Southern Illinois University at Carbondale
LESSON PLAN
Teacher's Name: Lori C. Reed
School: Murphysboro High School Grade Level: 11-12
Title/Theme: Bacterial Transformation Kit (molecular biology and genetic recombination)
Project Description: Students will learn about the process of moving genes from one organism to another with the aid of a plasmid. The process this lab illustrates is transformation. A pGLO plasmid which contains the GFP gene also contains the gene for beta-lactamase which provides resistance to the antibiotic ampicillin. The beta-lactamase protein is produced and secreted by bacteria that contain the plasmid. The students insert the pGLO gene into the bacteria E. coli. Bacteria that are transformed would then contain the pGLO gene on the plasmid. Only transformed bacteria that contain the plasmid and express beta-lactamase can survive on plates which contain ampicillin.
Major: ll.A.5c, 12.1.5b,
Lesson/Unit Outcomes: One of the biggest challenges for first time student of biotechnology or molecular biology is that many of the events and processes they are studying are not visible to the naked eye. This lab provides the students with the opportunity to see the results from genetic recombination of a bioluminescent jellyfish gene and its Green Fluorescent Protein (GFP)-when expressed in a competent bacterium, E. coli. If the bacterium is transformed, it does indeed fluoresce a brilliant green when viewed with a hand held long wave ultraviolet light.
ILS Applications of Learning
Solving Problems
Using Technology
Working on Teams
Making Connections
Technology Utilization
Other (please list): lab investigation (aseptic technique) culturing bacteria
Lesson Overview/Activities:
lntroductory Activity: Students must properly learn aseptic technique before doing this lab. They must also be familiar with recording accurate procedures and observations in their lab notebook. Before doing this lab, the instructor should cover these key terms and concepts: genetic transformation, uses of genetic transformation in plant resistance, bioremediation and medicine.
Students are introduced to the lab. Students use a procedure to transform bacteria with a gene that codes for Green Fluorescent Protein (GFP). This gene, normally found in Aequorea Victoria, causes the jellyfish to fluoresce and glow in the dark. Following this procedure, the bacteria will do the same under an ultraviolet light.
Students learn about the process of mixing genes from one organism to another with the aid of a plasmid pGLO. pGLO plasmid encodes for the gene for GFP and a gene for resistance to the antibiotic ampicillin. pGLO also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in the transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells nutrient medium. Selection for cells that have been transformed with pGLO, is accomplished by growth on antibiotic plates. Transformed cells will appear white (wild type phenotype) on plates not contain arabinose and fluorescent green when arabinose is included in the nutrient agar medium.
Daily Activities and Timeframe:
Three lab sessions carried out in consecutive 50 minute periods.
Day one: Setting the Stage: Lecture and discussion of transformation.
Day two: Transformation Laboratory: Transform cells and spread plates: Day Three: Data collection and analysis: Observe transformants and controls, analyze and interpret results.
Enabling Skills: Introduction and practice of aseptic technique.
Method of Student Assessment (such as rubric, observation, performance assessment, or paper/pencil): Laboratory results and laboratory write up on those results.
Student Artifacts:
Notebooks/Journals
Other (please list) Laboratory techniques: collection and analyzing of data
Resources (museums, experts, print material etc.)
Teacher Resources: pGLO transformation kit by BIO-RAD: Catalog Number 166-0003EDU
Student Resources: Materials provided by teacher
Materials & Supplies Needed
Kit materials: K-12 BIO-RAD Catalog Number 166-0003EDU
1-800-4biorad
OTHER MATERIALS:
UV lamp-long wavelength
Clock to time seconds
Microwave, pressure cooker or autoclave
37* incubator
temperature controlled water bath
thermometer
1 L flask
500 mL graduated cylinder
distilled water
crushed ice
10 mL bleach
permanent markers
Contact Information:
Name: Lori C. Reed
Address: 10670 Hickory Ridge Road
City: Murphysboro State: IL Zip: 62966
Email: lcreed@accessus.net