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GENETIC ENGINEERING
This unit was developed by
California Foundation for Agriculture in the Classroom
2300 River Plaza Drive
Sacramento, California 95833
Telephone: (800) 700-AITC
cfaitc@cfbf.com
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.
1
st
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 1997
2
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 for
Agriculture in the Classroom.
Calgene, Inc. is an agricultural biotechnology company which develops improved varieties of plants
and 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 and
the development and marketing of value-added food and fiber crops, as well as crop protection
products, performance materials, food ingredients and pharmaceuticals.
Bio-Rad Laboratories is a multinational manufacturer and supplier of products for life science
research and education.
The California Farm Bureau Federation actively represents, protects and advances the social and
economic interests of farm families and California communities by organizing productive agriculture
to provide group benefits and manage issues which affect its membership.
The California Foundation for Agriculture in the Classroom is dedicated to fostering a greater public
knowledge of the agricultural industry and seeks to enlighten students, educators and leaders in the
public and private sector about agriculture's vital, yet sometimes forgotten, role in American society
and 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. Their
comments 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 Kennedy
Science Teacher
Armijo High School
Fairfield-Suisun Unified School
District
Fairfield, CA
Suzanne Weisker
Science Teacher
Will C. Wood High School
Vacaville Unified School District
Vacaville, CA
Editor
Pamela Emery
Curriculum Specialist
California Foundation for
Agriculture in the Classroom
Sacramento, CA
Field Testers
Dorothy Reardon
Science Teacher
Del Campo High School
San Juan Unified School District
Fair Oaks, CA
Francesca Lowe
Science Teacher
Concord High School
Mount Diablo Unified
School District
Concord, CA
Kathleen McCarthy
Science Teacher
Concord High School
Mount Diablo Unified
School District
Concord, CA
Illustrators
Patricia Houk
Maceo Montoya
Layout, Typing and Design
Margaret Anderson
Karin Bakotich
Sherri Hughes
Tami Gutschall
Rae Lehutsky
Cinamon Vann

California Foundation for Agriculture in the
Classroom 1997
3
Curriculum Advisory and Review Committee
David Anderson, Ph.D.
Director of Cotton Biotechnology
J.G. Boswell Company
Corcoran, CA
Carol Bastiani
Research Assistant
Department of Medical Biological Chemistry
University of California
Davis, CA
Beth Brookhart
Freelance Journalist
Bakersfield, CA
Lucas Calpouzos, Ph.D.
Former Dean of Agriculture
California State University
Chico, CA
Steve Clark
Science Teacher
Monterey High School
Monterey Peninsula Unified School District
Monterey, CA
Norma Clawson
Science Teacher
Cajon High School
San Bernardino Unified School District
Blue Jay, CA
Constance Coman
Biology Teacher
Winters High School
Winters Joint Unified School District
Winters, CA
Lane Conn
Chairman
Stanford Human Genome Center
Stanford University
Palo Alto, CA
Jenny Cuccinello
Science Teacher
Florin High School
Elk Grove Unified School District
Sacramento, CA
Judy Culbertson
Manager of Programs and Services
California Foundation for
Agriculture in the Classroom
Sacramento, CA
Jerry Delsol
Agriscience Teacher
Woodland High School
Woodland Joint Unified School District
Woodland, CA
Jim Elam
Animal Nutritionist
Agricultural Technology, Incorporated
Solvang, CA
Richard Engel
Project Coordinator
California Foundation for Agriculture
in the Classroom
Sacramento, CA
John Fedors
Science Education Consultant
San Diego, CA
Mary Jo Feeney, MS, RD, FADA
Director of Education
California Beef Council
Pleasanton, CA
David Hammond
Educational Consultant
Sacramento, CA
Carolyn Hayworth
Manager, Investor and Public Relations
Calgene, Inc.
Davis, CA
Catherine Houck, Ph.D.
Vice President, Product Development
and Variety Development
Calgene, Inc.
Davis, CA
Lauren Hubbard
Graduate Student Researcher
Plant Molecular Genetics
University of California
Berkeley, CA
Gay Jividen
Senior Director of Research
Cotton, Inc.
Raleigh, NC
Andy Kennedy
Field Representative/Buyer
Colusa County Canning Company
Williams, CA
Jean Landeen
Agricultural Education Consultant
California Department of Education
Sacramento, CA
Jeanne Layton
Research Biologist
Monsanto Company
Chesterfield, MO
Mark Linder
President
California Foundation for
Agriculture in the Classroom
Sacramento, CA
Ron Mardigian
Product Manager
Bio-Rad Laboratories
Hercules, CA
Martina McGloughlin
Associate Director of Biotechnology
Biotechnology Division
University of California
Davis, CA
Craig McNamara
Farmer
Sierra Orchards
Winters, CA
Donna Mitten
Genetic Engineering Consultant
Woodland, CA
Jeffrey ONeal
Extension Coordinator
Biotechnology Program
University of California
Davis, CA
Janette Oaks
Research Scientist
Calgene, Inc.
Davis, CA
Maria Osborn
Biology Teacher
Foothill High School
Grant Joint Union High School District
Sacramento, CA
Frank Plescia
Manager, Government Affairs
Monsanto Company
Roseville, CA
Pam Schallock
Fourth Grade Teacher
Sandrini Elementary School
Panama-Buena Vista Union School District
Bakersfield, CA
Claudia Sellers
Graduate Student Researcher
Department of Plant and Microbial Biology
University of California
Berkeley, CA
Wynette Sills
Farmer
Pleasant Grove Farm
Pleasant Grove, CA
Roger Sitkin
Farmer
Old Dog Ranch
Linden, CA
Barbara Soots
Assistant Education Director
Center for Engineering Plant Resistance
Against Pathogens
University of California
Davis, CA
Nancy Stevens
Biology Teacher
San Rafael High School
San Rafael City High School District
San Rafael, CA
KarenBeth Traiger
Science Resource Teacher
Graystone Elementary School
San Jose Unified School District
San Jose, CA
Denise Van Horn
Fourth Grade Teacher
McSwain Elementary School
McSwain Union Elementary School District
Atwater, CA
John Vogt
Middle School Science Teacher
Creekside Middle School
Cotati-Rohnert Park Unified School District
Rohnert Park, CA
Thea Wilkins, Ph.D.
Associate Professor
Department of Agronomy & Range Science
University of California
Davis, CA
Mary Yale
Middle School Science Teacher
Grange Middle School
Fairfield-Suisun Unified School District
Fairfield, CA
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California Foundation for Agriculture in the
Classroom 1997
INTRODUCTION
The Science Framework for California Public Schools emphasizes the need to make science
education more meaningful to students so they can apply what they learn in the classroom to their
daily lives. Since all students eat food and wear clothing, one natural connection between science
and the real world is agriculture. Advances in agricultural technology, especially in genetic
engineering, are continually making headlines and are an excellent way for educators to connect
current 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 rural
areas become urbanized, more challenges exist to maintain and improve the quality of life on the
planet and feed the people of the world. It is extremely important to educate students about their
environment, agriculture and the current technologies and research that continue to make the Earth a
viable planet. Genetic engineering is a relatively new science that has blossomed over the last 40
years. Most genetic engineering research relates to agriculture and medicine. This unit will focus on
plant genetic engineering.
Genetic Engineering in Agriculture is a thematic high school unit that can be used at the end of a
genetics unit or an advanced unit on the cell. Throughout the unit, students are provided with the
scientific principles of genetic engineering and are encouraged to use their knowledge to think
critically, creatively and freely about the viability and ethics associated with genetic engineering and
agriculture. The introductory lessons present biotechnology terms as the students are presented
with a hypothetical scenario associated with two major environmental concernsfood and fiber
quality and the efficient use of resources. Students prepare interactive reports and perform DNA
transformation laboratory activities that teach genetic engineering concepts, such as how genetic
information is transferred from one organism into another through the use of plasmids and tissue
culture. The students then use their acquired knowledge to prepare a presentation to a hypothetical
research 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 Foundation
for 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 this
unit. Please contact the Foundation for assistance with furthering the integration of agriculture into
your curriculum. Comments on this unit and on other Agriculture in the Classroom resources are
always welcome and appreciated.
California Foundation for Agriculture in the
Classroom 1997
5
UNIT OVERVIEW
BRIEF DESCRIPTION
This interdisciplinary thematic unit consists of six lessons on genetic engineering in agricultural crops
along with the background knowledge needed to complete the lessons. This unit is designed for
college preparatory biology or physiology students who have a basic understanding of genetics and
advanced knowledge of cell structures and functions. The lessons are designed to introduce
students to biotechnology and genetic engineering in a format that supports critical thinking and
problem-solving on current issues associated with these sciences.
As indicated in the first lesson, one agricultural challenge is presented to the studentsthe presence
of a chemical called gossypol in cottonseed. This chemical acts as a defense mechanism for the
plants, but can be potentially harmful to animals, including humans, if consumed in large quantities.
Through background information gathered from hypothetical, yet realistic, memos, meeting minutes
and newsletters, the students are asked to address the gossypol challenge. The lessons guide the
students to investigate gossypol using a genetic engineering approach and to test their procedures in
the 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 this
project.
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 of
amino 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 of
student homework. It is possible, however, to perform only selected lessons from this six-lesson
unit. Long-range planning is required by the instructor so appropriate materials can be ordered and
prepared.
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California Foundation for Agriculture in the
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SCIENCE CONCEPTS
According to the 1990 Science Framework for California Public Schools, the students should
understand 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 be
predicted (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 is
inserted 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 and
technologies (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 of
others, decision-making, compromising, resolving conflicts and leadership skills (p. 21).
California Foundation for Agriculture in the
Classroom 1997
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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 different
sources.
 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 other
organisms.
 Understand how bacterial DNA can be transformed, having different genotypes than they
once 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 are
discussed in this unit. Definitions for many of these words are incorporated into the lessons. A
partial 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

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California Foundation for Agriculture in the
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BEFORE YOU BEGIN
1.Review the lessons to gain understanding of the unit. Determine which lessons you will use and
when you will teach them.
2.Order the appropriate supplies needed for the DNA transformation activities your students will
perform. 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 83
for 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 are
described 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 been
made so your students can complete the activity without becoming overwhelmed. Several of the
facts and generalizations are discussed below. Share this information with your students as you
feel appropriate.
 The gossypol content in cottonseed is an actual issue in agriculture today. Many attempts have
been made to resolve this issuefrom producing glandless cotton plants that do not contain
gossypol to detoxifying the gossypol in the cottonseed. Unfortunately, to date, none of the
methods have been successful at a commercial level.
 In this unit, references are made to a gossypol gene. In actuality, there are numerous genes
involved in gossypol production. Gossypol production is not a single gene trait.
 Gossypol is actually a secondary metabolite (sesquiterpene), a chemical compound that deters
chewing insects from eating the cotton plant. Gossypol is not a protein.
 Gossypol synthase is the name of the gene which encodes for gossypol synthase enzyme
production. 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 the
vegetable parts of the plant, but not in the cottonseed.
California Foundation for Agriculture in the
Classroom 1997
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TEACHER BACKGROUND INFORMATION ON
GENETIC ENGINEERING
While the sciences of genetics, genetic engineering and biotechnology are complicated, there are
many components that can easily be incorporated into the classroom. The following information can
help 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 useful
products, processes and services. Production can be carried out by intact organisms, such as yeast
or 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 another
example of simple biotechnology. In the 1970s, a new type of biotechnology began genetic
engineering or recombinant DNA technology.
What is genetic engineering?
Genetic engineering is a process where genetic material (DNA) is taken from one organism and
inserted into the cells of another organism. Genetic engineering also can be the rearrangement of
gene location or the removal of genes. The altered organism then makes new substances or
performs new functions based on its new DNA. For example, the protein insulin, used in the
treatment of diabetes, now can be produced in large quantities by genetically engineered bacteria
and yeasts. Insulin was formerly extracted from pigs or cows. Some say the genetic engineering of
plants 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, an
adjustment in the amino acid balance in a particular corn variety improves the corns ability to be
stored.
It can suppress, or stop, an organism from doing something it already does. For example, the gene
that codes for the softening of tomatoes is turned off in a genetically engineered tomato variety so
the tomatoes do not soften as quickly.
It can make an organism do something new. For example, particular bacteria and yeasts have been
genetically 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 living
things. DNA is found in all cells, usually in the nuclei. In bacteria and viruses, which do not have
nuclei, the DNA floats within the cell. DNA is composed of six molecules: sugars, phosphates and
four bases. A gene produces a specific protein or has an assigned function.
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California Foundation for Agriculture in the
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What is a protein?
Proteins are chains of amino acids that perform the necessary functions of living organisms. When
a gene is expressed, that means it is transcribed into mRNA which is used as a template for
translation into a protein. Some of these proteins perform specific functions themselves (such as
becoming insulin or muscle); others participate in the production of cell components (such as
becoming enzyme proteins that assist in making carbohydrates and fats); still others are regulatory
and modify gene expression.
What are some examples of genetically engineered products?
 Human growth hormone, normally produced in the human pituitary gland, can be made in
bacteria 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 without
harming 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 governmental
agencies, have allowed the United States to maintain its safe food record. In fact, the United States
has the safest food supply in the world. The following information will help you better understand the
genetic engineering food safety guidelines.
Before any plant food developed through biotechnology is made available to the public, it undergoes
a safety evaluation. In 1992, the Food and Drug Administration (FDA) issued testing guidelines for
genetically engineered foods. The specific policies are under the title Foods Derived From New
Plant Varieties. There are other policies for products other than plants. The genetically engineered
plant 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 than
on the methods used to produce them.
 Any new food additive produced via biotechnology will be evaluated for safety employing the
same guidelines used for a traditional food additive (such as food coloring).
California Foundation for Agriculture in the
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 Any food product that is found to contain material that could render it unsafe will not be allowed to
enter commerce.
 If the introduced product contains an allergen, or if the production of the food has altered its
nutritional value, the FDA may require informational labels.
As in the case for any food product, any genetically engineered plant food found to contain
substances not in keeping with the safety guidelines may be removed from the marketplace by the
FDA. The United Nations World Health Organization continues to debate the policies revolving
around 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 of
Agriculture (USDA) oversees all field testing of genetically engineered products. Before a new crop
can move into commercial production, the USDA reviews the field-testing results. Field-testing
results and studies must demonstrate that plants altered using biotechnology react with ecosystems
in 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 engineering
practices include the possibility that modifications in the genetic make-up of the plant could result in
some type of unknown toxin. The odds of that occurring in normal plant breeding and selection are
far greater than that occurring in genetic engineering. Genetic engineering involves only the
movement of specific genes with specific functions. In traditional plant breeding, crosses between
different varieties and wild relatives result in the transfer of many genes. The science of genetic
engineering is carefully monitored and the risks associated with any products and processes, such
as 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 must
constantly find ways to increase yields and lower production costs. As farmers continue to look for
renewable resources and safe ways to control pests and fertilize plants, genetic engineers continue
their 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 with
bromoxynil to kill weeds without affecting the cotton plant itself. This method of weed control
greatly reduces the amount of herbicide used on cotton while increasing the yield of cotton per
acre.

 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 plant
oils in canola and soybean plants can be used to produce biodegradable plastics.
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California Foundation for Agriculture in the
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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 to
verify that the replicated gene is precisely what is wanted.
e) GENE IMPLANTATION: Using a bacterium or other procedure, the desired DNA (gene) is
transferred into the chromosomes of the host plant cells.
f) CELL REGENERATION: Researchers select the plant cells that contain the new gene and
regenerate whole plants from the selected plant cells.
g) THE NEW PLANT TESTING: Laboratory and field testing occur to verify the function and safety
of the new plants.
h) SEED PRODUCTION: Seeds with the desired traits are produced using standards set for
specific crop production.
California Foundation for Agriculture in the
Classroom 1997
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LESSON 1: WHAT CAN WE DO?
(An Introduction to Biotechnology)
PURPOSE
The purpose of this activity is for students to realize that biotechnology, specifically genetic
engineering, 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 the
challenges 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)
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California Foundation for Agriculture in the
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TIME
Teacher preparation ..................................................... 10 minutes
Class activity ................................................................ One or two 50-minute sessions
Homework .................................................................... Two nights
BACKGROUND INFORMATION
BIOTECHNOLOGY: If you split up the word, it is easy to understand. Bio stands for biology, living
things. Tech stands for technology, the tools and techniques used to study things. Ology means
the study of. Biotechnology, in simple terms, means the study of living things using various tools and
techniques. Here, biotechnology is the study of how technology is used to impact the function of
living things. Or rather, biotechnology is the development of products using technology in biological
processes.
This lesson presents facts about cotton and current agricultural challenges associated with it. In this
hypothetical yet realistic scenario, students learn about one real challenge that faces the cotton
industrythe high gossypol content in cottonseed. Using this example, the students work as
scientists to confront this challenge through research and development.
Gossypol is a naturally occurring chemical found in the seeds and plants of cotton. Located in the
pigment glands of cotton plants and cottonseed, it deters chewing insects and other animals from
eating the plants. The presence of gossypol in cultivated cotton has been an issue in the cotton
industry for over 40 years.
Cottonseed is used as a food source for both cattle and other animals. Mature cattle can eat small
amounts of cottonseed as part of their diet and be unaffected by the presence of gossypol. The
problem with the presence of gossypol in cottonseed, however, is that it is toxic to animals in high
quantities. For this reason, only limited quantities of cottonseed are permitted in cattle and chicken
feed and it is not a widespread ingredient in human food. The processing methods used to detoxify
the gossypol in the seed meal have been unacceptable because they decrease the nutritional value
of 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 of
producing 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 of
thousands that are being researched today. This topic of study was chosen for many reasons, some
of which include its interesting history and current research possibilities. The issues addressed in
this unit are being addressed in the agriculture research community. To make this unit
understandable by high school students, however, many details have been left out of the research
process.
California Foundation for Agriculture in the
Classroom 1997
15
PROCEDURE
1.Discuss the idea of this unitto examine issues associated with agriculture and to investigate and
propose 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 list
are:
 Find wild strains of cotton that have gossypol in the vegetative parts of the plant, but not in the
seed. Selectively breed those plants.
 Find an economical way to detoxify the gossypol in the cottonseed through some type of
processing.
 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 of
cottonseed to produce human food and silage.
5.As homework, have the students complete a response letter to Mr. Davis. In small groups, have
the student share their response letters with one another.
CONCLUSION
There are many issues associated with agriculture that affect people and the environment. There
are also endless possibilities on how major challenges can be resolved through research and
investigation. 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 way
by 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 issues
associated with genetic engineering.
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M E M O
TO:Caitlin Noonan
Research Director
Agri-Gene, Inc.
FROM:Tom Davis
Senior Research Investigator
Cotton Research Associates
WHAT DO YOU THINK?
I recently attended the 20th Annual United States Department of Agriculture Symposium at the
University of California at Berkeley. It featured a lecture on specific issues in California
agriculture. One of the topics discussed was the gossypol content in cottonseed and cotton
plants and the issues associated with this naturally occurring chemical.
Cotton is an important California commodity and is currently the leading commodity in Fresno
and Kings counties. It is believed that the first cultivation of cotton was in India. The American
Indians grew cotton in the early 1500s with true American cultivation beginning in 1621 when
the English settlers were provided cottonseed from the West Indies. The history of cotton
cultivation and processing techniques is fascinating and I will be happy to talk with you about
this further, if you so desire.
The purpose of this memo, however, is to discuss a possible research partnership in the area
of gossypol reduction in cottonseed. The presence of gossypol in cultivated cotton has been
an issue in the industry for more than 40 years. Here are some facts we already know about
gossypol:
 Gossypol is a naturally occurring chemical found in the seeds and vegetative material
of cotton. This chemical exists in the pigment glands of cotton plants and in the
cottonseed. It deters chewing insects, such as caterpillars and other animals, from
eating 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 the
feed.

 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 relatively
tasteless, flavorings can be added to the seeds to produce items such as chocolate
flavored chips and imitation nuts.
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Classroom 1997
17
 It is the industrys goal to increase the saleability of cottonseed so this by-product from
cotton fiber production can be utilized more efficiently and effectively; therefore, it can
benefit the cotton growers financially and potentially be a currently untapped food
source 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 human
foods and in cattle and chicken feed. Extensive studies have not been done on
tolerance levels of gossypol; however, evidence shows that humans and cattle
exposed 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. Research
indicates that it may also suppress the spread of cancer cells, such as those which
result 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 of
gossypol are not as successful in the field.
Here is our challenge. Cotton Research Associates would like to develop a cultivated cotton
plant that has high amounts of gossypol in its vegetative material, but little or no gossypol in its
seeds. To date, we have not been successful in achieving this goal, but are interested in
providing the funding for research to improve cotton production and the stability of the
cottonseed market.
I have been asked by your Board of Directors to investigate the feasibility of this task. I need
to gather data to present at its next meeting. At that time, the board will determine if funding
will be provided for such a project. I request your input on how to reduce or eliminate gossypol
in 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 and
your continued support of scientific research in agricultural advances and look forward to
hearing from you soon.
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HERES AN IDEA Name(s)



TASK LIST
1) Brainstorm at least four potential ways gossypol levels in
cottonseed can be reduced or gossypol could become less toxic to
livestock and humans. During this brainstorming, you may use
your textbook or other references to help you obtain ideas.
Note: Think of scientific procedures and technologies used today that have resulted
in healthier, more flavorful or more desirable foods. Examples include technologies
used to develop leaner beef, low-fat cheeses, sweeter corn, tastier tomatoes and
seedless grapes.
2) Decide on two methods you think might be worth pursuing in regard
to the gossypol issue. For each method, provide a brief description
on how this could be achieved and the pros and cons for each
method.
3) On a large piece of butcher paper, prepare a visual aid that will
help express your ideas. Prepare a three-minute explanation that
describes 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 from
Agri-Gene, Inc., write a reply to Mr. Davis discussing the various
ideas presented by your class (research team). Be sure to discuss
which method you suggest Cotton Research Associates pursue.
The potential pros and cons of your recommendation should also
be addressed.
California Foundation for Agriculture in the
Classroom 1997
19
COTTON PLANT
20
California Foundation for Agriculture in the
Classroom 1997
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 to
gain background information and begin preparatory work regarding the genetic engineering activity
they will perform.
CONCEPTS
 Genetic engineering is one method used to alter
the genetic make-up of an organism.
 The genotype and the phenotype of an organism
can 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 strongly
suggested that your students have an understanding of the following concepts and vocabulary prior
to this lesson:
 the terms genotype and phenotype, and how the genotype of an organism affects its phenotype;
 how DNA codes for protein;
California Foundation for Agriculture in the
Classroom 1997
21
 transcription and translation, and how they are associated with cell replication and protein
synthesis;
 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 minutes
Student activity .................................................Three to four 50-minute class periods, plus homework
BACKGROUND INFORMATION
The meeting notes are incorporated into this lesson for two reasons. One reason is to continue the
scenario with the students so the upcoming lessons have more meaning. Secondly, the meeting
minutes inform students, in a unique way, much of the background information needed to complete
the upcoming bacterial transformation activity. Based on the prior knowledge of your students, you
may need to explain some of the terminology and concepts. Provide the necessary supporting
activities so your students have comprehended the listed terms by the conclusion of this lesson.
 amino acid
 antisense
 chimera
 complimentary
base pairs
 gene
 genome
 mRNA
 plasmid DNA
 promoter sequence
 protein
 selectable marker
 sense
 termination
sequence
 transcription
 translation
 vector
PROCEDURE
1. Discuss the previous activity, if necessary. Then, have each student individually read the letter
from 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 are
unclear. 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 public
relations newsletter.
4. Allow sufficient time in class for students to develop draft ideas of the newsletter. Also, allow
enough 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.
California Foundation for Agriculture in the
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6.Individually, have the students write a one-page summary describing the most interesting
concepts or facts they have learned. Clarify any misconceptions that have surfaced or plan
appropriate 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 as
ethical 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 that
discusses the information. Use video equipment, if available.
 Assign particular students the roles of the people in the meeting minutes. Have them orally
present 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 how
funding sources for those ideas are obtained.
 Working with the English, social studies, journalism, mathematics and computer teachers at your
school, 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 a
project.
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Classroom 1997
TOM DAVIS
Senior Research Investigator
COTTON RESEARCH ASSOCIATES
123 Leaf Way
Cottonseed, Missouri 54321
September 1, 1999
Caitlin Noonan
Research Director
Agri-Gene, Inc.
Cottontown, California 12345
Dear Caitlin,
Thank you for your suggestion to have an informational meeting about how to
reduce the gossypol content in cottonseed. I have included the minutes from one
meeting we had on this topic. The group of people attending the meeting were very
informative and enlightened me as to the processes our company will be involved in as
we pursue this new venture.
As you will see from the transcription, the Board of Directors decided that a
genetic 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 as
things progress.
Sincerely,
Tom Davis
Senior Research Investigator
Enclosures
cc:Alexandra Hoeppner
Gordon Spicer
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25
GOSSYPOL MEETING TRANSCRIPTION
8/20/99
Sacramento, California
Meeting Attendees:
Tom Davis, Senior Research Investigator, Cotton Research Associates
Alexandra Hoeppner, Research Geneticist, Calgene, Inc.
Gordon Spicer, Agronomist, University of California, Davis
Assistants to Tom Davis, Cotton Research Associates
Tom:Good morning. Thank you for attending our meeting. Our task today is to
investigate the feasibility of genetically engineering cotton plants to
contain cottonseed with little or no gossypol. There are several questions
we 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 be
used 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 and
protein production. Please explain this relationship to me in regards to
gossypol and cotton.
I would like to turn this forum over to Alexandra Hoeppner, who will
discuss how the work at Calgene, Inc. on the Flavr-Savr
®
tomato seeds
was accomplished. The company performed research associated with
this tomato for ten years prior to its development. The genetically
engineered tomatoes are presently sold in selected stores throughout the
nation under the name MacGregors
®
tomatoes.
Alexandra:Calgene developed the MacGregors
®
tomato in response to the tasteless
tomatoes usually available in the grocery stores during the winter months.
Normally, tomatoes consumed in the winter and spring months are picked
green so they can be stored and shipped without becoming too soft. The
tomatoes redden during shipping or in warehouses after being exposed to
ethylene gas, a gas naturally released by ripening fruit. The softening of
a tomato is associated with a single sense gene that causes the
development of a protein called polygalacturonase (PG). At Calgene,
we figured out how to place the backwards version of the PG gene, called
the antisense PG gene, into the tomato genome. The backwards
version is combined with the sense PG gene so replication and
expression of the softening PG gene cannot occur. Thus, the level of the
PG enzyme is reduced and the softening of the tomato is slowed. As a
26
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Classroom 1997
result, the tomatoes can be shipped vine ripened and have a more
flavorful taste.
Tom:How can you get the PG antisense strand to be expressed?
Alexandra:In any organism, whether it is prokaryotic or eukaryotic, transcription of
DNA to form mRNA must occur. In order for transcription to occur, an
enzyme RNA polymerase must bind to a specific recognition site on the
gene. This recognition site is called the promoter region. To reduce PG
enzyme production, the PG gene is removed using restriction enzymes
that cut DNA; then, the inverted gene, ASPG, is inserted next to the
promoter. As a result, the ASPG gene instead of the PG gene is
transcribed and translated. Thus, the polygalacturonase needed for fruit
softening is not synthesized.
Tom:How do you get the PG gene out of the genome and the ASPG gene into
the genome? It seems difficult and complex.
Alexandra:Developing the process was complex, but the process itself incorporates
the natural actions of plants. To accomplish this task, we made use of a
common reaction that occurs when a plant is injured. Frequently, when a
plant gets a cut or another type of wound, it may get infected by a soil-
borne bacterium called Agrobacterium. When the wound is infected by
this bacterium, some of the bacteriums DNA is transferred into the plant
and a tumor is formed. This particular tumor is called a Crown Gall
Tumor. We used this knowledge to our benefit.
We also made use of a natural feature of many bacteria. Many bacteria
contain genetic material called plasmids. Agrobacterium, like other
bacteria such as E. coli, contain two types of genetic material, one larger
chromosome and numerous smaller circular plasmids. Plasmids are
relatively easy to remove from bacterial cells. Using specialized
enzymes, called restriction enzymes, the PG gene is removed from the
tomato genome, flipped backwards and then placed into a plasmid. The
insertion of a gene is done with ligating enzymes. The backwards version
of this gene is called the antisense PG gene or the ASPG gene. The
plasmids containing the ASPG gene are put into living Agrobacteria.
Then, when a wound is purposely made on a tomato leaf, the
Agrobacteria containing the ASPG gene goes into the wound site and
creates a callusa scarand transfers the ASPG gene into the plant.
From this callus, tissue culture techniques are used to grow tomato plants
from a single cell of the callus. Hence, the tomato plants grown from the
callus have the new feature of the reduction of the non-softening PG
enzyme because it binds with the ASPG gene that entered the cell
through the plasmid. Its a complex process, but is relatively easy now
that we have the method down. (As Alexandra speaks, she is writing this
schematic diagram on the board.)
California Foundation for Agriculture in the
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Classroom 1997
Tom:It seems that this new tomato plant genome is composed of DNA from
several different sourcesthe original plant and Agrobacterium.
Alexandra:Yes, it is. This is called a chimera. Chimera means from several
sources. In this case, the genome is from the original tomato plant as
well as the Agrobacterium, which contains the antisense PG gene cloned
from a different tomato plant.
Tom:I heard that you grew the Agrobacterium in a solution that contained the
antibiotic Kanamycin. Why was this done and what effects will this
antibiotic have on the person who eats the tomato? Can they become
resistant to this antibiotic by eating your tomatoes?
Alexandra:It is common practice to use what is called a selectable marker when
transferring genetic material. Let me explain. . . When the plasmid is
made, it contains the promoter sequence, the ASPG gene, the selectable
marker and a terminating sequencea sequence on the DNA which stops
the formation of mRNA. When the mRNA is made during transcription,
both the ASPG gene and the selectable marker gene are transcribed at
the same time. The selectable marker for our tomatoes is the resistance
to the antibiotic Kanamycin. This antibiotic resistance serves several
purposes. It serves as a control so only the bacteria we want will survive.
You see, after the plasmid is placed back into the bacterium, the
bacterium is grown on a special medium plate which contains the
antibiotic. It will kill all of the bacteria except those that have been
transformedthose that have the softening gene removed and the
Kanamycin resistance gene. Secondly, it is easy to see antibiotic
resistance in bacteriathe bacteria either grow or dont. It is much more
time-consuming to see if the softening occurs or doesnt occur.
Therefore, when a new gene is inserted, a gene for antibiotic resistance is
also included. Sometimes two selectable markers are used.
CHIMERIC GENE CONSTRUCT DIAGRAM
And . . . to answer your questions about humans becoming resistant to
the antibiotic during the processthis is, frankly, not possible. When
people eat the tomatoes, they digest the DNA as well. The DNA is not
being inserted into the human cells. The antibiotic resistance is part of
the plant genome and is only recognized by plants.
Tom:What about the question of feasibility concerning the use of this
procedure for reducing the gossypol content in cottonseed? Gordon, can
you address this?
Gordon:I believe that it is theoretically possible to reduce the gossypol in
cottonseed without reducing the amount of gossypol in the adult plant.
Promoter
Sequence
Terminator
Sequence
Selectable
Marker
Gene
of Interest
California Foundation for Agriculture in the
Classroom 1997
29
This question intrigued me and I asked one of my graduate students to
begin researching this question. She found out that it is possible to
produce a gossypol-free plant. This has been done by breeding cotton
plants that are homozygous recessive for glandless cotton. These plants
will produce only plants that do not make gossypol. They are not grown
commercially at present because they do not resist chewing insects;
therefore, they are not as productive and may require more use of
chemical pesticides. These homozygous recessive plants, however, will
suit our research purposes since they produce no gossypol in their
genomes. I would suggest that we start by determining if a glandless
plant can make gossypol when a genetically engineered plasmid
containing the gossypol gene is inserted.
If this works, this is half our battle. We still need to somehow transform
the 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 cotton
that have a gene that regulates the production of gossypol synthase so
that gossypol is made only in an adult plant, but not the embryonic part or
seed. If we can isolate this gene, we can create glandless plants that can
be infected with a transformed bacterium that contains both a gossypol
gene and a regulator gene. This way gossypol will be made only in the
adult 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 gossypol
content or will you try to just control gossypol production?
Produce glandless gossypol plants
from wild strains
Isolate gossypol gene and
gossypol regulator genes from
cotton plants
Insert gossypol and gossypol
regulator genes into
glandless cotton
Use tissue culture techniques to
produce cotton plants which
have gossypol in the plants,
but not in the seeds.
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Gordon:Good question . . . At this point, I think it would be best to insert the
gossypol gene and then regulate it with a promoter sequence rather than
incorporate the antisensemirror image of the gene. Another thing, really
quick if I may . . . we are calling this gene the gossypol gene for
simplicity. In actuality, it is a group of genes that produce an enzyme
called gossypol synthase. This gossypol synthase is the protein that
causes gossypol production. Were after the genes that create this
enzyme. But for ease, lets continue to call the group of genes needed
the gossypol gene.
Tom:Do you have suggestions about how we can do some short transformation
experiments to see what might happen?
Gordon:Well, in fact, I do! Not only do I have a graduate student who has isolated
this gossypol gene, we think we have a selectable marker that can be
used as well. Last summer, a high school student, Jorge Villalobos, took
a five-week summer research internship with us. Jorge not only mapped
the gene adjacent to the gossypol gene, but in a serendipitous event,
determined that this adjacent gene becomes luminescent when grown in a
medium that contains a special sugar called arabinose. It would make
an easy second marker gene for you. You could immediately know if
transformation has occurred correctly just by looking at the bacteria grown
in the media containing arabinose. Since it glows beautifully under a UV
light, we named this plasmid the PGLO

plasmid.
The research that you are proposing will be quite involved. I propose that
we try to attach the gossypol gene to the glow marker, put these genes
into a plasmid and then insert the plasmid into E. coli or Agrobacterium. If
this is possible, we should be able to use tissue culture techniques to
grow 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 just
created. This will take quite a bit of research and will require a major
funding source.
Tom:That sounds great! It gives us a great place to start. I would like to
publish an account of all of this information in our PR newsletter. This will
keep the farmers abreast of what is happening in research and will inform
our staff and stockholders of potential products. Hmmm. . . I wonder what
environmental impact studies we will have to do as this progresses! Lets
keep this in mind.
Thank you everyone, for participating in this meeting. There has been an
extensive amount of information presented here. My committee and I will
assimilate it and ask further questions as they arise.
California Foundation for Agriculture in the
Classroom 1997
31
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 create
one 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 Research
Associates is involved in and to be forthright in discussing issues and challenges that
this particular research project addresses.
Your newsletter should have a minimum of six articles, plus any other information you
would like to add (pictures, upcoming events open to the public, etc.). Determine how
the newsletter tasks will be divided among your group members. Each member of your
group 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 engineering
venture 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 this
knowledge going to be applied to the cotton industry?
 What considerations need to be taken into account when placing a gene into a
plasmid?
 What are some ethical issues that surround genetic engineering and how
should 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?
32
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The following vocabulary should be included in your articles: Agrobacterium, amino
acid, biotechnology, chimera, chromosome, enzyme, restriction enzyme, gene, genetic
engineering, mRNA, plasmid, protein, promoter sequence, selectable marker,
terminating sequence, transcription, translation and vector.
_____ 1) Review the meeting minutes and discuss with your colleagues any areas
that confuse you. Assist your colleagues in understanding the key points.
_____ 2) Skim over newsletter samples you have at home and those provided by
your teacher. As a team, discuss format and content ideas you particularly
like 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 group
the contents of each article.
_____ 5) Write the draft articles individually.
_____ 6) Review the work of your colleagues for technical accuracy, grammar and
spelling.
_____ 7) Review the articles to make sure all required vocabulary words are
included.
_____ 8) Prepare the final version of the newsletter, making sure you follow any
rules and guidelines provided by your instructor regarding size, content and
style.
_____ 9) Share your newsletter with your classmates.
CREATING THE
NEWSLETTER
California Foundation for Agriculture in the
Classroom 1997
33
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 one
organism can be inserted into DNA of another organism. The focus is on the insertion of the
gossypol 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 a
promoter sequence, a gene of interest, a selectable
marker 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)
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TIME
Teacher preparation ..................................................... 15 minutes
Student activity ...............................................................One 50-minute session, plus a 15-minute
summary and review
BACKGROUND INFORMATION
Students should have a basic understanding of the functions of DNA and RNA including the
processes of transcription and translation. Transcription is the process in which RNA polymerase
synthesizes a messenger RNA chain by reading the code of a DNA sense strand. Translation is the
process in which the messenger RNA (mRNA) is decoded to produce amino acids. The amino acids
link together in specific sequences directed by the mRNA to form specialized proteins. These
proteins are then used somewhere in the cell.
A chimeric gene construct generally consists of a promoter sequence, the gene of interest, a
selectable marker and a terminator sequence. The promoter sequence indicates the place in which
DNA transcription for protein synthesis should begin. The promoter can also dictate where and
when the gene of interest is expressed. The gene of interest is the gene that one wishes to transfer
to another organism. Examples include virus resistance, color or added nutrition. Since it often is
difficult to determine whether a specific gene, such as increased nutrition, is transferred, a selectable
marker is used. A selectable marker is an easily identifiable gene, such as antibiotic resistance or
phosphorescense, that is attached to the desired gene so that it is obvious whether or not the genetic
transformation has occurred. The terminator sequence is a section of DNA that stops the mRNA
translation for protein synthesis.
CHIMERIC GENE CONSTRUCT DIAGRAM
Chimeric gene constructs are created by bringing bits of DNA together from various sources. These
sources may include DNA from bacteria, viruses, plants or animals. A list of genetically engineered
crops and the source of the gene inserted is located on page 81. Genetic engineers isolate desired
genes using restriction enzymes. Restriction enzymes identify specific sequences of DNA bases
and make a cut at these specific sites on a DNA molecule. Ligating enzymes glue bits of DNA
strands together.
By cutting the DNA molecule into small pieces using restriction enzymes, scientists are able to
discover and study genes. There are four restriction enzymes commonly used in genetic
engineering. Each type differs in the type of cut it makes. Over 175 different restriction enzymes are
known and are characterized with respect to their cleavage sites and the sticky ends that are
available for attachment. Sticky ends are the single strands of DNA left after a restriction enzyme
separates the base pairs. Some examples are described below. The Bam HI and Sac I restriction
enzymes are used to cut the polygalaturonase genes from tomato genomes. This method is used by
Calgene, Inc. to produce the MacGregors
®
tomatoa tomato that resists softening.
Promoter
Sequence
Terminator
Sequence
Selectable
Marker
Gene
of Interest
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Restriction Enzyme Recognition Site
Cleavage Site
EcoR I GAATTC G / AATTC
Hind III AAGCTT A / AGCTT
Bam HI GGATCC G / GATCC
Sac I GTCGAC G / TCGAC
/ = cleavage site
This activity shows the students how restriction and
ligating enzymes work. It is important for the students
to realize that where a gene inserts itself into another
genome is dependent on biochemical and
environmental factors as well as the availability of
sticky ends. Where a chimeric gene construct
chooses to attach itself can affect other processes of
the organism.
A typical gene insertion challenge is that scientists
want a gene to insert itself in a location that will not
affect other plant genes. One such example has
happened in the cotton plant. Researchers were able
to isolate a gene that could increase fiber strength.
The problem was that it always inserted itself in the
middle of a gene sequence that caused cotton boll
formation. The cotton boll is where the fiber grows.
Thus, when the gene for fiber strength was inserted
the mechanism for cotton boll formation was
interrupted and no cotton bolls would form. In
conclusion, the fiber strength gene could not be
expressed. Since this study, other fiber strength
genes have been identified and have been
successfully inserted into certain cotton varieties.
PROCEDURE
1.Read aloud the memo to Tom Davis from Alexandra Hoeppner (p. 39). Discuss how
communication 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 it
individually 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 occur
for DNA insertion into a plasmid. Have them save their ideas and amend them at the conclusion
of 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 as
well as actual research challenges.
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6.Have the students refer to their pre-activity sequencing maps. With their newly gained
knowledge from this activity, have them amend or rewrite the sequence of events necessary to
insert 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 ginning
process, the varieties of cotton, the economic impact of cotton on society, etc.
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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 reading
selection that discusses how DNA is added to a plasmid. I think it explains what we will
be doing. Remember, we will cut out the gossypol gene from a cotton genome and
then put it into E. Coli plasmids. This plasmid example depicts most of the preliminary
work that we will be testing.
The gossypol gene has already been mapped. We now need to determine which
restriction enzyme we can use to insert the gene into the plasmid genome. We are
working it out on paper prior to trying it in the lab. I hope you can join us in this
preliminary work. By participating in placing the plasmid into the bacterial cell, you will
gain understanding of the steps in this process which will help you prepare for your
presentation to your board.
I have attached an idea that I have used in the past. It shows how DNA is inserted into
a plasmid on the molecular level. I have used this idea during presentations, and it
may be something you can use with your Board of Directors. I hope it helps to clarify
our 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 small
circular pieces of DNA called plasmids. These
plasmids contain genes which code for proteins that
are beneficial to the survival of the cell.
The first plasmids discovered contained genes for
antibiotic resistance. Geneticists believe that plasmids
contain these genes because they neutralize the action
of an antibiotic on the bacterial cell. To counteract the
effects of antibiotics which kill the bacteria, large
quantities of the enzyme are required. More copies of
the antibiotic-resistant gene that produces the enzyme
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can be carried on several plasmids than can be
incorporated into one large chromosome.
Geneticists take advantage of prokaryotic plasmids
by incorporating the DNA of a desired gene into the
genome of the plasmid. When the plasmid replicates,
the desired gene is also replicated. This way the
information in the gene is passed from one generation
to the next as the bacterial cell divides. More
importantly, as the DNA in the plasmid is transcribed
into mRNA, the desired DNA from the implanted gene
also gets transcribed into mRNA so that translation
can occur. Translation is when the mRNA is decoded
to produce amino acids which attach together to make
the desired proteins.
Plasmids have been used in the process of gene
splicing in a variety of study areas, including
medicine and agriculture.
To perform the necessary procedure that will place
a piece of DNA into a plasmid, researchers use a
restriction enzyme. A restriction enzyme is a
specialized enzyme that cuts the DNA at a site where
the base pairs are arranged in a specific order. For
example, the restriction enzyme Bam HI cuts DNA
between 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 is
the same in all organisms, the same enzymes can be
used in both prokaryotic and eukaryotic cells.
When geneticists want to insert a gene into another
organism, they cut out the desired DNA from an
organism using restriction enzymes. Using the same
restriction enzyme, plasmids from a bacterial cell are
cut in one spot to open it up. When DNA is cut, or
spliced, this leaves the two open ends chemically
active. These chemically active ends are called
sticky ends. Because of DNAs complimentary
base-pairing rules, a sticky end will readily
recombine with another piece of DNA with
complimentary bases in order to chemically bond and
once again become stable. When the new DNA is
placed in with the cut plasmid, ligating enzymes are
used to seal the new connection. It is possible for
plasmids to recombine with themselves or with other
compatible sticky ends to get a genome arrangement
other than the one desired.
After the plasmid has been inserted into a
bacterium, the scientist grows the bacterium on an
agar plate to create the colony of bacteria with the
new genotype. A selectable marker is used to identify
the cells that have been transformed in the desired
way.
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GOSSYPOL DNA
1 2 3 4
A G G
GGGGG
C A T G

C
G C A
GGGGG
T A T G

C
A T T
GGGGG
A A T G

C
A T C
GGGGG
G T A G

C
A T G
GGGGG
C T A G

C
G C C
GGGGG
G T

A A

T
G C G
GGGGG
C T

A A

T
G C G
GGGGG
C A

T A

T
C G G
GGGGG
C A

T T

A
A T G
GGGGG
C A

T T

A
A T A
GGGGG
T C

G T A
A T A
GGGGG
T C

G T A
T A A
GGGGG
T C

G C G
T A A
GGGGG
T C

G G C
T A A
GGGGG
T T

A C G
A T T
GGGGG
A T

A C G
G C T
GGGGG
A A

T A T
G C T
GGGGG
A G

C A T
C G C
GGGGG
G G

C A T
C G C
GGGGG
G G

C G C
C G C
GGGGG
G G

C G C
A
GGGGG
T T
GGGGG
A G

C C G
A
GGGGG
T A
GGGGG
T A

T C G
A
GGGGG
T G
GGGGG
C A

T T A
A
GGGGG
T G C A

T T A
A
GGGGG
T G C T

A T A
Glow Gene Gossypol Gene
GGG

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PLASMID DNA
1 2 3 4
A T A T C  G A T
C G A  T C  G A T
G C T  A G  C A T
G C T  A A  T C G
G C T  A G C C G
T

A C  G A T T A
C

G C  G A T G C
A

T G  C T A G C
A

T C  G C G A T
C

G A  T G  C G C
G

C G  C G  C G C
G C C  G A  T G C
G C T  A T  A C G
G C C  G A  T T A
C G G  C T  A T A
A T A  T A T A T
A T G  C G C G C
G C A  T C G T A
G C T  A C G T A
C G C  G A T A T
C G C  G A T A T
A T C  G A T A T
A T G  C C G C G
G C T  A C G C G
G C A  T G C G C
G C T  A A T C G
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Promoter Sequence Ampicillin Gene
Terminator Sequence
RESTRICTION ENZYME REFERENCE PAGE
C
C
T
G
G
G
G
A
C
C
Ava II
C
T
C
G
A
G
G
A
G
C
T
C
Sac II
T
T
C
G
A
A
A
A
G
C
T
T
Hind III
G
G
A
T
C
C
C
C
T
A
G
G
Bam HI
G
G
C
C
C
C
G
G
Hpa II
G
A
A
T
T
C
C
T
T
A
A
G
EcoR I



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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 you
understand the workings and complexities of restriction and ligating
enzymes as they pertain to inserting a piece of DNA into plasmid DNA.
At the conclusion of this activity, you should be quite familiar with restriction
and ligating enzymes, sticky ends, DNA structure and chimeric gene
components.
II. MATERIALS
For each partnership:
 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 the
keys, find the areas needed to produce the desired chimeric gene and
then answer Question #1.
2.Observe the available restriction enzymes on the attached page.
Remember . . . restriction enzymes cut the DNA. Answer Question #2.
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3.Using scissors, cut the plasmid DNA and attach each strip to form one
long strand of DNA. Attach in the order that the lanes are numbered
1-2-3-4.
4.Attach the loose end of #4 to the loose end of #1 to form a circular
plasmid.
5.Cut and attach the plant DNA in a similar manner as you did the
plasmid DNA, but do not attach to form a circle. This represents the
piece of the eukaryotic gossypol DNA that will be placed into the
plasmid.
6.From the restriction enzyme page, locate the area on the plasmid where
each restriction enzyme will cut. Pencil in where the cut would be.
Repeat for the plant DNA. Complete the chart under Question #3 and
answer Question #4.
7.Using scissors, cut the plasmid in the same manner that the chosen
restriction 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 the
sticky 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 at
the sticky end sites. You should end up with a larger circular plasmid
than found in #4.
11.Double check to make sure that all of the needed parts of the plasmid
DNA are present for the desired protein to be made. Answer Questions
#5 through #8 and complete the Conclusion Statement as homework.
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IV. QUESTIONS (Write your answers on a separate sheet of paper)
1.Explain what areas of the plasmid DNA and the plant DNA will be
needed 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:
Restriction
Enzyme
Should
use?
(State yes or no)
Why? Give your reasons why the
enzyme 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 was
not?
6.If an analogy can be made about the scissors representing the
restriction enzyme, what would the tape represent?
7.Does the plasmid you just made contain all of the components needed
to make protein synthesis occur? Explain your answer.
8.Many diabetics use human insulin that is made from the bacterial cell
E. coli. How can a eukaryotic gene be placed in a prokaryotic cell?
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V. CONCLUSION STATEMENT

Write a summary statement for Mr. Davis that he can use as a handout for
his presentation to the Board of Directors.
Special thanks to John Fedors of San Diego, California, for his contributions to
this lesson.
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DNA INSERTION INTO A PLASMID
(Answer Key)
1.Explain what areas of the plasmid DNA and the plant DNA will be needed to create the
desired chimeric gene.
The glow gene and gossypol gene need to be cut from the plant DNA strand (requiring
two cuts). The plasmid gene will need to open up after the promoter and before the
terminator sequence (requiring one cut). The ampicillin gene on the plasmid DNA
must 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:
Restriction
Enzyme
Should use?
(State yes or no)
Why? Give your reasons why the enzyme should or
should 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 the
middle of the ampicillin gene.
Hpa II
Yes Cuts out the glow and gossypol genes in the
plant. Opens the plasmid in one spot and does
not interfere with the promoter sequence,
ampicillin gene or terminator sequence.
Bam HI
No Cuts plant DNA, but not plasmid DNA. Will
interfere with the glow gene.
EcoR I
No Does not cut either plant or plasmid DNA.