pGLO
Genetic Transformation of Bacteria
Abstract The transformation of bacteria was successfully carried out using a plasmid carrying a gene that codes for green fluorescent protein, which gives a signature green glow reminiscent of a jellyfish. This gene, however, is only active when the sugar arabinose is present. A gene coding for antibiotic resistance was also found within the plasmid and served as a means to verify that transformation had indeed taken place. The hypothesis was that the bacteria would be transformed and only those transformed bacteria in the presence of arabinose would fluoresce the characteristic green of the active protein. In addition, it was hypothesized that the protein, not the gene itself was responsible for the traits seen. The hypothesis has been accepted based on the data collected. This acceptance is largely based on two simple observations. Only those bacteria that had been transformed with the plasmid could grow in the face of the antibiotic. Furthermore, the only bacteria that could glow green was the bacteria that was exposed to the sugar arabinose which in this case has been proven to induce transcription of the GFP protein. This indicates that the molecule responsible for the observed green trait was protein. The transformation efficiency was found to be 793.75 transformants per microgram. The protein was filtered out of the cell and column chromatography was used as a means to isolate the protein by taking advantage of its hydrophobic properties. This was performed by washing the column containing the GFP protein amongst other proteins with buffers varying in salt concentrations. As expected, when a buffer of high salt concentration was used (binding buffer) the protein remained in the column. This was evidenced by the green glow of the column coupled with the non-glowing wash remnants collected. However, when the low salt buffer was used, the column no longer maintained the green glow and the wash collected
Abstract The transformation of bacteria was successfully carried out using a plasmid carrying a gene that codes for green fluorescent protein, which gives a signature green glow reminiscent of a jellyfish. This gene, however, is only active when the sugar arabinose is present. A gene coding for antibiotic resistance was also found within the plasmid and served as a means to verify that transformation had indeed taken place. The hypothesis was that the bacteria would be transformed and only those transformed bacteria in the presence of arabinose would fluoresce the characteristic green of the active protein. In addition, it was hypothesized that the protein, not the gene itself was responsible for the traits seen. The hypothesis has been accepted based on the data collected. This acceptance is largely based on two simple observations. Only those bacteria that had been transformed with the plasmid could grow in the face of the antibiotic. Furthermore, the only bacteria that could glow green was the bacteria that was exposed to the sugar arabinose which in this case has been proven to induce transcription of the GFP protein. This indicates that the molecule responsible for the observed green trait was protein. The transformation efficiency was found to be 793.75 transformants per microgram. The protein was filtered out of the cell and column chromatography was used as a means to isolate the protein by taking advantage of its hydrophobic properties. This was performed by washing the column containing the GFP protein amongst other proteins with buffers varying in salt concentrations. As expected, when a buffer of high salt concentration was used (binding buffer) the protein remained in the column. This was evidenced by the green glow of the column coupled with the non-glowing wash remnants collected. However, when the low salt buffer was used, the column no longer maintained the green glow and the wash collected