Hardware: Electroporation for Transformation
February 28, 2019
In this week's experiment, we used an electroporator in order to insert genes into bacteria. This process is called transformation. It works by shocking the cell membrane, which temporarily opens up holes in it. Plasmids with our genes of interest are then able to enter the cell. The plasmid we inserted was called pBlu, supplied by Carolina Biological Supply Company. It contains two genes-ampicilin resistance and beta-galactosidase. Beta-galactosidase breaks down galactose and its variants. Once we transform the cells, we plate them on agar containing a variant of galactose that turns a certain color when broken down, giving us a visual cue for whether or not the transformation was effective. The ampicilin resistance is necessary as the agar that we plate our cells also contains ampicilin, an antibiotic. This allows us to select for only cells that have the plasmid. Any cell that does not have the plasmid will die on the agar.
Unfortunately, we did not see any growth on our plates, indicating that we did not successfully transform our cells. This could be due to a number of reasons, but, as the entire class was unable to transform their cells, it was likely a systematic error. As the electroporator had been calibrated for a different species of cell and a different plasmid, it is possible we were using the wrong settings. It is also possible there was another technical flaw with the electroporator.
If we were to repeat the experiment, we might want to also consider transformation efficiency, as our settings on our electroporator might have vastly different results. We could do this by removing the ampicilin from the agar, which would allow the cells to grow whether or not they had the plasmid. We would then compare the number of colony forming units that were colored over the total number of colonies. This would give us an estimate of what percentage of cells were successfully transformed.