pGLO Transformation Experiment

Introduction
During the week of February 1st, Shreyan, Vikram, Vinay and I returned to the lab to perform an experiment involving transformation and protein production. Transformation is the process by which organisms take up new DNA, in this case in the form of plasmids, that change a trait or cause the production of new proteins. Plasmids are a form of DNA that are taken up by bacteria and then used to produce proteins in the cell. This is a useful biological process because it can help the bacteria to produce new proteins and integrate new genetic diversity into a population.

Genetic transformation is an extremely useful process for science, being used by pharmaceutical companies to produce large quantities of antibiotics or drugs, by agricultural companies to introduce new beneficial traits to organisms to help them grow, and even by doctors to cure sick people. For our experiment, the group is tasked with transforming a harmless strain of E. coli bacteria with a plasmid DNA that we are given called pGLO. The DNA contains genes for Green Fluorescent Protein (GFP), obtained from jellyfish, as well as a gene for resistance to the antibiotic ampicillin. We are hoping that the introduction of this plasmid will cause the bacteria to produce GFP as well as be resistant to ampicillin so that we can isolate the bacteria that have taken up the plasmid. Below is a diagram for the pGLO plasmid.

GFP, as the name implies, is a fluorescent protein produced by jellyfish to glow. However, the pGLO plasmid has been specially engineered to use a gene regulation system that controls the expression of GFP in cells with the plasmid. The gene that codes for GFP can be activated in the cell, but only when the cell is in the prescence of the sugar arabinose. Therefore, the cells will be ampicillin resistant, so will grow on agar plates with ampicillin, but will not glow unless activated by a plate with arabinose.

Knowing all of this from our discussions in class and reading, the lab group got to work.

Materials

• E. Coli Starter Plate
• Poured Agar Plates
• 1 with only nutrient broth
• 2 with nutrient broth and ampicillin
• 1 with nutrient broth, ampicillin and arabinose
• Transformation Solution
• Nutrient Broth
• Inoculation Loops
• Pipets
• Foam Microtube Holder
• Foam Cup Full of Ice
• 42 degree Celsius Water Bath
• 37 degree Incubator

Procedure

The first thing that the entire lab group did was put on safety equipment such as goggles and aprons so that we do not accidentally hurt ourselves with the E. coli we were experimenting with (Note: this step may or may not have been forgotten). Next, we labeled our microtubes, one as +pGLO and the other as -pGLO. Using a pipet, we then transferred 250 micoliters of transformation solution into each tube. Placing the tubes in our foam cup of ice, Shreyan and I used the inoculation loops to grab a single colony of bacteria from the starter plate and place it into each microtube, swirling the solution to make sure all bacteria were taken off the loops. 

Using a micropipet, the lab group removed 10 microliters of pGLO DNA and put it into the microtube labeled +pGLO. We made sure not to ass the plasmid DNA to the other microtube because it would ruin our experiment. Following this, the group iced the microtubes for 10 minutes.

While the tubes were icing, we labeled the 4 agar plates that we had, one as -pGLO LB (no plasmid, only nutrient broth), one -pGLO LB/amp (no plasmid, nutrient broth and ampicillin), one +pGLO LB/amp (plasmid with nutrient broth and ampicillin), and one +pGLO LB/amp/ara (plasmid, nutrient broth, ampicillin, and arabinose). 

After 10 minutes of icing, we took the microtubes out of the ice and put them into the 42 degree hot water bath for 50 seconds. This helps us perform what is called a heat shock. To get the bacteria to take up the new plasmid DNA, the bacteria is iced and shrunk, then the heat of the heat bath causes it to rapidly expand, sucking up fluid from the transformation solution that also just happens to contain new DNA for the bacteria. Thus, after the heat shock, the plasmids are inside the cells.

To keep the plasmids inside the cell and stop the cells from exchanging fluid with the surrounding solution, we put the microtubes back on ice to shrink the cells back down again. We iced the cells for 2 minutes, and then added 250 microliters of nutrient broth to the microtube solutions to help the bacteria survive. We then flicked the containers to mix the solution. 

Using a new pipet for each time, the lab group put some of the microtube solution on each plate. 100 microliters of the +pGLO solution was added to the plates marked +pGLO, and then 100 microliters of the -pGLO solution was added to the plates marked -pGLO. The group then used inoculation loops, a new one for each plate, to spread the solution around the agar and allow the bacteria to access the nutrients in the agar. 

Once this was finished, the group stacked our plates up, taped them together, and placed them all in the 37 degree incubator for a day so that the bacteria colonies could grow. Then the next day we checked our plates to see if our transformation was successful. 

Data/Observations

When we checked our agar plates, the lab group discovered that our experiment was not as successful as we had hoped. 

For our control plates, the results were as we had predicted. Unfortunately, no one in the group took pictures of our experiment or our agar plates, so I will use diagrams filled out in class to show the growth on each of the plates.

On the -pGLO LB plate, there was carpet growth of bacteria because the bacteria's growth was not impeded, as seen in the diagram above.

On the -pGLO LB/amp plate, there was no growth of bacteria, presumably because all of the bacteria were killed by the ampicillin. This is shown on the diagram above.

On the transformation plates, our results were extremely disappointing. For the +pGLO LB/amp plate, there was no growth at all. This can be seen in the diagram above.

On the +pGLO LB/amp/ara plate, there was little growth. Only one colony of bacteria grew on this plate, and when placed under UV light, this colony glowed. The growth on this plate can be seen in the diagram above.

In comparison to other groups' bacterial growth on their plates, our group's plates had extremely low growth. For example, here is a photo of the agar plates from Joe Ballard's group, shown under a UV light so we can see the group's GFP protein in the bacteria glow.

As you can see, there is considerable growth on the bottom plate, which contains nutrient broth, ampicillin and arabinose as seen by the glowing protein.

Conclusion

In conclusion, though our transformation was not completely successful, I believe that our experiment was a success. We were in fact able to transform bacteria, even tho in this case we only transformed a single bacterium. Whereas some people may call that unlucky, the lab group calls it lucky that we were able to only get a single bacterium to transform. In fact, one could argue that our group was more precise with our transforming, seeing as we were able to precisely change the DNA of one bacterium. 

Using our knowledge of mathematics and different values from the experiment, the group was able to calculate the transformation efficiency of our experiment. Using the number of cells growing on the agar plate divided by the amount of DNA spread on that plate, we determined that our group's transformation efficiency was 6.373 transformants/microgram of DNA used. When the entire class collected data of their own transformation efficencies, we found out that our efficiency is not a good efficiency. The other groups got inefficiencies of anywhere from 700 transformants/microgram to 10,000 transformants/microgram! 

Even though the group did the procedure exactly as it is listed in the manual, I think there are a few likely possibilities of where we went wrong. I think when rubbing the bacteria onto the agar plates, perhaps the group was too gentle and did not press down hard enough because we did not want to split the agar. Another time that we could have gone wrong is when we transferred the microtubes from the hot water bath back into the ice cup. Perhaps our tubes were not immersed in the ice far enough and therefore the bacteria did not shrink in time to keep the plasmids inside. Both of these errors are due to experimenter mishaps, which is very disappointing considering how hard we worked on transforming the bacteria. Moving forward, I believe that if had another shot at transformations and didn't make any mistakes as we had for this experiment, we could have an extremely high transformation efficiency.