Bacterial Transformation Lab

As we learn more about DNA, there are any different concepts we can question and explore. One such concept is genetic transformation, which we explored as a class. This was done through a lab experiment in which we introduced plasmid into an E. coli bacteria colony in an attempt to alter the DNA. To gain comparable data, the following conditions were tested in separate agar plates:

1. +pGLO (plasmid), LB (nutrient), and Ampicillin (antibiotic)

2. +pGLO, LB, Ampicillin, and Arabinose (sugar)

3. -pGLO (no plasmid), LB, Ampicillin

4. -pGLO, LB

To begin the lab, we used a sterile pipette to transfer 250 ul of transformation solution into separate tubes containing +pGLO and -pGLO. These were then put on ice. Using a sterile loop, a colony of bacteria was added to each tube while they sat on the ice. Plasmid was then added to +pGLO only, not -pGLO. Both tubes were then incubated on the ice for 10 minutes. After this time, the tubes were transferred  to a heated water bath for 50 seconds, then immediately placed back on the ice for 2 minutes. 250 LB nutrient broth was added to each tube, then incubated for another 10 minutes at room temperature. 100 ul of the transformation and control suspensions were added onto the appropriate plates. The suspensions were spread evenly around each plate, and then incubated, upside down, at 37* C, for 24 hours. 

After placing the plates in the incubator, but before data was collected and analyzed we were asked to answer the following questions:

1) On which plates would you expect to find bacteria most like the original non-transformed E. coli colonies originally observed?

I would expect to find bacteria growth most similar to the original colonies in the plate "-pGLO, LB". I predict this because it has the bacterial cultures, but doesn't need to fight the ampicillin in order to grow.

2) If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located?

I predict that any genetically transformed cells would exist on the plate "+pGLO, LB, Amp, and Ara". This is because the bacteria has to fight the ampicillin while still being able to survive, which would only be possible if the bacteria had transformed.

3) Which plates should be compared to determine if any genetic transformation has occurred?

All of the plates should be compared to plate "-pGLO, LB" to determine transformation, as "-pGLO, LB" most closely resembles the original colonies. 

4) What is meant by "control plate"? What purpose does it serve?

A control plate is a plate that is not affected by any variables; it's results can be relied on to compare transformation data. 

The results from the experiment are displayed below.

The chart above details which dishes had cellular growth as well as which had the ability to glow. As you can see, the data aligned with the original hypothesis. 

Another hypothesis proven correct was that the +pGLO, LB,Amp, and Ara plate would contain the transformed bacteria. The reasoning behind the hypothesis is the same reason as to why it was proven correct.

It is very difficult to see in this picture, due to the lighting, however, this plate shows significant bacterial growth; nearly the entire plate is covered. 

It is amazing to see how genetic transformation occurs, and what factors determine the traits of bacteria cultures. This was a very interesting process that was incredible to witness first-hand. It has allowed me to understand genetics with a different perspective than I had had before.