The plasmid pGLO contains an antibiotic-resistance gene, ampR, and the GFP gene is regulated by the control region of the ara operon. Ampicillin is an antibiotic that kills E. coli, so if E. coli, so if E. coli cells contain the ampicillin-resistance gene, the cells can survive exposure to ampicillin since the ampicillin-resistance gene encodes an enzyme that inactivates the antibiotic. Thus, transformed E. coli cells containing ampicillin-resistance plasmids can easily be selected simply growing the bacteria in the presence of ampicillin-only the transformed cells survive. The ara control region regulates GFP expression by the addition of arabinose, so the GFP gene can be turned on and off by including or omitting arabinose from the culture medium.
The purpose of this lab was to understand bacterial transformation, how it occurs, and to make DNA glow.
If the transformed E. coli is mixed with the ampicillin resistance gene, it will be able to grow in the ampicillin plates, but the non-transformed E.coli will not.
Two microcentrifuge tubes
500 uL of ice cold 0.05 CaCl2
E. coli bacteria
A sterile plastic loop
A sterile P-20 micropipette
10 uL of pAMP solution
A water bath
500 uL of Luria broth
A spreading rod
Day before lab
1. Streak E. coli host cells for isolation.
2. Prepare six source plates.
Day of lab
1. Get two microcentrifuge tubes, which should each contain 200 uL of cold CaCl2 solution. Label one tube with your initials and a (+) and the other tube with your initials and a (-).
2. Transfer 2-4 large colonies using a sterile plastic loop to each microcentrifuge tube and completely resuspend. Do not transfer any agar. Put the tip of the loop into the CaCl2 solution and spin until there is not any cells on the loop.
3. Close each of the tubes and put them in ice.
4. Ask your teacher to use a P-20 micropipette to add pGLO DNA to your transformation mix.
5. Add pGLO DNA to the (+) labeled microcentrifuge tube.
6. Incubate both microcentrifuge tubes on ice for fifteen minutes.
7. Take both tubes out of ice and immediately place in incubator at 42٥C for 90 seconds.
8. After place both tubes back in the ice for two minutes.
9. Add 200uL Luria Recovery Broth to both microcentrifuge tubes.
10. Let both the tubes rest at room temperature for 10 minutes.
11. During the 10 minutes, get the LB agar and LB+AMP agar plates ready. Mark your plates with the transformation tube mixture to use (+ or -), the lab group names, and the date on the top of the dishes.
12. Add 100ul of the pGLO transformation cell mixture to the center of the agar surface of the corresponding LB agar and LB+AMP plates.
13. Use a sterile plastic loop to distribute the cell suspension evenly on the plate by “skating” the loop back and forth across the LB agar plate several times.
14. Use the same loop and technique to spread the same cell suspension (+) on the LB+AMP agar plates. Dispose of
the sterile loop in a beaker of germicide.
15. Repeat the procedure by spreading the (-) transformation cell mixture to each of the (-) labeled LB and LB+AMP plates. Be sure to use a fresh plastic loop for the ‘None’ transformation mix.
16. Stack your group’s set of plates on top of one another and tape them together. The plates should be left upright position to allow the cell suspension to be absorbed by the agar.
17. Place the plates in an inverted position (agar side on top) in a 37٥C bacterial incubation oven for overnight incubation (15-20 hrs.).
Day after lab
1. Lower the lighting in the room and use a long wave U.V. light to visualize the transformed cells that will glow due to the expression of the green or blue fluorescent proteins.
The bacteria treated with the pAMP solution developed a resistance to ampicillin and were able to grow on the ampicillin plate. Those that were not treated with the pAMP were not able to grow on this medium. The plates with no ampicillin served as a control to show how the bacteria would look in normal conditions. Transformation is never fully effective, Only cells that are competent enough are able to take up the foreign DNA. Therefore, the ampicillin+ plates showed less growth that the control plate.
1. Record your observations about the color and growth (number of colonies) of bacteria on the Petri plates. If you have so much bacterial growth that you can’t count individual colonies, this is referred to as “lawn.”
2. Calculate the transformation efficiency of your transformation experiments. Transformation efficiency refers to the number of cells transformed per microgram (ug) of DNA. The transformation efficiency of my transformation experiments is 0.0125 cells transformed per microgram (ug) of DNA.