Shreya+B

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

__ Introduction: __ The process of purification is commonly done in labs dealing with proteins.

__ Materials & Methods: __ First the DNA was transformed, which was done by adding 25 milliliters (ul) of the bacteria (BL21(DE3)) to the tubes with the DNA. These tubes were spun down and then 2ul of the plasmid, pGEM-gbr22 was added to these tubes. The tubes were then put on heat shock and 200 ul of SOC media was added. The mixture from the tubes was then added to Amp plates and put in an incubator. After the tubes sat in the 37 ° C incubator overnight, ampicillin and some bacteria colonies which grew from the night before were added to LB broth and put on a shaking incubator. This culture was then centrifuged the next day for 10 months at 4 ° C at 5000 rpm. 2.5 ml of the 1x PBS solution was added to the 50 ml conical with the cell pellet and then put on a vortex to take out any clumps. Lysosome was then added to the cell pellet and then put in the -20 °C. Then the cells were purified. This was done by added 2 ul of either Benzonase or Cyanase to the 50 ml conical. The conical was then centrifuged for 20 minutes at 14,000 rpm. The supernatant was then put into 15 ml conical tubes and two buffers, wash (1x PBS mixed with 20 mM imidazole) and elution (1x PBS mixed with 250 mM imidazole), were prepared. The supernatant culture (lysate) was filtered through a syringe filter. Then 0.5 ml of Ni-NTA resin/buffer was added to the lysate. The mixture was then run through the Econo column, as were the two different buffers. Waste, wash and elution 1 and 2 samples were collected from these runs. The syringe was stripped with 2 runs of 10 cv water and then 10 cv .5 M NaOH. Samples of the elution were put through the Nanodrop spectrophotometer at both 280 and 574 nm. The last part of this lab is the characterization of protein. The cells were centrifuged and then a 6x loading buffer was added to the samples. The tubes were put on a heat block for 5 minutes at 95° C. The samples were then loaded into an electrophoresis well and run for 25 minutes at 200 volts. The first well had the standard, a protein ladder, and the next 6 contained 6 samples from the other protein labs. Lastly, the gel was stained with Imperial protein stain and dried in a vacuum at 75°C at 1 and ½ hours on the gradient cycle.

__ Results: __ Figure 1a- The experimental plate. My experimental plate had some problems and did not grow up the bacteria as expected.

Figure 1b- The control plate. My control plate had a swab from the floor on it.

Figure 2- The flask above contains a bacterial culture with LB and ampicillin. Figure 3- The picture above is of the cell pellet which results from harvesting the cell. My wet pellet weight was 0.63 grams. Figure 4- This is an image of the spectra from the first Nanodrop spectrometer reading. The absorbance is at 280 nm. Figure 5- This is the second run of the Nanodrop spectrometer. The absorbance is also at 280 nm. Figure 6- This Nanodrop run was done at the extinction coefficient obtained from the magazine article and compares that result to the results from 280 nm.

Figure 7- This picture shows the gel before it went through the drying process. The well with the protein ladder contains the standard and then every well after that increases in sample number (1 to 6). Figure 8- This is the prestained protein ladder used to determine the molecular weight. It was obtained from the Fermentas website.

__ Discussion: __ Q- Lysosome is used to break down bacterial cells walls in order to gain access to DNA in bacterial cells. Bensonase/Cyanase is used to digest the DNA and/or RNA in the lysate and reduce the viscosity of the lysate as a result. Q- Sample 1 contains a cell culture with LB and ampicillin. Sample 2 contains a lysate with Bensonase/Cyanase. Sample 3 contains the lysate with about 1 ml of 1x PBS. Sample 4 contains the wash buffer with 20 mM imidazole in 1x PBS. Sample 5 contains the lysate with the first run of the elution buffer, which has 250 mM imidazole in 1x PBS. Sample 6 had the second run of the elution buffer in the lysate. Q- The wash buffer has 20 mM imidazole in 1x PBS and the elution buffer has 250 mM imidazole in 1x PBS. Q- The histadine tag attaches itself to the C terminal of the protein with a vector marked for this terminal. The tag then tracks the movement and actions of the protein.

__ Conclusions: __

__ References: __