Bacterial Fluorescence Lab Report

During the second day of experimentation, the contents of the stored microtubes from the previous week exhibited fluorescence prior to any further procedural steps. At the beginning of class, the microtubes were centrifuged for 10 minutes and the resulting supernatant was collected and the bacterial pellet was discarded, at this time the microtube exhibited fluorescence. Next, 250 µl of the binding buffer and supernatant mixture was added to the column and exhibited fluorescence under UV light, while the collection tube, FT, did not. After the addition of 250 µl wash buffer, the contents collected in tube W did not glow, while the column did. Next, 750 µl was added to the column and was allowed to collect in the microtube labeled E. This

coli cells before the experiment began, confirming that the plasmid DNA was successfully transformed by the bacterial cells during the growth process. Following the centrifugation and removal of supernatant, the bacterial pellets remaining in the tubes were observed to fluoresce because the GFP was still present within the bacterial cells. When the lysozyme and TE solution were added to the bacterial pellet, the specimen continued to demonstrate fluorescent properties under UV light because GFP was still within the microtube, although the lysozyme had begun to degrade the cell walls during this

Following centrifugation, the microtube fluoresced because GFP was still present in the solution of supernatant, lysozyme, and TE solution. After transferring 350 µl of the supernatant, taking care to avoid the collection of the bacterial pellet, the microtube containing only the supernatant solution exhibited fluorescence under UV light, while the bacterial pellet did not. This observation was caused by the degradation of the bacterial cell walls and membranes in the presence of lysozyme and freezing temperatures over the week of storage. The bacterial debris did not glow because GFP was only in the supernatant and not the pellet. When 250 µl of supernatant and binding buffer were allowed to drain through the column, microtube FT did not fluoresce under UV light, while the column did because all constituents besides the GFP had passed through the column. The GFP remained in the column because both the protein and the column matrix were hydrophobic, resulting in affinity chromatographic interactions between the protein, but not other non-hydrophobic