GFP LAB - lab PDF

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GPF Isolation Results The goal of this experiment was to understand the uses of marker or reporter genes in molecular biology experiments and

GFP isolation is achieved in two steps; bacterial cell lysate preparation and GFP isolation using HIC(hydrophobic interaction chromatography ) . During cell lysate,

4. The result of all the tubes can be seen in figure 9 with the fluorescence only ended up in tube 4 meaning that all of the molecules that were still bounded were removed.

Discussion Overall, after the bacteria lysate is introduced right above the matrix making sure to not deturbed the beads in (results displayed in tube 1) the green fluorescent protein is clinging to the top beads. After the low salt solution is added the green fluorescent protein remind clinging and the other proteins dripped out. (results displayed in tube 2 ) and after the no salt buffer was added to the column that is when you get the green fluorescent protein start moving down ,( tube 3 ). And

after more TE no salt buffer was added you can see the florescence green in the collection tube for tube 4. We know that the green fluorescence protein is hydrophobic, so when we add our bacterial lysate to this. The green fluorescent protein is going to cling to the sides of the beads in the column because it is always hydrophobic, especially in a high salt buffer. Then after you add the low salt buffers this removes all the other proteins that the bacteria produces this causes the release of the green fluorescence protein , allowing it to be captured. Overall this experiment proves the point that if we can add a foreign gene into a bacteria and it produces a lot of protein then the GFP HIC procedure can be used to purify it and the protein can be used to do what scientist want them to do.

Heat stability Results The goal of this part of the experiment is to understand the highest temperature at which GFP remains functional, so that GFP can be used in research at a right temperature. This part of the lab aims to design and “conduct” an experiment to test GFP’s heat stability. The experiment was separated into two parts, positive control, and negative control. The negative control tubes had 20 uL of distilled water and 20 uL of GFP from tube 4. For the positive control tube one tube of the purified GFP was used. After the incubation of two tubes of GFP each at the chosen temperatures of 37℃, 50 ℃, 70 ℃, and 100℃ were used for 10 min and the results are shown in figure 9 under a UV transilluminator. The negative control indicated that the fluorescence thrives at 37℃, and 50 ℃,. For the positive control the fluorescence only thrives at 70℃ and completely fades away at 100 ℃,.

Discussion The results from this experiment indicates that the fluorescence does not thrive at temperatures that are 100 ℃ and higher. The significance of the GFP heat stability is important because you must know the temperature limits when it comes to doing experiments with this protein. For example, if the GFP heat stability was tested we would not know if it was possible to do the previous HIC experiments under the temperature conditions that we had it. What if the GFP only worked in super low or super high temperatures, or if certain temperatures expedite or slows down the awaiting results. The only thing I would do differently in this experiment is to test wider ranging in temperatures.

Figures

figure 1 : Before the start of the experiment Ecoli in normal room lighting

figure 2 : Bacterial culture observed under the UV light

figure 3 : After centrifugation, pellet and supernatant of bacterial culture observed under UV light

Figure 4 : After three cycles of freeze/thaw and centrifugation, bacterial culture was observed under UV light

Figure 5: Column and collection tube 1 observed under UV light

Figure 6: Column and collection tube 2 observed under UV light

Figure 7: Column and collection tube 3 observed under UV light

Figure 8: Column and collection tube 4 observed under UV light

figure 9 : all tubes on a UV transilluminator Top left to right: Negative control, 37oC, 37oC, 50oC, 50oC Bottom left to right: Positive control, 70oC, 70oC, 100oC, 100oC...