Ling+W

__ Bacterial Protein E.P.C (Expression, Purification and Characterization) __

__ Introduction: __ Bacteria expression is used in labs to have a visual confirmation that the goal of the experiment was met. The three parts to the process include transforming competent bacterial cells with DNA plasmid, purifying the plasmid to get rid of the “other” substances, and characterization. The strain of E.coli used was BL21(DE3) with the plasmid pGEM-grb22. The pGEM-grb22 encodes for a fluorescent protein that glows purple, originally cloned from coral from the Great Barrier Reef. To ensure that the correct plasmid being tested is present, the substance will be placed into a spectrophotometer at 280nm and 574nm after purification, because most bacteria can be detected at 280nm and the purple protein can be detected at 574nm. Throughout the process the presence of the purple protein will be tested, if it is inserted into BL21(DE3), then the bacteria will appear purple, because the protein itself is fluorescent.

__ Materials & Methods: __ A plate of the bacteria with the insert was grown out into colonies with the initial start of 25microliters. A bacteria colony was placed in water bath at 37degress Celsius with LB-ampicillin media. After about a day, 50mililiters samples of the bacteria were put into two conical tubes and centrifuged by an Allegra X-15 benchtop (Beckman Coulter, Inc. Brea,CA). The fluids were removed from the centrifuge and decanted. After, Lysozyme and 2.5mililiters of 1X PBS was added to the tubes and stored at -20 degree Celsius.

The next step is purification. 2microiliters of benzene was added to the sample. The lysate from part one was centrifuged and decanted. The supernatant was filtered through a PES syringe filter and the precipitates were discarded. Then, the solution left was put into an Econo column and added Ni-NTA. During the first wash, the elution buffer was used to remove excess Ni-NTA that did not stick to the histidine on the protein. A second elution that contained pGEM-gbr22 was added to the solution to move the protein. Lastly 1ml of nanopure water with 30% ethanol was added. Then a sample after elution 1 was measured at 280nm and 574nm for its absorbance with a nanodrop spectrophotometer.

The final step is characterization by the use of electrophoresis. Each of the 6 samples were given 6x gel-loading buffer and placed into a heat block for 5 minutes at 95 degrees Celsius. After, sample one was placed into well 2 and so on until all 6 were loaded into their designated well. The gel was placed into the SDS for 90 minutes. The gel was stained with imperial dye and washed. In between each wash the gel was placed onto an orbital shaker. After, the gel was dried and analyzed.

__ Results: __

__ Discussion: __ Part one of the methods, the colonies did not grow, so someone else's were used to substitute. The reason for this could have been an error of heat shocking the bacteria too long that it killed most of it.

During purification, the spectrophotometer shows that at 280nm the bacteria was present by graph one and at 574nm, graph two, the protein of interest was present based on its absorbance.

In figure 1 wells two through six were mine, while the rest belong to someone else. looking at well seven, two lines showed up meaning that there were two proteins present. During the purification process, not all of the other substances were taken out so it was not only the protein of interest. __ Conclusions: __ Through the experiment, there were many failures, but also the better experience of how working with bacteria, a living organism, can be challenging. The first two part were the most tricky, because it was working with the bacteria itself, and manipulating it so the protein of interest was left. The last part would be considerably simple since it wast just taking the already made samples and putting them into a machine for the gel to run. The results showed that the hypothesis if BL21(DE3) is inserted into the bacteria, then the bacteria will appear purple, because the protein itself is fluorescent. That was true observing other's successful bacteria colones grown out in LB broth. But that does not necessarily mean it carried out through the experiment, because the protein could have been pulled out during purification.