pGLO Transformation Lab

Introduction:

In this lab, we will be performing a procedure known as genetic transformation. It involves inserting a gene into an organism in order to change the organism’s trait. We will use genetic transformation to transform the bacteria E. Coli  with a gene that codes for Green Fluorescent Protein (GFP). GFP causes organisms to become bioluminescent. This gene does not occur naturally in E. Coli. We will use a plasmid which encodes the gene for GFP and a gene for resistance to antibiotic penicillin. Bacteria such as E. Coli have the ability to transfer plasmids back and forth, which in return allows genes to be shared, in this case, it would be GFP and antibiotic resistance. However, there are many safety measures we need to take in order to do this lab safely.

Purpose:

The purpose of this lab is to observe gene transformation in a bacterial organism. We did this with a single celled organism in order to facilitate the process. The E. Coli should be able to glow and be antibiotic resistant, depending on which plate it is on. Each plate has different properties that will affect the growth and gene expression of the glowing colonies.

Materials:

• Sterilized pipettes (5)
• Transformation solution (250 μl exactly)
• Foam microtube holder/float
• LB nutrient broth (250 μl exactlly)
• Inoculation loops (7)
• E. coli starter plate (1)
• Poured agar plates (4)
• Container full of crushed ice (1)
• Marking pen (1)
• Rehydrated pGLO plasmid (1 vial)
• 42 degrees Celsius water bath (1)
• 37 degrees Celsius incubator (1)

Procedures:

1. Label one of the two closed micro-test tube +pGLO and another -pGLO. Label both tubes with the group’s name and place them in the foam tube track.
2. Open the micro-test tubes and using a sterile transfer pipette, transfer 250 μl of transformation solution (CaCl2) into each of the tubes.

3. Make sure you then put the tubes in the ice bath.

4. Using a sterile loop, pick up a single colony of bacteria from the starter plate.

5. Then pick up the +pGLO tube and immerse the loop (containing the bacteria) into the transformation solution at the bottom of tube.
6. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution. Make sure there is no floating chunks.

7. Place the tube back in the ice. Repeat for the -pGLO tube.
8. Examine the pGLO DNA solution with the UV lamp and note your observations.
9. Immerse a new sterile loop into the pGLO plasmid DNA stock tube. Get a loopful, making sure that there is a film of plasmid solution across the ring.

10. Mix the loop-containing-plasmid into the cell suspension of the +pGLO tube. Close the tube (both +pGLO and -pGLO) and return them to the rack on ice.
11. Incubate the tubes for 10 minutes on an ice bath. Make sure that to push the tubes down so that only the bottom of the tubes fully stick out and make contact with the ice.
12. In the meantime, label your four LB nutrient agar plates on the bottom as follows

• LB/amp plate: +pGLO
• LB/amp/ara plate: +pGLO
• Second LB/AMP plate: -pGLO
• LB plate: -pGLO

13. Using the foam rack as a holder, transfer both the +pGLO and -pGLO tubes to a water bath set at 42 degrees Celsius for 50 seconds. Make sure that the tubes are all the way down to ensure contact with the warm water.

14. After 50 seconds, place both tubes back on ice for two minutes. Try to make the transition from the warm water to the ice bath as quick as possible.
15. After two minutes, remove the tubes from the ice bath and place them on the bench top.
16. Open a tube and insert 250 μl (using a new sterile pipet), of the LB nutrient broth to the tube (+pGLO) and reclose it. Repeat for the other tube (-pGLO) and incubate both tubes for 10 minutes at room temperature.
17. Tap the closed tubes with you finger in order to mix.
18. Using a new sterile pipet for each tube, withdraw 100 μl of the transformation and control suspensions onto the appropriate nutrient agar plates.
19. Using a new sterile loop for each plate (this is very critical, for the risk of cross-contamination are high), spread the suspensions evenly around the surface of the LB nutrient agar. Do not press too deep into the agar and it should be a quick skating of the flat surface back and forth.

20. Close the plates, stack them and tape them together. Write the group name and class period at the bottom and place the stack of plates upside down in a 37 degree Celsius incubator until the next day.  

Day 2:

1. Observe the four plates under normal room lighting. Obtain a ultraviolet light and run it over the plates (make sure the lights are turned off for this part).
2. Record data and observations.

Data:

Analysis:

In an ideal setting, we should have observed no growth whatsoever on the -pGLO LB and -pGLO LB/Amp plates, growth on the +pGLO LB/Amp plate, and growth/glowing on the +pGLO LB/Amp/ara plate. Our plates did not exactly match what they should have been ideally. There was growth on one of the plates that was not supposed to have any. This was most likely due to us not using a different loop to spread the broth on the plate after having used it to place E. Coli on another plate. It would lead to cross contamination of the plate and therefore result in growth. For future reference, we should ensure that a different loop was used for every plate.

The -pGLO LB plate was supposed to demonstrate how LB alone cannot foster E Coli. and assist in the growth of colonies. This is because there was no pGLO plasmid present, no “food” for the E Coli. to eat and thrive off of and no ara to make the E Coli. glow, if it were to show any growth. That means it would only be logical to see little to no colonies on this plate. However, for us there a few colonies on the plate. As previously stated, this was most likely due to cross contamination due to not using different loops for every plate.

Likewise, the -pGLO LB/Amp plate was meant to show the same concept, and was a way to prove that the Amp was working in this environment. Amp is an antibiotic, so unmodified E. Coli would be killed by it. The ones on this plate did not receive the pGLO, so they will not be antibiotic resistant. There should be no growth due to the amp killing off the E. Coli.

The +pGLO LB/Amp plate should have shown some growth as well as taken in the plasmid, however, it should not glow underneath a UV light since there is no ara present which is necessary for the glow effect.  On our plate we observed two odd clusters of growth. This indicates that the “food” for E. Coli. did in fact assist in the growth of colonies. Amp is an antibiotic that should have prevented growth of colonies, but since the ones on this plate have antibiotic resistance it did not have an effect on them. It also shows that while the E. Coli. have the genes to be fluorescent, they are still not activated until they react with ara.

The plate containing +pGLO LB/Amp/ara should have shown growth and glowed under a UV light. This is because the sugar (ara) allows the plasmid the E Coli. should have taken in to react and glow. Each dot seen on the plates was an E. Coli colony. It may have been difficult to see some of them due to there being too much broth. If there is too much broth, it will take a longer time for the E.Coli to eat though, there may have been colonies present that we were not able to see because of this. It could have also clouded up the glowing colonies. Calculating the transformation efficiency, it is evident that the the efficiency of the DNA transformation was very low, meaning it wasn’t efficient at all. This could have been a reason as to why we had so few E. Coli colonies, and why very few were able to glow.

Conclusion:

Through this lab, we were able to observe the an organism being genetically modified to express a trait it shouldn’t be able to have naturally. E. Coli. was chosen to make our work more efficient. The lab would have taken a much longer time if it’d been another kind of organism. This was based on bacteria’s ability to reproduce quickly and being able to exchange these genes through replication. Specifically, the E. Coli. received genes that gave it the ability to glow and be antibiotic resistant. This lab took a lot of safety precautions, both to protect ourselves and the results of the experiment. We had a few errors because of not properly following the directions, such as not using a different loop when transferring E. Coli to a plate. There were also errors due to things not in our control, such as there being too much “food” for the E. Coli to eat through, so much to the point that some colonies can’t be seen. We believe this was an error on the part of whoever prepared the plate.