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One major accomplishment in the corn industry during this century has been the development of Bt Corn. This variety of corn is genetically modified with a gene that produces a highly selective toxin, designed to control for European Corn Borers. How did scientists know this toxin was indeed being produced by the altered corn variety? Scientists take samples of the new plants and purify the protein they are trying to produce in order to confirm their overall results and determine whether they were successful in achieving their overall goals. Purifying proteins from cells is a multi-step process and it is dependent, in part, on the properties of the protein themselves. During this lab students will purify the Green Florescent Protein (GFP), which was previously transformed into a strain of E. coli bacteria during the previous lab, Bacterial Transformation, which is Part 3 of Feeding the World – DNA to the Rescue. They then break down the genetically altered cells to extract the newly created proteins from their freshly grown bacteria cultures.
Science
Students who demonstrate understanding can
Language Arts
Math
Ag Competencies: 018.Agriscience in Our World
The E coli strain used in this lab is not a pathogenic organism. It has been genetically modified to prevent its growth unless grown on an enriched medium. However, handling of the E. coli strain requires the use of standard Microbiological Practices. Work surfaces should be decontaminated once a day and after a spill of viable material. All contaminated liquid or solids wastes are decontaminated before disposal. All persons must wash their hands after they handle material containing bacteria and before exiting the lab. Do not eat or drink in the lab. Protective eyewear and gloves are strongly recommended.
Anything that comes into contact with the bacteria should be placed into a 10% bleach solution for at least 20 min for proper sterilization if an autoclave is not available. Once sterilized any plates that are used can be double bagged and treated as normal trash. Safety glasses are recommended when using bleach solutions. Ampicillin may cause allergic reactions or irritation to the eyes, skin, respiratory systems. In case of contact, rinse area immediately with plenty of water. Ampicillin is a member of the penicillin family and those with penicillin allergies should avoid contact with ampicillin.
UV radiation can cause damage to eyes and skin. Short-wave UV is more damaging than long-wave UV light.
Length of Time for Preparation: 1 day
Length of Time for Classroom Teaching: 4-5 days
Preparation Procedure/Instruction
One major accomplishment in the corn industry during this century has been the development of Bt Corn. This variety of corn is genetically modified with a gene that produces a highly selective toxin designed to control for European Corn Borers. In this case, the donor organism is a naturally occurring soil bacterium, Bacillus thuringiensis, hence the abbreviation Bt Corn. The toxin produced, Bt delta endotoxin, is very specific in its targeting of the Lepidoptera larvae. In this stage of development, Lepidoptera larvae create the most damage to growing crops. Once the larvae ingest the protein, Bt delta endotoxin, it binds to the organism’s gut and the larvae stop eating, which prevents further damage. It also decreases growing populations of the European Corn borer by causing the larvae to die. This is due to the release of normal gut bacteria from the organism into the body, which causes septicemia and eventual death. The increase in death rate and decrease in birth rate of the organisms ultimately leads to overall population control, all by simply adding one protein into the corn genome. The need for the widespread spraying of pesticides for this pest is eliminated, and it leads to an increase in yield due to less crop destruction by the pests themselves.
How did scientists know these effects would occur? How did they know there were sufficient levels of the protein being produced in the newly genetically altered corn crops? Multiple tests conducted over many years must be performed to confirm and purify the intended protein. Purifying proteins from cells is a multiple-step process and is dependent, in part, on the properties of the protein themselves.
In this lab students will be extracting and purifying the GFP protein they transformed into E. coli cells in the Bacterial Transformation lab, which is Part 3 of the lab previously performed, Kansas Corn: Feeding the World – DNA to the Rescue (available on kansascornstem.com). Students begin by using one colony of transformed bacteria from each condition of the Bacteria Transformation lab. The colonies are added to nutrients to grow, then incubated. This produces many exact copies of one genetically unique organism. After 24-48 hours, students will put the bacteria through many physical and chemical processes to release the produced protein GFP by breaking down the bacteria cell walls and removing bacterial debris from the sample. The remaining proteins are then purified by going through a chromatography column treated with different buffers to retain the GFP protein and rinse away any other proteins present. The final rinse in the process releases the GFP proteins into a collection tube.
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 questions below can be used to help facilitate small group discussions of two to four people or as post-lab review.
Refer to the GFP Procedure – Quick Guide handout from Bio-Rad Laboratories, Inc.
Tips
Resources
For analysis during the lab, use the black light pen that was provided in the kit. You should see a light green line in the chromatography column that will be located towards the top. This will remain until the last step of adding solution into the chromatography column where you will see the green fluorescent line move through the column and eventually into the final test tube. The green fluorescent line is the protein produced by the E. coli that was made in the following lab: Bacterial Transformation, which is Part 3 of Kansas Corn – Feeding the World – DNA to the Rescue.
Review chromatography and how we used it in this lab. What purpose did each buffer serve when running the chromatography?
Why is it important to purify proteins in this manner? Do we do anything like this today? Reviewing some of the questions in the background can be done here. How did scientists know that there were sufficient levels of the protein being produced in the newly genetically altered corn crops?
Critical Takeaways: Creating new varieties of any crop through genetic modification is a complex multiple-step process. All along the way there are many checks and balances to ensure that products are safe and that the results follow the original intent of the program. Millions of dollars, and sometimes decades of work, can go into a single variety of a genetically modified crop. We are not playing gene-roulette and randomly selecting genes to add into these varieties. The cost and time associated with that type of approach to genetic modification would be a huge obstacle to the development of any new variety. Students must understand that the selection of donor genes depends on the needs of the industry as a whole. Genes that convey drought tolerance are much more critical in arid regions of the world than genes that provide resistance to types of fungi. Careful analysis of the genes occurs to ensure the genes selected fit not only the organism but also produce the appropriate traits in the crop for specific regions. A central message to students must include the careful analysis of not only the host organism but the selected donor gene as well.
We see this cautious approach being used in the multitude of tests newly created varieties must pass before they reach the wide spread market. As a part of these tests, scientists would analyze what new proteins are being created by the new variety. This method of protein purification can be utilized to ensure not only what protein is being produced but also that the appropriate levels of protein are present in the new varieties. After the extraction, purification, and collection of these new proteins, additional tests would then be undertaken to ensure the products safety years before it would be available for use in the open market.
The production of Bt Corn was a great accomplishment and the trait has been expanded to other lines of crops as well. However, this agricultural innovation has created one downfall. By introducing this trait into crops produced in large-scale agriculture we have artificially increased its prevalence in the environment. This has caused the evolutionary process of natural selection to speed up in the case of pests. The effectiveness of this trait was so great that most pest populations remaining are resistant to toxins such as those found in Bt corn. As a result, new strategies and lines of defense must be discovered in order to continue to protect our crops from destruction from these newly evolved pests. For this we will need highly skilled individuals with an eye for detail and a passion for laboratory investigation. 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 https://agexplorer.ffa.org/.
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 https://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.
This lesson is the work product of the Kansas Corn Commission. Our lessons are written in collaboration with Kansas teachers for use in the classroom. Teachers may copy and share this curriculum. Use of this product for commercial or promotional use is prohibited without express permission of Kansas Corn.
As the need to produce more food with less resources grows, it is important that future generations have a better understanding of agriculture and how corn farming and agriculture fit into our daily lives. That’s why the Kansas Corn STEM program provides lessons to teach science through the lens of corn. We are committed to providing free materials and training to support educators.
