TRANSFORMATION WITH A FIREFLY GENE
Talent Unlimited High School, Manhattan
Summer Research Program for Science Teachers
After completing this lab a student should be able to do the following:
a. a. Understand the process of bacterial transformation.
b. b. Be able to describe the function of 2 important genes on the pBestLuc plasmid.
c. c. Be able to explain how to induce competence in E. coli cells.
Per group of 2 students
- Microcentrifuge tubes
- Calcium chloride
- Plasmid pBestLuc
- Inoculating Loop
- microcapillary pipette that allows for 10- 100 ml
- A beaker with ice
- 2 Luria agar plate that contains ampicillin
- 2 nitrocellulose membrane
- Water bath, hot plate and beaker
- Thermometer to check temperature of water bath to be 42°C
- 500 ml Luria Broth (250 for control and experimental group)
1) A plasmid is a circular form of DNA. In our lab today, we want to grow bacteria on plates that contain the antibiotic ampicillin. Explain what this plasmid must contain in order to allow this growth to occur.
2) We want to have to plates of bacteria: 1. Control group vs. 2. Experimental. Explain what conditions the control group vs. the experimental will have in order to grow on these ampicillin plates.
1) Using a sterile pipette put 250ml of ice-cold calcium chloride in a microcentrifuge tube.
(Calcium chloride is needed to induce E. coli to become competent.)
2) Using aseptic technique, transfer a loopful of E. coli from the culture tube to your tube of calcium chloride. (Be careful to not transfer any agar. You may have to twirl your loop in the solution to dislodge the bacteria.)
3) Using a microcapillary pipette, add 10ml of the plasmid pBestLuc to your tube. (You are adding the plasmids to your solution now!)
4) Close your microcentrifuge tube.
5) Gently tap the side of the tube with your finger to mix the plasmid into the solution.
6) Incubate the tube on ice for 15 minutes.
7) Obtain a Luria agar plate that contains ampicillin. Using forceps, carefully place a nitrocellulose membrane on the surface of the agar. (Lift the lid of the petri dish only enough to place the membrane on the agar and replace the lid as soon as you are done in order to minimize the chance of contamination.) (*** the nitrocellulose membrane is VERY fragile…so handle it gently***)
8) Place your microcentrifuge tube with the bacterial cells into a 42°C water bath for 75-90 seconds. (This “heat shocks” the E. coli increasing the likelihood that they will take up the plasmid.)
9) Remove the tube from the water and place back on ice for 2 minutes.
10) Using a sterile pipette, add 250ml Luria broth to your tube.
11) Close the tube and tap gently to mix.
12) Using a sterile pipette, transfer 200 ml of your transformed bacteria to the agar plate with the nitrocellulose membrane. Gently rock the plate to ensure the solution thoroughly covers the surface of the membrane.
13) Label the lid of the agar plate with your name, date and contents.
14) Leave the plate in an upright position for 1 hour.
15) Invert the plate and place in a 37°C incubator for 48 hours.
Repeat steps 1- 15 above in order to create your control plate, but leave out step # 3.
1) Remove the lid of the plate and place upside down on the lab bench.
2) Add 0.5ml of 1mM luciferin to the center of your lid.
3) Using forceps, carefully remove the nitrocellulose membrane containing the transformed colonies of E. coli from your plate and place on top of the luciferin solution.
4) Observe the luminescence in a dark area!
1. Draw the bacterial colonies observed in the control and experiment plates above.
2. What percentage of bacteria were able to glow in the dark in the experimental plate? (# of bacteria that glowed/ total # of bactieria)
1) How could this form of bacterial transformation benefit humans?
2) Under the right conditions bacteria have shown the ability to incorporate genes and DNA from its surroundings. How is this a danger to humanity and what sort of genes will bacteria need to incorporate in order to make this world a less than beautiful place to live?
Standard 1: Analysis, Inquiry, and Design- Students will use mathematical analysis, scientific inquiry, and engineering design, as appropriate, to pose questions, seeks answers, and develop solutions.
Standard 4: Science- Students will understand and apply scientific concepts, principles, and theories pertaining to the physical setting and living environment and recognize the historical development of ideas in science.
Standard 6: Interdisciplinary Problem solving – Students will apply knowledge and thinking skills of mathematics, science, and technology to address real life problems and make informed decisions.