Stephanie Bohr
pGlo Bacterial Transformation Lab
Period A
Introduction
The purpose of this experiment is to see how bacterial cells be genetically
transformed with plasmid DNA containing a jellyfish gene.
If the bacteria contain arabinose it will make green fluorescent protein (GFP),
then it will glow. The independent variable is the GFP and the dependent variable is
whether or not it will glow.
Plasmid occurs naturally in many bacteria. Plasmid is small circular DNA
molecules. They contain few genes are much smaller than the bacterial chromosomes
which are highly compacted structures and share many properties with their eukaryote
counterparts. Plasmid DNA is replicated inside bacterial cells. You will also be using
pGLO which is a recombinant plasmid. It has been engineered with many different
genes, including one form a bioluminescent (glowing) jellyfish. There is also GFP or
known as green fluorescent protein. It is a protein that glows a green color when exposed
to ultraviolet lights. It is seen only when in the presence of sugar arabinose which is a
sugar that activates the production of GFP.
Materials

UV Lamp

Two micro centrifuge tubes

Foam tube rack

Marker

Calcium chloride solution

5 sterile transfer pipettes

Plastic cup for biohazard waste containing 10% bleach solution

Crushed ice

2 foam cups

Starter agar plate with colonies of E. coli

6 sterile inoculating loops

pGLO plasmid DNA

hot and cold tap water

thermometer

LB nutrient broth

4 agar plates

Clock or watch

Incubator
Procedure
1. Before starting, observe the starter plate (don’t remove the Petri dish lid)
with colonies of E. coli.
2. Shine the UV lamp on the colonies and observe them
3. Label one closed micro test tube +pGLO and another –pGLO.
4. Label both tubes with your group’s name.
5. Place them in the foam tube rack.
6. Open the tubes and using a sterile transfer pipette, transfer 250µ of
transformation solution (CaCl2)
7. Place the tubes on ice
8. Use a sterile loop to pick up a single colony of bacteria from your starter
plate.
9. Pick up the +pGLO tube and immerse the loop into the transformation
solution at the bottom of the tube.
10. Spin the loop between your index finger and thumb until the entire colony
is dispersed in the transformation solution with no floating chunks.
11. Place the tube back in the rack in the ice.
12. Using a new sterile loop repeat for the –pGLO tube
13. Examine the pGLO plasmid DNA solution with the UV lamp and note
observations.
14. Immerse a new sterile loop into the plasmid DNA stock tube.
15. Withdraw a loopful
16. There should be a film of plasmid solution across the ring
17. Mix the loopful into the cell suspension of the +pGLO tube.
18. Close the tube and return it to the rack on ice.
19. Incubate the tubes on ice for 10 minutes
20. Make sure to push the tubes all the way down in the rack so the bottom of
the tubes stick out and make contact with the ice
21. When the tubes are sitting on the ice, label the four agar plates
22. Using the foam rack transfer the +pGLO and –pGLO into the hot water
bath set at 42 degrees Celsius.
23. Wait 50 seconds and place both tubes back on ice for two minutes
24. Remove the rack containing the tubes from the ice and place on table.
25. Open a tube and using a sterile pipette, add 250µ of LB nutrient broth to
the tube and recluse it.
26. Repeat for other tube
27. Let sit at room temperature for ten minutes
28. Tab the closed tubes with your finger to mix
29. Using a new sterile pipette for each tube, add 100µ of the transformation
and control suspensions onto the appropriate plates.
30. Use a new sterile loop for each plate and spread evenly around the surface
of the agar
31. Stack up your plates and tape them together
32. Store upside down in the 37 degrees Celsius incubator until the next day
Next Day
1. Observe the colonies of E. coli cells on the four plates you prepared
2. Observe the E. coli starter plate
3.Use the hand held UV light to look for the presence of the fluorescent
protein.
Plate
Appearance Under UV light
LB/amp –
No Glow
LB –
No Glow
LB/amp/ara +
Glow
LB/amp +
No Glow
Variable held constant: Tubes holding pGLO, bin of cold water, hot bath
Observations
-pGLO
LB/amp
No Glow
-pGLO
LB
No Glow
+pGLO
LB/amp/ara
Glowed
+pGLO
LB/amp
No Glow
Conclusion
In conclusion, we discovered that only bacteria containing arabinose will glow
because arabinose is the sugar that activates the production of green fluorescent proteins.
The plate that contained the LB/amp/ara was the only one that glowed because it was the
only one that contained the arabinose.
Our experiment was not valid or reliable because when it came time to spread the
pGLO onto the plates we spread it onto the lid not allowing the bacteria to eat off of the
stuff inside each plate, this doesn’t allow any bacteria to grow. When we consulted with
another group that did it correctly it showed that only the LB/amp/ara glowed where as if
we were to look at our results we wouldn’t have been able to see that which would have
thrown off all of our results. Other than the problem of the placement of the pGLO, our
group followed all of the other instructions properly and was successful. Everything was
accurately measured and distributed correctly to each plate, and was held on the ice, heat
and room temperature for the correct amount of time. If this experiment were to be
repeated and the pGLO was put on the proper side of the plates, the results would come
out correctly