Kansas College and Career Ready Standards

Science

• HS-LS1-1: Construct and explanation based on evidence for how the structure of DNA determines the structure of proteins which carry out the essential functions of life through systems of specialized cells.
• HS-LS3-1: Ask questions to clarify relationship about the role of DNA and chromosomes in coding the instructions for characteristic traits passed from parents to offspring.
• HS-LS3-2: Make and defend a claim based on evidence that inheritable genetic variations may result from (1) new genetic combinations through meiosis, (2) variable errors occurring during replication, and/or (3) mutations caused by environmental factors.
• HS-LS3-3: Apply concepts of statistics and probability to explain the variation and distribution of expressed traits in a population.
• HS-ETS1-1: Analyze a major global challenge to specify qualitative and quantitative criteria and constraints for solutions that account for societal needs and wants.
• HS-ETS1-2: Design a solution to a complex real-world problem by breaking it down into smaller more manageable problems.

Language Arts

• RST.11-12.1: Synthesize information from a range of sources (e.g., texts experiments, simulations) into a coherent understanding of a process, phenomenon, or concept, resolving conflicting information when possible.
• WHST.9-12.1: Write arguments focused on discipline-specific content.

Math

• MP.2: Reason abstractly and qualitatively.
• HSS-IC.B.6: Evaluate reports based on data.

Ag Competencies: 018.Agriscience in Our World

• 1. Define and relate agriscience to agriculture, agribusiness, and renewable natural resources. (LA)
• 2. Connect biology, chemistry, and biochemistry to agriscience. (S)

Learning Objectives

• To demonstrate the universality of DNA and its expression.
• To explore the concept of phenotype expression in organisms.
• Understand how to use phenotypic expression of DNA to screen for a gene of interest and the importance of marker genes.
• Investigate how horizontal gene transfer is a mechanism by which genetic variation is increased in organisms.
• Explore the relationship between environmental factors and gene expression.
• To investigate the connection between the regulation of gene expression and observed differences between individuals in a population of organisms.
• Learn the importance of sterile techniques and how to properly and safely handle bacteria and the decontamination process.

Materials

• Printable version of Kansas Corn: Feeding the World- DNA to the Rescue
• Paper Plasmid Modeling
• Plasmid Modeling Technology-Based
• Kansas Corn: Feeding the World- DNA to the Rescue PowerPoint

Materials for pGLO Transformation Lab (Class):

• pGLO Quick Guide
• pGLO Student Sheet
• pGLO Lab Kit:
  • Microtubes
  • Floating foam micro tube racks
  • Disposable sterile pipettes
  • Disposable sterile inoculating loops
  • Sterile disposable plates
  • LB powder
  • Ampicillin
  • Arabinose sugar
  • DNA plasmid
  • E. coli
• Spray bottles containing 10% bleach solution
• Disposal/waste containers with 10% bleach solution
• Water bath
• Incubator (if available)
• Parafilm
• UV Light

Materials for Each Lab Group:

• Disposable gloves
• Safety goggles
• 2 different colored micro tubes
• 1 floating micro tube rack
• 6 disposable sterile pipettes
• 1 package of sterile loops
• Foam cup with crushed ice
• 4 prepared plates (1 LB, 2 LB & Amp, 1 LB, Amp & Ara)
• Fine tipped Sharpie
• Waste container with 10% bleach solution

Materials for Assessments:

NSTA Lab Report Rubric

Safety Considerations

The E. coli strain used in this lab is non-pathogenic, meaning it will not directly cause illness in humans, but it is important to teach the students good sterile technique and safe disposal of bacteria or anything that could be contaminated.

• Gloves and safety glasses are to be worn at all times during this experiment.
• Be cognizant of all surfaces, wiping down the surfaces with the 10% bleach solution before the lab to prevent growing anything you aren’t intending to grow.
• Use a 10% bleach solution to wipe down the benches at the end of the experiment as well.
• Disposal beakers with a 10% bleach solution to place all contaminated or used loops or pipettes should be provided to students in a convenient location at each lab station.
• Wash hands well with warm soap and water before leaving lab.

To dispose of contaminated material:

Immerse all disposable pipettes, tubes, and loops that have come in contact with bacteria in 10% bleach solution for at least 20 minutes before draining, rinsing, and disposing of in the trash.

Immediately after use of the pipette or loop, place it directly into the waste beaker. Remind them that these items are designed to be used one time only. When you are finished with the lab, collect all petri dishes, open, and immerse in a 10% bleach solution to kill all bacteria. This can be easily accomplished by flooding all the plates with a few ml of the bleach solution. Allow materials to stand in bleach solution for 20 minutes or more. Drain excess solution, seal materials in a plastic bag, a large zip lock bag works well to contain any liquid that may seep from the plates. Place all bags containing the materials and double bag in trash bags then simply place in the trash for final disposal.

Procedures for Instruction

Length of Time for Preparation: 3-5 days

Day 1: Pour plates
Day 2: Cure plates (room temp.)
Day 3: Inoculate starter plates with E. coli and incubate
Day 4: Complete transformation; begin incubation (overnight in incubator, or 2 days on lab table)
Day 5: Review lab results

Length of Time for Classroom Teaching: 2-3 days

Day 1: Complete transformation; begin incubation (overnight in incubator, or 2 days on lab table)
Day 2: Review lab results

Classroom Discussion

Introduce the topic and assess students for prior understanding. Let students discuss their ideas, and guide the discussion without telling them if they are right or wrong.

“The Problem of the Past: How do we grow enough food to be able to stay in one place and support our growing population?”

• Write the problem above on the board.
• Put students in groups of 2-3 to discuss and (potentially) research how humans solved the problem of the past.
• Additional Guiding Questions:
  • What food sources should we try and grow for ourselves?
  • How do we make sure our crops will survive to harvest?
  • How do we make sure our crops will produce enough food?

Solutions from the Past: The Human-Guided Evolution of Corn

• Have students research the plant teosinte and its modern day relative corn.
• Create a Venn Diagram showing the similarities and difference between the two crops.
• Note: As an alternative activity, have students create a trait time line, showing how the plant itself changed overtime from what teosinte was in the past to corn we know today. Paying close attention to what specific traits changed over time (ex: number of kernels, protective outer layer on seed…).

“Today’s Problem: The Food and Agriculture organization of the United Nations estimates that in order to support the world population we will need to produce 70% more food than current levels now by the year 2050. How can we do this?”

• In the same groups, discuss the new question above – do you see any crossovers?
• Additional Guiding Questions:
  • How can we increase the yield of our crops?
  • How can we decrease the amount of pesticide used in agriculture?
  • How can we produce more food with the same amount of land?

Potential Crossover Points:

• Domestication of plants and animals leads to more food security.
• More food security leads to the ability to have larger populations in one fixed area.
• Intentional production of new lines of plants and animals for desirable traits through cross breeding and artificial selection.

Explain to the class that domestication of plants and animals has always been about trying to meet the needs of humans based on our available resources. This is how civilization as we know it developed. We have used domestication methods of breeding and hybridization in the past with great success. Now we are growing our population more and more, so the demand for food is larger. These traditional methods of breeding are no longer able to provide the large amount of food needed to support our civilization. However, biotechnology allows us to do the same things we did with traditional breeding techniques, with higher success rates and more direct trait selection.

Background Information

What is a plasmid? A plasmid is a small circular piece of DNA, which is often passed between bacteria. This allows genetic traits to move from one bacteria to another. A great example of this is the growing number of bacterial infections that are developing antibiotic resistance. The DNA that codes for this antibiotic resistance can sometimes be found on plasmids; once this plasmid has been developed it is passed from bacteria to bacteria taking with it the ability to resist certain antibiotics. The plasmid DNA is incorporated into the new bacterial DNA and all of the future offspring of that one bacteria (which is an asexual producing organism) now possess the gene for this antibiotic resistance.

Scientists have used these plasmids to intentionally transmit genes that code for specific traits from one host organism, where the advantageous genes have been found, into a new organism (this process is called “transformation”). This new organism is now transgenic. Transgenic organisms have genes from the new host organism and their own genes. This process has been a great tool for humanity. We have used it to create crops that contain genes that will protect themselves from pest, resist herbicides, and survive in areas where the environment is not hospitable to traditional crop varieties.

The process of making plasmids involves multiple steps:

• Step 1: Find a gene that codes for the trait you want to add to the new crop variety from a host organism.
• Step 2: Find a restriction enzyme that will cut that gene from the host organism’s DNA and will cut the plasmid you have chosen to insert the gene into.
• Step 3: Join both the source DNA with the desired trait with the plasmid DNA.

You then have to introduce the plasmid into the crop’s DNA and carefully select the cells where the new gene has been successfully inserted into the genome. This step can be difficult to do, because of this most scientist also include marker genes into their plasmid that allows scientists to easily determine if the transformation process occurred in the cells. One such marker gene will cause bacterial cells that have been successfully transformed to glow under ultra violet light.

For student activities, utilize the Paper Plasmid Modeling handout and the Plasmid Modeling Technology-Based.

Procedure for Lab

(Pre-Lab: 20-30 minutes and Lab: 45-60 minutes )

Complete the Bacterial Transformation Lab as outlined with the instructions from the Bio-Rad Laboratories, Inc. Transformation Kit – Quick Guide. Below, on (pg. S5-S6, you will find the pGLO Quick Guide for students (also available online at kscorn.com). This visual reference should be utilized in the pre-lab with students.

Procedure for Pre-lab

Each student should have a printed copy of the pGLO Quick Guide.

• Have each student highlight the actions taken during the lab, go through the entire guide and look for verbs.
• Once you have isolated the actions you must take, rewrite the instructions next to the picture of each step. Use abbreviations and as few words as possible. Write these in a different color (red, bright blue, pink….) next to each picture.
• Pay special attention to times and temperatures listed on each step. The times required for the heat shock are especially important.
• Count up the number of sterile pipettes and loops you will need in total for the lab. Write a large “P” next to each step that requires the use of a new sterile pipette, write a large “L” next to each step that requires the use of a new sterile loop. If you are using multiple loops or pipettes you can use a short hand notation, “2xP” or “2xL”, using the total number needed in place of the “2” when necessary.

Procedure for Lab

Follow the instructions on the pGLO Quick Guide that you have highlighted. It is important to make sure all students have done the pre-lab before you complete the actual lab, as there are time sensitive elements that must be followed correctly for the best results. Refer to the pGLO Quick Guide handout from Bio-Rad for lab instructions.

Teacher Resources

• Indiana Public Media  How Did Corn Evolve? 
  Short article or audio commentary on the evolution of corn.
• Cold Spring Harbor Laboratory  Genetic origins of corn on the cob 
  Article identifying the gene that is likely responsible for corn on the cob as we know it.

Reflection and Conclusion

Refer to the pGLO Student Sheet for post-lab activity.

After the incubation of the transformed plates, you will have 4 observations to make. There are guiding assessment questions to answer on the following document as well as a place to record data. This can be printed or can be sketched into a science journal.

Do not forget to use this as an opportunity to expand student ideas about how we would then use these genes. In corn, one of the first transgenic varieties contained a gene from the bacteria, Bacillus thuringiensis (Bt), which causes the plant to create a toxin which stops insects from eating the plant and killed any insect that ingested the plant. This allowed the newly transformed plant to be less susceptible to damage from insects; thus increasing yield overall and decreasing the use of additional pesticides. As an extension activity, pose the following questions:

• Is this the only type of gene we could insert?
• If you were a scientist, what other genes would you be looking for to help develop new varieties of crops?

Assessments

Plasmid Modeling

Final plasmid can be checked for accuracy. This is easily accomplished if done via the technology-based version. The paper version, Paper Plasmid Modeling, can be visually inspected as well.

Bacterial Transformation Lab

In order for students to achieve transformation it is critical that they work through the lab using the precise steps and times outlined in the pGLO Quick Guide (pg. S5-S6, or available online at kscorn.com). If they have completed the lab correctly, one plate and one plate only should have glowing bacteria, the + LB/Amp/Ara. If their results do not show this, allow students the opportunity to analyze where their mistake may have taken place and determine what they would do differently if they could complete the procedure again.

Overall Assessment

Written or oral assessments on how and why this transformation lab is considered genetic modification is a fantastic way to assess the overall understanding of the students’ knowledge. This can be done via a presentation or through a paper explaining each step in the process, the creation of plasmids, the transformation process, and how it relates to the overall goals of food stability. While performing assessments, refer to NSTA’s Lab Report Rubric.

Science and Agriculture Careers

Almost all new seed varieties being created today have some level of biotechnology involved in their development. From plant tissue culturing to genomic analysis and alteration, all rely on the primary skills of sterile technique and basic understanding of the genetic code. Several different careers in agriculture are fundamental in the development of new varieties of crops: botanists, horticulturalists, biochemists, biological engineers, climatologist, ecologists, food scientists, geneticist, microbiologists, plant pathologist, and an army of lab technicians are all involved in the development of each and every variety.

To learn more about agriculture careers visit agexplorer.com. You can also find career profiles at Careers in Corn.

Sources

• Ohio Corn and Wheat curriculum – http://ohiocorneducation.org/
• pGLO Quick Guide – Courtesy of Bio-Rad Laboratories, Inc., © 2018
• NSTA Lab Report Rubric
• Food and Agriculture Organization of the United Nations  2050: A third more mouths to feed. News article discussing the need to increase food production by 70% in order to meet the growing demands for food
• Teosinte to corn, Weed to Wonder.
• University of Utah Learn Genetics  The Evolution of Corn 
  Great article explaining the genetic relationship of teosinte and modern corn.
• Harvard University Graduate School of Arts and Sciences  From Corgis to Corn: A Brief Look at the long History of GMO Technology. Great graphic of the history of genetic modifications.
• The University of North Carolina at Chapel Hill  How to make a transgenic plant. 
  Step-by-step  guide to inserting DNA from one organism into a plant to develop a transgenic plant.
• NSTA  NSTA Minimum Safety Practices and Regulations for In-Person and Virtual Sessions
  General safety guidelines for laboratory experiences.

Disclaimer

Any educator electing to perform demonstrations is expected to follow NSTA Minimum Safety Practices and Regulations for Demonstrations, Experiments, and Workshops, which are available at http://static.nsta.org/pdfs/MinimumSafetyPracticesAndRegulations.pdf, as well as all school policies and rules and all state and federal laws, regulations, codes and professional standards. Educators are responsible for abiding appropriate legal standards and better professional practices under a duty of care to make laboratories and demonstrations in and out of the classroom as safe as possible. If in doubt, do not perform the demonstrations.