Transformation is a genetic exchange mechanism by which “naked” DNA is taken up by bacteria. This newly acquired DNA may include genes that enable bacteria to perform tasks that were previously impossible. For example, a gene that enables the microorganism to digest oil spills or perhaps more simply, as seen in this experiment, to produce the Green Fluorescent Protein (GFP). The gene coding for GFP comes from the bioluminescent jellyfish, Aequorea victoria, and resides on a DNA plasmid called pGLO.
The pGLO plasmid also contains a gene encoding for beta-lactamase. Beta-lactamase is an enzyme that will allow transformed bacteria to be resistant to antibiotics that have a beta-lactam ring. Thus, in order to identify bacteria that have been transformed with pGLO, we select for their growth using a media that contains an antibiotic with a beta-lactam ring, ampicillin. The use of media containing ampicillin allows for the selection of successful transformants, however it is only when the sugar arabinose is also added to the media that it will be possible to observe the bright green fluorescence. This is because the GFP protein is under the control of an arabinose operon. Thus, arabinose acts like a switch turning on the section of DNA that codes for GFP.
Collect 1 LB, 2 LB/Amp, and 1 LB/Amp/Ara plate from the side bench. Also collect 2, sterile microcentrifuge tubes.
1. Label the plates as follows:
 |
 |
 |
 |
|
"-DNA" LB |
"- DNA" LB/Amp |
"+DNA" LB/Amp |
"+DNA" LB/Amp/Ara |
2. Label one microcentrifuge tube "- DNA" and the other "+ DNA".
3. Place the microcentrifuge tubes on ice.
4. Using a P-20 pipetman, carefully pipette 10 microleter of pGLO plasmid into the microcentrifuge tube labeled "+DNA". Make sure you see a tiny drop of fluid in the bottom of the tube before going on.
Collect a tube of competent Escherichia coli from the TA or instructor. These cells will be frozen. It is important to let the cells thaw on ice and then proceed with step 5 as soon as they thaw.
5. Using a P-200 pipetman, add 100 microliters of competent E. coli to the "-DNA" tube. Place the tube back on ice.
6. Using the P-200, add 100 microleter of competent E. coli to the "+ DNA" tube and place it back on ice.Tap the "+ DNA" tube gently to mix.
7. Incubate both tubes on ice for 20 minutes.
8. Heat shock the cells by placing both tubes in a 42° C water bath for 30 seconds. Place both tubes back on ice.
9. Using a P-1000, add 500 microleter of LB/20mM glucose media to each tube.
10. Allow the cells in both tubes to recover from the heat shock by incubating them in a 37° C water bath for 30 minutes
11. Using a P-200 pipetman.
-Pipette 100 microleter from "- DNA" tube onto the LB plate.
-Pipette 100 microleter from "- DNA" tube onto one LB/Amp plate.
-Pipette 100 microleter from "+ DNA" tube onto the other LB/Amp plate.
-Pipette the remaining volume from "+ DNA" tube onto the LB/Amp/Ara plate.
12. Using a flamed, sterile loop, spread the volume evenly over the agar surface by
running the loop side to side across the plate. Rotate the plate 90° and repeat this
pattern. Do thisseveral times to ensure that the entire surface of the plate is covered.
- Be sure to flame the loop before and after spreading each plate.
13.Allow the plates to dry for 5 – 10 minutes and place all four plates upside down in the 37°C incubator for 24 hours.
Observe the results on these plates both with and without UV light
-DNA on LB/Amp
-DNA on LB
+DNA on LB/AMP (B)
+DNA on LB/Amp/Ara (A)
