This lesson compares and contrasts prokaryotic and eukaryotic cells and examines the form and function of the plasmid found in prokaryotic cells. Students will then use these principles to simulate how a desirable gene can be isolated and inserted into a plasmid as one step in the process of creating a bioengineered (GMO) plant variety. Grades 9-12

biotechnology: the use of living systems and organisms to develop or modify products or processes

chromosome: a threadlike structure of nucleic acids and protein found in the nucleus of most living cells, carrying genetic information in the form of genes

deoxyribonucleic acid (DNA): deoxyribonucleic acid; a self-replicating material present in nearly all living organisms as the main constituent of chromosomes; the carrier of genetic information

eukaryotic cell: cells containing membrane-bound organelles such as the nucleus

genetically modified organism (GMO): any organism whose genetic material has been altered using genetic engineering techniques

marker genes: a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA

nucleus: an organelle present in most eukaryotic cells containing genetic material

plasmid: a genetic structure in a cell that can replicate independently of the chromosomes; typically a circular DNA strand in the cytoplasm of a bacterium or protozoan

prokaryotic cell: cells that do not contain membrane-bound organelles

restriction enzyme: DNA-cutting enzymes that recognize one or a few target sequences and cuts DNA at or near those sequences

transgenic: containing a gene that has been transferred from one organism to another and acts as a synonym for genetically modified

Most eukaryotic cells contain one or more chromosomes found in the nucleus of animal and plant cells. Chromosomes are made of strands of DNA, which contains the genetic code of an organism. A plasmid is a genetic structure in a cell that can replicate independently of the chromosomes. Plasmids are a small circular strand of DNA found in the cytoplasm of prokaryotic cells such as bacterium or protozoan. While both structures contain DNA, plasmid and chromosomal DNA differ. First, plasmids have relatively few genes (less than 30) compared to chromosomal DNA. Second, plasmid DNA is not usually essential to the survival of the cell (bacteria), but instead carries genes that confer a selective advantage on their host such as a resistance to bacteria, fungi, or antibiotics. In contrast, chromosomal DNA carries essential information for the life of the cell. Third, while chromosomal DNA replicates as part of mitosis or meiosis, plasmids complete their own replication process completely separate from the chromosomes. In nature, plasmids can be passed between two bacterial strains by a set of transfer genes located on the plasmid. The proteins produced by the transfer genes bind to the DNA within the plasmid. When a break or “nick” occurs in the plasmid, the DNA unwinds and the new DNA can enter the strand bridging the gap in the plasmid DNA and potentially allowing the plasmid to inherit the new trait.²

Plasmids have become useful tools in research and biotechnology due to their ability to move genes from cell to cell. Researchers use plasmids to carry marker genes, allowing researchers to trace successful inheritance of desired traits in processes such as cloning or genetic engineering. They are also used to transfer genes from one organism to another. These scientific processes are used to produce and improve products in the areas of medicine and agriculture.

In agriculture, plasmids are used in the scientific process of creating a genetically modified organism (GMO). A GMO is an organism that has DNA that was introduced artificially using biotechnology. GMOs are also referred to as bioengineered. There are 10 bioengineered crops that have been approved for commercial production in the United States.³ Each plant has a gene giving the plant a genetic characteristic such as resistance to a pest, resistance to a specific chemical herbicide, etc. Each of these genetic traits were originally found in the environment in another plant or a bacteria. After a desired trait was identified in the genome of another organism, it was isolated and then inserted into a plasmid using enzymes which cut the circular shaped plasmid open and allowed the new DNA to bridge the gap and become part of the plasmid. The plasmid with the new gene is then introduced into the genome of the plant. This is only one step of the entire process. To learn all of the steps in creating a bioengineered plant variety, see Activity 2 of The Science of a GMO.

1. Display the crop collage picture for students to see. Ask, “What do these 10 crops have in common?” If needed, identify each crop from left to right as corn, soybeans, cotton, canola, sugar beets, alfalfa, papayas, squash, apples, and potatoes. After students offer their ideas, tell them that each of these 10 crops has at least one variety that contains a gene from another genome. These are the 10 crops in the United States that have a commercialy available bioengineered varieties available for farmers to grow.
2. Draw on students’ prior knowledge of genetics reminding them that each genetic trait possessed by an organism is coded in their DNA. Review examples of traits determined by genetics. Examples of genetic traits in plants could include flower or leaf color, size, resistance to disease, insect resistance, herbicide tolerance, etc.
3. Provide further examples by referring back to the bioengineered crop collage. Point to the corn, soybean, and cotton plants and explain that the bioengineered variety of these crops has a gene making it resistant to specific insects that kill or damage the plant. Without spraying insecticide, the plants are resistant to insects that could otherwise destroy the crop. Next, point to the corn, canola, alfalfa, soybean, cotton, and sugarbeet. These six crops have a bioengineered variety that is resistant to a specific type of herbicide (chemical used to kill weeds.) This allows farmers to spray the plants with an herbicide to kill the weeds (unwanted plants) without killing the crop. Last, point to the squash and the papaya. These two crops are resistant to specific plant diseases.
4. Summarize by drawing a simple DNA double helix on the board. Point out that every genetic trait (such as the ones we just pointed out) an organism possesses is found in a section of its DNA structure. Using biotechnology, these useful genes can be identified and transferred to benefit other organisms. In these examples, the genes benefit plants used for our food supply, to feed livestock that provide us with meat, eggs, and milk, and to produce the fiber (cotton) we use for our clothes.
5. Ask students, “How is a desired gene transferred from one organism to another?” Tell your students that the answer is in a specific kind of cell structure and they will be finding out soon.^{Image source www.gmoanswers.com}

 Activity 1: Comparing Cell Types

1. Ask students, “Where are cells found?” Students should recall from their prior knowledge that all living things are made up of cells. They are found in anything living, including plants of all types, animals, and humans.
2. Tell students that there are two major types of cells. They are called prokaryotes and eukaryotes.
3. Give each student 1 sheet of blank paper or have them open to a new page of their interactive notebooks. Also, give them 1 copy of the attached Prokaryote vs Eukaryote Interactive Notebook Cutouts.
4. Instruct students to cut out the layered circle tabs and the diagrams of each cell type. This step may also be completed as bell-work as students come into class to save time.
5. Once the notebook page/worksheet has been assembled, review it as a class and discuss the differences and similarities of each type of cell. Use the following questions as a formative assessment:
   • Which type of cell is relatively larger than the other and usually part of a multicellular organism? (Eukaryote)
   • Which type of cell has a membrane-bound nucleus and organelles? (Eukaryote)
   • Which type of cell is usually a bacteria and is responsible for strep throat? (Prokaryote)
   • Which type of cell has DNA in a circular structure called a plasmid? (Prokaryote)
   • Which type of cell has DNA that is linear and found mostly in the nucleus? (Eukaryote)
   • Which type of cell makes up the structure of a plant or animal? (Eukaryote)
   • Which type of cell divides through mitosis or meiosis? (Eukaryote) 

Activity 2: What is a plasmid?

1. Ask students to look closely at the diagrams of their eukaryotic and prokaryotic cell from Activity 1. Ask, “ Is there a structure in the prokaryotic cell that you have not seen or heard of before?” Students may identify the flagellum or pilus. These answers are correct, but have them keep looking. When they identify the plasmid, tell them that they have found the cell structure that was an important tool in creating the genetically modified plants you introduced them to in the beginning of the lesson.
2. Have students place and label the plasmid diagram (from the printout used in Activity 1) on their notes page and write, “What is unique about a plasmid?”
3. Drawing on student’s knowledge of chromosomal DNA, discuss the unique characteristics of a plasmid and the DNA it contains.

Activity 3: Plasmid Problem Solving

1. Divide students into groups of 2-3, and give each group one copy of the Plasmid DNA packet, scissors, clear tape, and three different colored pencils or highlighters.
2. Instruct students to cut out the strips from page 1 of their packet (titled Plasmid DNA) and tape them consecutively into one, continuous strip (first strip 1, then strip 2, etc.). Then, tape the end of strip 6 to the beginning of strip 1 to make a ring. Check to be sure that none of the bases were covered up in the process of taping. (Pages 2 and 3 of the packet will be set aside until step 7)
3. Once the paper plasmid has been created, review with students what they learned about plasmids in Activity 1 and Activity 2. Ask questions such as:
   • What kind of cell would contain a plasmid? (prokaryote)
   • What is a plasmid composed of? (DNA)
   • What kind of traits does plasmid DNA typically code for? (traits giving the cell a selective advantage over others such as resistance to a strain of bacteria, fungi, or antibiotics.)
4. Instruct students to keep the last question in mind (plasmid DNA can code for resistance traits) as you introduce them to a scenario.
5. Project the attached Plasmid Problem Solving Scenario sheet for students to see. Ask students to brainstorm some solutions to this farmer’s problem. Ask, “How can the rootworm be controlled?” If time allows, give students a few minutes to research methods of rootworm control on their own. If time is short, inform students that farmers can use insecticides to kill the pest or they can rotate their crops from year to year to break the life cycle of the rootworm.
6. Ask students to think deeper about what they have learned about plasmids. Could science (specifically biotechnology) provide a solution? (Yes)
7. Ask students to find page 2 of their Plasmid DNA packet titled, “DNA Strip for Rootworm Resistance.” Instruct students to cut out the DNA strips and tape strip 1 to strip 2 (make sure students do not circle the DNA segment like they did the plasmid.) As students are cutting and preparing their DNA strip explain that this section of DNA came from a bacterial cell of the species Bacillus thuringiensis which we will call Bt for short. The shaded portion codes for the production of a protein that is toxic to corn rootworm larvae.
8. Inform students that their goal is to insert the shaded portion of DNA into the plasmid using the following steps:
   1. First, the plasmid has to be cut open. In it’s current circular shape, new DNA cannot be inserted. Ask, “How can a plasmid be cut?” (with a restriction enzyme) Prompt students to the correct answer by having them take a look at the remaining page (page 3) of their Plasmid DNA packet titled, “Restriction Enzymes.”
   2. Students should evaluate the available restriction enzymes to determine which enzyme is the best for this situation. The best restriction enzyme will be able to cut the plasmid, as well as the desired DNA strand in order to create “sticky ends” in the nucleotide base that can fit together like a puzzle. Direct students to review the restriction enzyme patterns within the cut area. Students should use three different colored pencils or highlighters to highlight the base pattern that matches enzyme cut area if the enzyme was used.
      • For example: Students will highlight CTAG in the enzyme Bam HI, GGCC in the enzyme Xma I, and CTG in the enzyme AVA II.
   3. Have students search the plasmid for the patterns highlighted in the restriction enzymes. Students should highlight these with corresponding colors. For example, if you highlighted CTAG in Bam HI green, highlight this base sequence in the plasmid green as well.
   4. Repeat the process in the DNA strand for rootworm resistance. After students have completed this step, ask which enzyme will cut both the plasmid and the DNA strand for rootworm resistance. (Xma I is the correct answer)
   5. Have students cut the shaded portion of the DNA strand at the cut sites for the enzyme.
   6. Open the plasmid at the enzyme cut sites and place the DNA strand inside the plasmid. 
9. Once students have created their new plasmid with the desired (Bt) gene, explain that if the bacteria cell “takes” the transformed plasmid and the DNA in the bacterial cell successfully integrates into the corn plant’s DNA, the resultant corn crop will produce the Bt protein which is toxic to the rootworm and other damaging insects. The resistance trait will be passed from generation to generation with traditional plant breeding (cross pollination) and the rootworms will be controlled without the use of insecticide (a substance used to kill insects).
    

A common misconception is that bioengineered crops have increased the use of pesticides. It is important to understand that the word pesticide refers generally to substances used to kill harmful organisms. For example, herbicides kill selected unwanted plants and insecticides kill unwanted insects, but both are generally referred to as pesticides. As students come to understand the function of Bt corn they should recognize that it virtually eliminates the need for insecticides. When speaking specifically, the use of genetically modified Bt corn decreases the need for insecticides in crop production.

After conducting these activities, review and summarize the following key concepts:

• Prokaryotic cells are smaller, contain smaller amounts of DNA, and are found in bacteria and protozoan.
• Eukaryotic cells are larger, contain more complex DNA (than prokaryotic) and are found in typical plant and animal cells.
• Plasmids are found in prokaryotic cells and are useful tools in biotechnology due to their containing smaller amounts of DNA compared to the DNA found in chromosomes and eukaryotic cells.
• Plasmids are a tool used in creating bioengineered (GMO) plant varieties.

1. https://bitesizebio.com/13522/who-found-the-first-plasmid/
2. http://www.encyclopedia.com/science-and-technology/biology-and-genetics/genetics-and-genetic-engineering/plasmid
3. https://gmoanswers.com/current-gmo-crops
4. Pharmacogenomics. (n.d.). Retrieved October 11, 2016, from https://www.boundless.com/biology/definition/pharmacogenomics/.
5. Biotechnology in Medicine - Boundless Open Textbook. (n.d.). Retrieved October 11, 2016, from https://www.boundless.com/biology/textbooks/boundless-biology-textbook/biotechnology-andgenomics-17/biotechnology-119/biotechnology-inmedicine-480-11702/.
6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5250645/
7. “By Planting Insect resistant Cotton, Researchers Hope.” (n.d.). Insect-resistant Crops Through Genetic Engineering. Retrieved October 11, 2016, from http://www.aces.uiuc.edu/vista/html_pubs/biotech/insect.htm.
8. Green, J. M., & Owen, M. D. K. (2011). “Herbicide-Resistant Crops: Utilities and Limitations for Herbicide-Resistant Weed Management.” Journal of Agricultural and Food Chemistry, 59 (11), 5819–5829. http://doi.org/10.1021/jf101286h.
9. “What Is Bioremediation?” (n.d.). Retrieved October 11, 2016, from http://www.hawaii.edu/abrp/biordef.html.

Activity 3 was written by Heather McPherson and Mariel Sellers as part of the Syngenta Summer Fellowship Educator's Guide. The remaining activities were developed and added with permission by the National Center for Agricultural Literacy.

• Genetic Engineering (Grades 9-12)
• How Can CRISPR Improve Food?
• Learn GMO
• New Plant Variety Safety Evaluation Project
• Plant and Animal Cell Overview video
• Targeted Genome Editing
• The Facts and Knowledge of BT Corn