pGLO

Topics: Protein, Green fluorescent protein, Molecular biology Pages: 6 (1313 words) Published: December 5, 2013
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 However, when the low salt buffer was used, the column no longer maintained the green glow and the wash collected was glowing green. This indicated that the GFP protein was removed from the column. Gel electrophoresis was then used to separate the GFP protein from the other proteins. Laemmeli buffer was added to equalize the charge of the amino acids found within the proteins, allowing the proteins to be separated solely on the basis of their size. Upon completion, a standard curve was used to estimate the molecular weight of the GFP protein separated. By using the y-intercept method, a molecular weight of 23,900 Daltons was estimated. Compared to the known molecular weight of the GFP protein, this is a percent error of 11.0%.

Materials and Methods
Week 1: pGLO Transformation
Materials:
E. Coli starter plate
Four agar plates
Transformation solution
LB nutrient broth
Loops for colony collection
Pipets
pGLO plasmid
Method:
Two microtubes were obtained and labeled +pGLO and –pGLO. 250 microliters of calcium chloride were added to each tube to serve as the solvent. These tubes were iced and bacterial colonies were collected from the startup plate using the sterile loops provided. The bacteria were inserted into each of the tubes by placing the loop into the solvent and twirling it between the fingers vigorously. We then added pGLO plasmids to the +pGLO tube only, using a similar procedure with the sterile loop and allowed the tubes to sit on ice for ten minutes. During this time, four agar plates were obtained and labeled as follows:

Heat shock was then performed as the two tubes were added to a hot water bath at 42⁰ C for 50 seconds. Immediately afterward, the tubes were rushed to be put on ice and allowed to sit for 2 minutes. Then 250 microliters of LB nutrient was added to each of the two tubes. The suspension contents of the tubes were added to their appropriately labeled agar plates and the plates were incubated. Week 2: GFP Purification

Materials:
Transformation plates from previous week
Culture tubes with growth media
UV light...
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