Teacher Notes for "Genetic Engineering Challenge – How can scientists develop a type of rice that could prevent vitamin A deficiency?"1

This analysis and discussion activity begins with an introduction to vitamin A deficiency, rice seeds, and genetic engineering. Next, several questions challenge students to design a basic plan that could produce a genetically engineered rice plant that makes rice grains that contain pro-vitamin A. Subsequent information and questions guide students in developing an understanding of the basic techniques of genetic engineering. Students use fundamental molecular biology concepts as they think about how to solve a practical problem. This activity can be used to introduce students to genetic engineering or to reinforce basic understanding of genetic engineering.

Before students begin this activity, they should have a basic understanding of DNA, proteins, and transcription and translation. To provide this background, you may want to use:

the hands-on activity "From Gene to Protein – Transcription and Translation" (http://serendip.brynmawr.edu/sci_edu/waldron/#trans) or

the analysis and discussion activity “From Gene to Protein Via Transcription and Translation” (http://serendip.brynmawr.edu/exchange/bioactivities/trans).

These Teacher Notes propose several follow-up activities, including: “Golden Rice – Evaluating the Pros and Cons” (which engages students in evaluating the

arguments and evidence in favor of an opposed to the development of genetically engineered Golden Rice)

“Cut it out! Editing DNA with CRISPR-Cas9” (which introduces a powerful new genetic engineering tool).

Learning Goals Promote student understanding of molecular biology concepts, e.g.:

Genes code for proteins (including enzymes). The genetic code is universal. Transcription of genes is the first step in producing proteins. (Almost) all the cells in an organism have the same genes in their DNA, but different

types of cells have different amounts of the various proteins, which results in the different characteristics of the different types of cells.

Differences in the rate of transcription of specific genes are a major cause of the differing amounts of specific proteins in different types of cells.

Promoters at the beginning of each gene play a crucial role in regulating the rate of transcription of each gene in different types of cells.

Introduce genetic engineering concepts, including plasmids and recombinant DNA Develop an understanding of the basic steps of genetic engineering to produce genetically

modified food plants

In accord with the Next Generation Science Standards2: Students prepare for the Performance Expectation HS-LS3-1. "Ask questions to clarify

relationships about the role of DNA and chromosomes encoding the instructions for characteristic traits passed from parents to offspring."

1 By Dr. Ingrid Waldron, Department of Biology, University of Pennsylvania, 2017. These Teacher Notes and the related Student Handout are available at http://serendip.brynmawr.edu/exchange/bioactivities/geneticengineer .

2 Next Generation Science Standards, available at http://www.nextgenscience.org/next-generation-science-standards 1

Students learn the following Disciplinary Core Ideas: "Genes are regions in the DNA that contain the instructions that code for the formation of

proteins." (LS1.A – Structure and Function) "The instructions for forming species' characteristics are carried in DNA.… Not all DNA

codes for a protein; some segments of DNA are involved in regulatory or structural functions… " (LS3.A – Inheritance of Traits)

Students engage in recommended Scientific Practices, including "constructing explanations (for science) and designing solutions (for engineering)".

This activity provides the opportunity to discuss two Crosscutting Concepts: "Cause and effect: Mechanism and explanation" and "Structure and function".

This activity will also help students meet Common Core English Language Arts Standards for Science and Technical Subjects, including "determine the central ideas or conclusions of the text; summarize complex concepts, processes or information presented in a text by paraphrasing them in simpler but still accurate terms".3

Suggestions for Discussion and Background InformationTo maximize student participation and learning, I suggest that you have your students work individually or in pairs to complete groups of related questions and then have a class discussion after each group of related questions. In each discussion, you can probe student thinking and help them develop a sound understanding of the concepts and information covered before moving on to the next group of related questions. I recommend that you have a class discussion after question 4, at the end of the section on "Inserting the Desired Genes in the DNA of Rice Plants", and at the end of the section on "Ensuring that the Genes for the Enzymes to Make Pro-Vitamin A are Active in Rice Grain Cells".

A key for this activity is available upon request to Ingrid Waldron, iwaldron@sas.upenn.edu. Some additional background information and suggestions for discussion are provided below.

(continued)

3 From Common Core Standards Initiative, http://www.corestandards.org/ELA-Literacy/RST/11-12 2

Pro-vitamin A, also called beta-carotene, is found in many plant foods (e.g. deep orange and dark green vegetables such as carrots, sweet potatoes and spinach). As shown in the diagram below, beta-carotene can be enzymatically split to form retinal (a form of vitamin A that can be converted to the other forms of vitamin A used in the body). Vitamin A is found in some animal foods (especially liver). Excess vitamin A in the diet can be toxic. Pro-vitamin A may be a safer dietary source of vitamin A since increased intake of beta-carotene results in decreased conversion of beta-carotene to vitamin A.

Vitamin A deficiency is widespread in children in poor countries, resulting in an estimated 250,000-500,000 cases of blindness and up to 2.5 million deaths each year.

White rice has been milled and polished to remove the hull, bran and germ, leaving only the endosperm which contains thousands of cells, abundant starch and some protein. In the figure in the Student Handout, the endosperm is labeled as "white rice". The major advantage of white rice is the removal of most of the oils, which tend to become rancid when stored, especially at warm temperatures. Brown rice has the hull removed, but keeps the germ and bran. The major advantage of brown rice is higher levels of B vitamins and vitamin E. Neither white rice nor brown rice provides pro-vitamin A or vitamin A.

Question 2 will serve to remind the students that genes code for proteins, including enzymes that synthesize other needed molecules such as pro-vitamin A.

With respect to question 3, genetic engineering to produce recombinant DNA in transgenic organisms is only useful because the genetic code is universal, so any type of organism can transcribe and translate a gene from any other type of organism.

Question 4 is designed to get students thinking about the problems that scientists confront when developing a genetically engineered plant. Question 4a may be challenging for students if they do not have any background information about genetic engineering; it is hoped that having students discuss this question in pairs will stimulate them to come up with some possible strategy, even if it is only injecting DNA in the rice cells' nuclei. With respect to question 4b, some students may recognize that it will be easier to transform a small group of embryonic cells that can replicate the inserted gene every time the cells divide and thus produce a rice plant that will have the inserted gene in every one of the thousands of cells in each rice grain.

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The bacterium discussed in the section "Inserting the Desired Genes in the DNA of Rice Plants" is Agrobacterium tumefaciens. This bacterium can inject a crucial part of the DNA in its plasmid into plant cells to form recombinant DNA in the plant cell nucleus. The bacterial part of this transgenic DNA contains genes that code for enzymes to make opines (modified amino acids that are synthesized by the plant cells and leak out where they are consumed by the bacteria as food) and genes that code for the production of plant hormones that stimulate plant cell division and the production of Crown galls (as shown below; the plasmid is called Ti or tumor-inducing because it induces the formation of Crown galls). These Crown galls provide abundant food for the bacteria to grow and multiply. To make use of the genetic engineering capabilities of this bacterium, scientists need to insert the genes for the enzymes to make pro-vitamin A into the T-DNA of the Ti plasmid and remove the Crown gall-inducing bacterial genes from the T-DNA of the Ti plasmid.

http://www.btny.purdue.edu/Extension/Pathology/PHM/BD/picts/agrobacteriumlc.jpg

Useful sources of additional information are "Gene manipulation in plants" (http://www.open.edu/openlearn/science-maths-

technology/science/biology/gene-manipulation-plants/content-section-2.1) "The Microbial World: Biology and Control of Crown Gall"

(http://archive.bio.ed.ac.uk/jdeacon/microbes/crown.htm).

This is just one example of how scientists frequently make use of the specialized capabilities of biological systems to carry out genetic engineering. Another example is the use of viruses to carry DNA into human cells for gene therapy.

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The section, “Ensuring that the Genes for the Enzymes to Make Pro-Vitamin A are Active in Rice Grain Cells”, engages students in thinking about the regulation of genes; this introduces an important general concept and also provides the basis for understanding one aspect of genetic engineering. The specific promoter used for genetically engineering rice plants that produce pro-vitamin A in rice grains is a promoter for an endosperm-specific storage protein; this ensures that the transformed genes are transcribed in rice grains.

Depending on your students, you may want to use the following diagram to discuss the role of the promoter. Of course, the regulation of genes in eukaryotic cells involves much more than just a promoter, but it is the promoter that needs to be incorporated at the beginning of the coding region of the gene as it is prepared for use in genetic engineering.4

http://www.citruscollege.edu/lc/archive/biology/PublishingImages/0262l.gif

The class discussion of student responses to question 12 should link back to issues, concepts and problems that were included in your discussion of question 4. You may find the figure on the next page useful for this discussion, although it shows some additional information not included in the Student Handout and the plant it shows is not a rice plant. If you have your students complete the Extension Activity (see next page), your class discussion of this extension activity should refer back to your discussion of question 12. Even if you do not have your students complete the Extension Activity, you may want to read the second reference, which provides helpful additional information.

4 Additional information about preparing the genes for genetic engineering is provided in "How to Make Transgenic Plants: Animation Demo" (http://cls.casa.colostate.edu/transgeniccrops/animation.html). Explanations of the regulation of transcription are available at http://www.nature.com/scitable/topicpage/gene-expression-14121669 and http://sandwalk.blogspot.com/2008/09/how-rna-polymerase-binds-to-dna.html An excellent brief video description of regulation of eukaryotic DNA transcription is available at http://www.hhmi.org/biointeractive/regulation-eukaryotic-dna-transcription. "Regulating Genes" (http://www.pbs.org/wgbh/nova/education/interactives/regulating-genes/) is a useful introduction to Evo Devo (how changes in gene regulation influence development and how these changes have contributed to evolution).

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(http://classes.midlandstech.edu/carterp/Courses/bio225/chap09/09-18_TiPlasmid_1.jpg )

Follow-up ActivitiesThe genetically engineered rice plants that produce pro-vitamin A in rice grains are called Golden Rice. The discussion activity, "Golden Rice – Evaluating the Pros and Cons" (available at http://serendip.brynmawr.edu/exchange/bioactivities/GoldenRice), engages students in evaluating the evidence and arguments related to Golden Rice and other possible strategies for preventing vitamin A deficiency. Students use this information to develop evidence-based conclusions about Golden Rice and other proposed strategies. Students also develop questions that could provide important additional information for evaluating the arguments in favor of and opposed to Golden Rice and other policy proposals. In addition, students analyze how two reasonably accurate articles can present totally opposing points of view on a complex policy issue.

Recently, researchers have developed a much more efficient method of genetic engineering. This method is described in a four-minute video, “Genome Editing with CRISPR-Cas9” (https://www.youtube.com/watch?v=2pp17E4E-O8). “Cut it out! Editing DNA with CRISPR-Cas9” (http://sciencecases.lib.buffalo.edu/cs/collection/detail.asp?case_id=915&id=915) is an analysis and discussion activity that develops student understanding of current research on gene editing to treat muscular dystrophy in mice. For a high school class, you might want to use the first two pages of this case study with their links to two resources that should be appropriate for students who have a high school background in molecular biology. You may want to omit the last two pages of the case study since they depend on reading and understanding a technical review of the molecular biology of gene editing.

An Extension Activity is shown on the last page of these Teacher Notes. This extension activity introduces students to many of the complexities and specific molecular procedures used in the type of genetic engineering discussed in this activity, including:

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the use of restriction enzymes to make recombinant plasmids and the use of transgenic bacteria to clone the gene of interest

the need for a marker that will allow scientists to select for plant cells that have been transformed

the need for a termination sequence for the coding region of the gene an alternative method of transforming plant cells (biolistic transformation using a gene

gun) the need to cross the transgenic rice plants with local breeds that have desirable

characteristics such as adaptation to local weather conditions and resistance to a variety of plant pests and diseases.

Some additional steps are needed before a genetically engineered food plant can be released for agricultural use (e.g. testing the safety of the food and testing for possible adverse environmental effects).

The second recommended source in the Extension Activity also illustrates the iterative nature of scientific research; this reading describes a few of the multiple steps involved in continuously improving the effectiveness of the genetic engineering techniques by repeatedly trying different approaches and using the results of these tests, together with new ideas, to develop new approaches to be tested. This iterative process has included:

trying different techniques to transform rice plant cells, research on the biosynthetic pathway for producing beta-carotene which has shown that

only two enzymes need to be genetically engineered into the rice plant, research to find specific versions of the genes for these enzymes that result in production

of higher levels of beta-carotene. Using this extension activity will also illustrate the iterative nature of learning; we need to begin with some basics, including a basic conceptual framework, and then repeatedly improve our understanding by incorporating new information and concepts with our previous understanding.

Additional information about some of the technical aspects of genetic engineering that are not included in this activity is available from: "Development of Recombinant DNA" (http://ocw.mit.edu/courses/biology/7-01sc-

fundamentals-of-biology-fall-2011/recombinant-dna/development-of-recombinant-dna/) "Basic Mechanics of Cloning: Restriction Enzymes and Cloning Vectors"

(http://ocw.mit.edu/courses/biology/7-01sc-fundamentals-of-biology-fall-2011/recombinant-dna/basic-mechanics-of-cloning/)

To learn about Cloning Animals, see http://learn.genetics.utah.edu/content/cloning/, which includes "What is cloning?", "Click and Clone", "Why clone?", "Cloning Myths", etc.

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Extension Activity – Learning More about How Scientists Do Genetic Engineering

Scientists have developed a genetically engineered type of rice called Golden Rice which produces rice grains with substantial quantities of pro-vitamin A. The actual process has been significantly more complex than the basic steps described in the activity you have completed. For useful additional information read and view: "How do you make a transgenic plant?" (including an animation demo from the University

of Nebraska at Lincoln; http://cls.casa.colostate.edu/transgeniccrops/how.html) (http://cls.casa.colostate.edu/transgeniccrops/animation.html)

"Gene Manipulation in Plants" (http://www.open.edu/openlearn/science-maths-technology/science/biology/gene-manipulation-plants/content-section-4.3)

As you read these sources, note any additional steps or modifications of the procedures needed to genetically engineer rice plants that produce pro-vitamin A in their rice grains.

Prepare a revised and expanded description of the steps needed to produce rice plants that make significant quantities of pro-vitamin A in their rice grains.

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