Since man first started raising crops, we have quested for stable varieties with high yield, no matter what the conditions may be. Our early ancestors developed many techniques such as cross breeding to develop some of our earliest crops. Humans have been seeking out plants that produced high yield fruit and grains, and nurturing specific varieties through selective breeding, until we developed the very crops we know today. Corn was once a small plant, which is today considered a weed, teosinte. Teosinte had very few grains on each fruiting body, and all grains had very hard outer seed cases that would protect the nutrition to be found inside. Through humans selecting varieties from the wild and cross breeding them by hand, we developed the many maize varieties we have built our civilization on. Today, we face an ever increasing challenge of producing more and more food for a growing human population. We must go back to our roots and continue to develop varieties, just as our ancestors before us, to meet this demand. Through the expanded biological understandings of genetics and new techniques of genetic modification we have to create varieties that increase yield, decrease herbicide and pesticide use, and ultimately grow more food. In this exercise students will learn more about the origins of plant varieties, and how we continue to develop new varieties by utilizing new technologies.
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.
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.
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
Day 1: Complete transformation; begin incubation (overnight in incubator, or 2 days on lab table)
Day 2: Review lab results
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.
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.
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:
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.
(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.
Each student should have a printed copy of the pGLO Quick Guide.
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.
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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:
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.
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.
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.
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.
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.
Investing in teachers is a priority therefore the Kansas Corn Commission is committed to providing materials and training to help teachers excel in the classroom. Teachers who seek to expand their knowledge and skill of connecting science with agriculture are encouraged to attend a Seed to STEM workshop.