culture Sequencing Activity answer key is provided onpage 75.

CONCLUSION

• Genetically identical plants can be produced using tissue culture techniques.

VARIATIONS

• Assign a tissue culture textbook reading assignment to students.

• Have the students perform actual tissue culture activities.

EXTENSIONS

• Visit a biotechnology company which uses tissue culture as a propagation technique.

• Investigate how hydroponic set-ups are used in tissue culture.

• Infect tobacco plants with Agrobacterium and observe gall formation.


• Observe galls in your environment—oak galls are common in California.


TISSUE CULTURE IN PLANTS Name ______________________

Read the following information and answer the questions.

Tissue culture includes methods of asexual propagation. Such methods allowresearchers to produce numerous genetically identical plants from one cell. Thismethod can involve the placement of sterilized terminal shoots, flower or leaf buds ontoa sterile agar gel or nutrient medium. The medium must contain proper nutrients andgrowth-regulating chemicals. Most media contain auxin, the hormone responsible forroot formation, and cytokinin, the hormone responsible for shoot formation.

Tissue culture procedures must be performed in a sterile environment in order toprevent infection. The work is done in a laminar flow hood that is equipped with analcohol lamp and dissecting instruments sterilized in 95% ethyl alcohol. Before placingplant tissue into the hood, the plant is treated with a solution of 10% bleach and 70%ethyl alcohol or hydrogen peroxide for 10 to 20 minutes. After washing, a few cells areremoved and plated onto the agar gel. A callus (ball of undifferentiated cells) will beginto grow. The callus is the scar tissue of the plant. This callus can then eventuallydevelop into a new plant.

Tissue culture can be used to propagate valuablegenetic lines or to regenerate tissue. Many plantshave the unique ability to regenerate via tissueculture. This is called totipotency—the ability of asingle cell to regenerate into a complete organism.Tissue culture is one of many laboratory techniquesused by many plant biotechnology companies.

QUESTIONS

1. Is tissue culture a method of sexual propagation or asexual propagation? Whatdoes this mean?

2. For successful growth, what must be present in the growing medium and why?

3. Why do you suppose tissue culture needs to be performed in a sterile environment?

4. How are the new plants produced from tissue culture related to the “parent” plant?

5. Explain how the parent tissue is disinfected.

6. What is a callus?

7. Give some examples of how you think tissue culture could be used in agriculture.

8. Write one question or concern you have about tissue culture.


M E M O

TO: Tom Davis


RE: Follow-up Research

I hope that it was beneficial for you to work with us last month. The first partof our project was successful—getting the gossypol gene and glow genesuccessfully into the plasmid. We have found, however, that Agrobacteriumis easier to work with than E. coli. The transformation procedures worksimilarly in both bacteria. I am including a short blurb about Agrobacteriumthat was written for another project.

We now need to determine if this newly transformed bacteria can be placedsuccessfully into the cotton plant and still function appropriately. If we aresuccessful at creating a glandless cotton plant that is genetically engineeredto produce gossypol, we will be ready to move forward with fundingproposals which will allow us to do more research on the gossypol regulatorgene.

I have included some information about what we need to do next. Yourartists will find it useful as they sketch out a storyboard for your Board ofDirectors. Good luck with your presentation. I’m rooting for your success!


AGROBACTERIUM SUMMARY SHEET(Week 3)

Editorial Note: This brief description is one of an eight-part series of summaries intendedto provide all staff members of Agri-Gene an overview of biotechnologicalconcepts. It corresponds with our once-a-month lunch lecture series onspecific topics.

Agrobacterium is a bacterium that naturally lives in the soil and causes a plant diseasecalled crown gall. Agrobacterium causes this disease by putting some of its plasmid DNAinto the plant’s DNA and tells the plant to grow the gall. The gall supplies the bacterium withfood to continue growing and dividing, thus spreading the disease. When a susceptibleplant is wounded by poor cultivation practices or animals, Agrobacterium can transfer itsDNA into the plant’s DNA. In the laboratory, wounds are purposely caused by cutting theplant.

The mechanisms of Agrobacterium and crown gall disease are used in tissue culture.Currently, we are trying to insert a gossypol gene and a glow gene into the genome of aglandless cotton plant. In other words, we are taking a plant that does not have the ability toproduce gossypol and trying to give it the ability to make gossypol. If this works, we willseek funding from Cotton Research Associates for further research to find a regulator genethat will allow gossypol to be made only in the leaf and stem portions of the plant, but not inthe cottonseed. The success of this venture will be discussed at an upcoming brown baglunch lecture.

When genetic engineers use Agrobacterium, they remove the DNA between the geneswhich causes a gall to grow and replace it with new desired DNA sequences and introducea selectable marker, such as an antibiotic resistance gene.

The selectable marker, such as an antibiotic resistance gene, gives a bacterium the abilityto grow in an antibiotic that would normally kill it. Approximately two out of 1,000 plantsproduced through tissue culture will be transformed plants. These “transformed” plantscontain the desired trait, such as the gossypol producing gene, and the selectable marker,such as the antibiotic resistance gene.

To get a gene into a plant, a genetic engineer needs to use both tissue culture techniquesand the natural DNA capabilities of Agrobacterium. After a seed germinates and thecotyledon leaves form, the cotyledon leaves are cut into pieces and placed onto an agarfeeder plate that conditions the cotyledons overnight. The cotyledons are then placed intoan Agrobacterium solution for about five minutes. The Agrobacterium used has its DNAthat causes crown gall growth removed, and it contains the chimeric gene that scientistswant to incorporate into the plant DNA. The cotyledons are then removed from this solutionand placed back on to the feeder plate for two days. After about five days, the cotyledonsstart to form offshoots and some callus (scar tissue) forms. The offshoots are transplantedonto an agar plate. As they mature, they are transferred onto a final agar plate thatcontains rooting solution and antibiotic. The antibiotic selects for only those plants which


have been transformed. Finally, the transformed plant is planted into soil for further growthand more tests.


TISSUE CULTURE SEQUENCING ACTIVITY(Student Activity Sheet)

INTRODUCTION

The purpose of this activity is for you to visualize the process a laboratory technician performsto create numerous plants made of genetically identical DNA. This sequencing activity you areabout to perform illustrates the replication of glandless cotton plants that have the gossypolproducing gene inserted into their cells.

PRE-ACTIVITY QUESTIONS

1. What is a crown gall?

2. Why are cotyledons used in this experiment?

3. Why is an antibiotic used in the nutrient solution?

4. Is it possible for Agrobacterium to be put in a solution and be absorbed through theroots? Explain.

5. Why are new plants containing a chimeric gene developed using tissue culturetechniques?

6. What is the chimeric gene your group has developed?

MATERIALS

• Tissue Culture Sequencing Activity Sheet

• Scissors

• Glue

• Ruler

• 12” x 18” paper

• Markers

PROCEDURE

1. Review the Follow-Up Research Memo and the Agrobacterium Summary Sheet.

2. Study the pictures on the attached Tissue Culture Sequencing Activity Sheet.

3. Using scissors, cut out the boxes and determine the correct sequence—start to finish.

4. Place the boxes, in order, on the paper provided, leaving space for a caption that explainseach picture.

5. Number each picture and glue it on the paper. Write a caption for each picture explainingthe sequential steps of this tissue culture procedure.


CHIMERIC GENE SEQUENCING ACTIVITYAgar plate with rooting solution

Transformed Not Transformed (roots) Healthy (no roots) grows poorly

Clipped Cotyledon

Cotyledons Cotyledon offshoots

Overnight conditioning of Cotyledons Co-Cultivation

Cotyledon with growth hormone, AgrobacteriumKanamycin, nutrients

Transplanted offshoots Seed with nutrient agar

Feeder plate Offshoots transferred to a pot with soil


CHIMERIC GENE SEQUENCING ACTIVITY(Answer Key)

Agar plate with rooting solution

Transformed Not Transformed (roots) Healthy (no roots) grows poorly

Clipped Cotyledon

Cotyledons Cotyledon offshoots

Overnight conditioning of Cotyledons Co-Cultivation

Cotyledon with growth hormone, AgrobacteriumKanamycin, nutrients

Transplanted offshoots Seed with nutrient agar


Feeder plate Offshoots transferred to a pot with soil


LESSON 6: THE PRESENTATION(Presenting Your Research and Seeking Further Funding)

PURPOSE

The purpose of this lesson is to encourage the students to think about the biotechnology activitiesthey have performed and to present their findings to the class (the Board of Directors of CottonResearch Associates).

CONCEPTS • Biotechnology, specifically genetic engineering, is a complicated subject both scientifically and

socially.

• Collaborative efforts between industry, educators, agriculturalists and the government areadvantageous and crucial in today’s technological society.

• In today’s technological society, it is important for all people to have an understanding of scienceand to have problem-solving and critical thinking skills.

MATERIALS

For each team:

• Presentation Task Sheet (p. 79)

• Butcher paper

• Markers

• Supplies to prepare visual aides

TIME

Teacher preparation ............................................... 10 minutesStudent presentation preparation ........................... One or two 50-minute sessions, plus homeworkStudent presentations and class discussion ......... Two 50-minute sessions



Throughout this unit, you have provided students with opportunities and tools to learn about thescience of biotechnology, specifically genetic engineering. It is important that you provide time foryour students to process what they experienced and share their knowledge with others.Many issues associated with genetic engineering are constantly arising in the news. Through theirexperiences in the lab, your students will be better prepared to think critically about such issues. It isimportant to remind students to think critically, considering the author(s) or source(s) of theirinformation, before making decisions of their own.

PROCEDURE

1. Divide students into groups of three to four. Provide a Presentation Task Sheet to each groupand discuss the purpose of the presentation they are to prepare and the procedures they are tofollow.

2. Provide enough time in class for your students to prepare the drafts of their presentation scriptand visual aids. The final products should be created at home with time in class to finalize grouppresentation plans.

3. Have each student group present reports to the class. Discuss appropriate concerns and askappropriate questions. Assess students on the thoughtfulness of the topics and on the quality oftheir presentations.

4. Conclude this unit by having the students write an evaluation of the unit . . . what they liked, whatthey did not like, what they learned, what challenges they had, what opinions they now have onthe subject of genetic engineering in plants, etc.

VARIATIONS

• Assign one group to prepare a presentation which would not support further funding of theproject.

• Have each student or student group prepare a written funding proposal to Cotton ResearchAssociates. Provide specific grant-writing guidelines for the students to follow.

• Develop a role-play where students act as different members of a Cotton Research Associatesfunding meeting. Some students could prepare a presentation, other students could support thefunding, others could oppose funding and some students can act as the voting members of theboard.

• Invite parents to the oral presentations.

• Have the students apply what they learned to another situation, such as producing sweeterstrawberries or drought tolerant lettuce.

EXTENSIONS

• Have the students write a follow-up proposal to the one they just completed. What researchwould they like to pursue next?


• Invite various people into class to discuss their professions—research scientists, funddevelopment coordinators, city board members, biotechnological industry representatives, etc.

Discuss farmers’ concerns and appreciation of genetically engineered crops.


PRESENTATION TASK SHEET

INTRODUCTION

As Cotton Research Associates representatives, you have now gathered the information you needfor your presentation to the Board of Directors of Cotton Research Associates. The purpose of thispresentation is to gather support for the funding needed to complete the development of a new strainof cotton that still resists pests, but no longer has gossypol in the seed. Your group will develop andpresent an oral presentation to the Board of Directors.

PROCEDURE

1) Discuss with your teammates the importance of developing a strain of cotton that does nothave gossypol in its seed.

2) Review the process by which a chimeric gene can be inserted into a cotton genome.Remember to discuss the functions of Agrobacterium, tissue culture, selectable markers,promoter and terminator sequences and restriction and ligating enzymes.

3) Discuss how the desired phenotype is expressed after protein synthesis.

4) Discuss bioethical issues pertaining to this particular process. What groups may supportthis project and why? What groups may oppose this project and why?

5) Prepare your presentation to the Board of Directors requesting financial support of thisproject. The presentation should focus on the information reviewed and discussed insteps 1–5 above and clearly state how further funding will be used. Use the followingchecklist to determine the completeness of your presentation.

Does your presentation include information discussed in #1 through #4 above?

Have you explained the research already performed and what research still needsto be funded before this project is successful?

Does your presentation clearly state how further funding will be used?

Is your presentation organized in a sequential fashion clearly expressing yourknowledge?

Are your visual aids eye catching, easy to follow, attractive to potential viewers andlegible?

Is the name of your project, with the author’s names, clearly visible?

Is the scientific information accurate?

Did you proof for grammar and spelling errors on all written work?

Have all members of your team contributed in an equal manner to the completion of the project?

Do all group members have a part in the oral presentation?

Did you practice your oral presentation? Do all members of your group agree that the presentation, preparation and

display are complete?


WHERE DO GENES COME FROM?This chart lists a variety of agricultural crops that have been genetically engineered. It also providesyou with information on the sources of the genetic materials, the names of the genes anddescriptions of how the new traits are expressed.

Crop Source of Genes Name of Gene New Trait

Tomato Tomato

Virus

Bacteria

Bacteria

Antisense enzyme

Coat protein

Sugar

Vitamin A, B-carotene

To soften slowly to allow it toremain on the vine longer

Virus resistance

Extra sweet

Extra nutrition

Canola Various plants

Various plants

Enzymes for oil

Synthesis

Lower content of saturated oils

Special oil compositions forproducing shampoo, syntheticlubricants and shortenings

Squash,Cantaloupe

Virus Coat protein Virus resistance

Potato Bacteria Starch Increased nutrition

Soybean,Sunflower,Canola

Legumes and nuts Storage proteins Increased protein in the plantby-products so they can beused for nutritious animal feed

Chrysanthemum Bacteria Antisense pigment genes Pure white petal color

Strawberry,

Raspberry Plants Ripening genes Increased size and firmness

Papaya Plants

Virus

Ripening genes

Coat protein

Increased flavor and firmness

Virus resistance


CAREERS IN BIOTECHNOLOGY

A career in biotechnology offers an individual a wide range of career choices. A strong backgroundin science is recommended. The diagram below shows the connection between biotechnology andthe sciences.

A MOSAIC OF BIOTECH FIELDS AND CAREERS

Anthropologist

Agriculture

BIOTECH

Biochemical Genetics

PlantGenetics

ProteinBiochemist

Biochemistry

EcosystemsEcologist

Geographer

Horticulturist Botanist

DevelopmentBiologist

DevelopmentGenetics

Neurobiologist

Behavioral Genetics

Euthenics

Sociology

MolecularBiology

Physiologist

MolecularGenetics

PharmacologistPathologist

Medical Genetics

Genetic Counselor

General Genetics

Biologist

Ecologist

Paleontologist

Systematics

Law

Oceanographer

Ecologist

Cytology

Cytogenetics

Evolution

EvoluntionaryGenetics

MicrobialGenetics

Animal Scientist

Medicine


TEACHER RESOURCES AND REFERENCES

Ag Access: Agricultural Book Source, P. O. Box 2208, Davis, CA 95617; (916) 756-7177.Request a catalog of agricultural books.

Agriscience—Fundamentals and Applications textbook, Elmer L. Cooper, Delmar Publishers,Inc., 1990. A general high school agriscience textbook written in an easy-to-read format.

“Agricultural Biotechnology: A World of Opportunity” video, National FFA Center,5632 Mt. Vernon Memorial Highway, Alexandra, VA 22309-0160;(703) 360-3600. This video describes the various career opportunities available inagricultural biotechnology.

American Society for Microbiology, ASM Finance Department, 1325 Massachusetts Avenue, NW,Washington, DC 20005-4171; Fax: (202) 942-9347. Members of this organization receivenewsletters and journals. Contact the association for membership information.

An Introduction to Biotechnology: A Junior High Unit, Monsanto Fund and National ScienceFoundation Mathematics and Science Education Center, 8001 National Bridge Road,246 Benton Hall, St. Louis, MO 63121; (314) 553-5552. Educational lessons designed toteach about biotechnology, including genetic engineering.

Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547; (800) 424-6723. Thecompany provides many biotechnological classroom kits, including the DNA transformationkit recommended for use with this unit. Request a free catalog.

“Biotechnology—Careers for the 21st Century” video, National Association of Biology Teachers,11250 Roger Bacon Drive #19, Reston, VA 22090; (703) 471-1134. This video sharesinterviews of people involved in the many careers associated with biotechnology.

Biotechnology Education Program, University of California, Davis, CA 95616; (916) 752-3260.Request information specific to your needs, or a packet of general information onbiotechnology.

Biotechnology: Microbes and the Environment pamphlet, Steve Nitt, Center for ScienceInformation, 1990. Provides information on how microbes can be used as a biotechnologytool.

Blazing a Genetic Trail pamphlet, Howard Hughes Medical Institute, 6701 Rockledge Drive, Bethesda,Maryland 20817. A pamphlet on human genetics and medicine.

“Building a Better Tomato” article, Ricki Lewis, High Technology, May 1996. A highly detaileddiscussion of tomato biotechnology.

Calgene, Inc., Public Affairs, 1910 Fifth Street, Davis, CA 95616; (916) 753-6313;Fax: (916) 753-1510. A genetic engineering company that has press packets available onsome of their products, including plant oils, tomatoes and cotton.


California Foundation for Agriculture in the Classroom, P.O. Box 15949, Sacramento, CA95853; (800) 700-AITC. Provides low-cost educational resources and lessons whichincorporate agriculture into existing curricula. Request a Teacher Resource Guide and askto be put on the mailing list.

California Science Teachers Association, 3550 Watt Avenue, #120, Sacramento, CA 95821-2666; (916) 979-7004. This association provides newsletters and journals to Californiascience educators about ideas, issues and trends in science education.

Carolina Biological Supply Company, 2700 York Road, Burlington, NC 27215; (800) 334-5551,ext. 5310. This company has a wide variety of science supplies, including DNAfingerprinting and transformation kits. Request a catalog.

“Cotton’s Journey From Seed To You” kit, The Alaca Company, P.O. Box 55, Tranquility, CA93668; Fax: (209) 698-5190. Contains booklet, teaching guide, student manual, video,cotton samples and other materials to teach students the journey of cotton from the farm tothe home. Approximate cost is $70.

“Food Biotechnology” video, Dr. Christine Bruhn, Center for Consumer Research, University ofCalifornia, Davis, CA 95616; (916) 752-2774. A video and worksheet which educatesconsumers about food biotechnology and the current issues associated with it.

Genetics Heads for the Supermarket pamphlet, World Book Encyclopedia, 1560 ShermanAvenue, Suite 1111, Evanston, Illinois 60201, Publ #5285. An easy-to-understand readingon food biotechnology.

Industrial Biotechnology Association, 1625 K Street NW, Suite 1100 Washington, DC 20006;(202) 857-0244. A variety of basic and detailed information on biotechnology, includinggenetic engineering.

Logal Science and Math Software, 125 Cambridgepark Drive, Cambridge, MA 02140;(800) 564-2587. A variety of CDs and other software programs are available. The MolecularBiology Explorer 3.0 CD ROM is a good supplemental tool for studying molecular biology.Request a catalog.

National Cotton Council of America, P.O. Box 12285, Memphis, TN 38182; (901) 274-9030.Several resources are available on United States cotton production.

Plant Molecular Biology, A Practical Approach textbook, C.H. Shaw, IRL Press Limited, 1988.Provides practical, simple information on plant molecular biology.

Recombinant DNA: A Short Course textbook, J. Watson, J. Tooze and D. Kurtz, ScientificAmerican Books, W. H. Freeman and Company, New York, 1983. Provides illustrations andgraphics of recombinant DNA technology.

Science Framework for California Public Schools, K-12, 1990. Bureau of Publications, SalesUnit. California Department of Education, P.O. Box 271, Sacramento, CA 95812-0271;(916) 445-1260. The state guidelines for teaching science. Other subject matter frameworksare also available.

The Agricultural Dictionary book, Ray V. Herren, and Roy L. Donahue, Delmar Publishers, Inc.,1991. Provides definitions of agricultural terms.


The Cartoon Guide to Genetics booklet, Larry Genick, Harper Collins Publishers, 1991. Can beused to add humor to your genetics lessons.

University of California Cooperative Extension, Refer to your local telephone book in thegovernment or county section for address and phone number. Practical and researchpublications are available on agriculture. One such newsletter is called the California CottonReview.


GLOSSARY

AGROBACTERIUM—A pathogenic bacterium that causes crown gall disease. Used in geneticengineering to put chimeric genes into plants.

AMINO ACID—The building blocks of proteins. Codons code for amino acids.

BIOTECHNOLOGY—A number of technologies involving living organisms used to produce usefulproducts, processes and services.

CHIMERIC GENE—A gene that contains DNA from at least two different sources.

CHROMOSOME—DNA strands responsible for the determination and transmission of hereditarytraits.

CLEAVAGE SITE—The site on a DNA strand that restriction enzymes recognize and then cut.

CODON—A triplet of bases within a molecule of DNA or mRNA that codes for a particular aminoacid.

COTYLEDON—Often the first “leaves” on a new plant; part of a seed that provides nutrients andprotection to a plant embryo.

E. COLI—A bacterium naturally found in the environment in a variety of strains. Selected strains areused in genetic engineering.

ENZYME—A protein that functions as a biological catalyst.

GENE SPLICING—A procedure by which one DNA molecule is attached to another DNA molecule.

GENES—Sections of DNA that code for a specific trait.

GENETIC ENGINEERING—Alteration of an organism by inserting genes from another organism intoits genome.

LIGATING ENZYME—Enzymes used to fuse or “glue” DNA strands together.

mRNA—Messenger RNA; codes for the production of amino acids.

MUTATION—A random change in the genetic material.

PLASMID—Circular DNA molecules found in bacteria that are not part of a chromosome.

PROTEIN—Chains of amino acids that perform the necessary functions of living organisms.

PROMOTER SEQUENCE—A sequence of bases within a DNA strand that initiates transcription byRNA polymerase.


RECOMBINANT DNA—A DNA strand comprised of pieces of DNA from two or more differentsources spliced together.

RESTRICTION ENZYME—An enzyme that cuts a DNA molecule at a specific nucleotide sequence.

SELECTABLE MARKER—An identifiable gene used to determine whether or not a genetictransformation has taken place.

TERMINATOR SEQUENCE—A sequence of bases on a DNA molecule that stops protein synthesis.

TISSUE CULTURE—A method of asexual propagation which allows genetically identical plants tobe propagated from one cell.

TRANSCRIPTION—The process by which the genetic information contained in DNA is converted toa molecule of RNA.

TRANSLATION—The process by which genetic information encoded in mRNA in the form ofcodons is converted into a sequence of amino acids that form a protein chain.

Documents

GENETIC · 2016-01-05 · • Genetic make-up is received from both parents, and is expressed as traits which can be predicted (pp. 128-130). • The genetic code instructs the production