Standards Aligned

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-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.

Math

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

Ag Competencies: 018.Agriscience in Our World

• Define and relate agriscience to agriculture, agribusiness, and renewable natural resources. (LA)
• 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

• Materials for pGLO Transformation Lab (Class):
• pGLO Quick Guide (pg. S1-S2)
• pGLO Student Sheet (pg. S3- S4)
• 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 assessment:

• NSTA Lab Report Rubric (pg. T8)

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 Part 1: Activating Prior Knowledge and Anticipatory Discussion

Classroom Discussion

It is recommended to complete the Paper Plasmid Lab as an introductory activity. Also visit the lab for classroom discussion suggestions.

(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. S1-S2, you will find the pGLO Quick Guide for students. 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.

• Plate Prep Video

Refer to the pGLO Student Sheet (pg. S3-S4) 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?

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. 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 (pg. T8).

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.

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 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.