NANCY+I

= Gbr22's Random Walk = Need a scientific title
 * Introduction: **

Recombinant protein is a protein that has been produced from recombinant DNA that has been made from combining many different pieces of DNA [1]. Being able to produce recombinant proteins using a host cell such as bacteria E. ColiBL21 (DE3) allows researchers to determine how the protein of choice would respond to different inhibitors, ligands or potential drugs [2]. Gbr22 was the protein chosen, and the plasmid pGEM-gbr22 was utilized to insert the recombinant DNA coding for the protein into the chemically modified E. coli BL21 cells. Plasmids are generally loops of DNA that function by entering the host cells and telling the cell to express its protein [3]. The plasmid used will spur the BL21 to synthesize gbr22. The pGEM-gbr22 plasmid has a gene that encodes ampicillin resistance. In order to ensure that only transformed cells were expressed, the BL21 cells were grown on an agar plate containing ampicillin. Te antibiotic will kill any non-transformed BL21, cells that do not have the gene coding for ampicillin resistance, while the transformed cells will succesffully grow under favorable conditions. The objective of the lab was to overexpress the protein Gbr22 in E. coli cells by using the plasmid pGEM-gbr22, lyse the cells open and purify the protein using a combination of batch and column chromatography, then characterize it through gel electrophoresis.


 * Materials & Methods: **

LAB 1 Protein Expression

25 uL of E. Coli BL21(DE3)- the host cell-(New England BioLabs, Ipswich,MA),was transferred to 2 transformation tubes to prepare for cloning. pGEM-gbr22 plasmid was added to the DNA tube and both tubes were heat shocked at 42 degrees Celsius for 45 seconds. SOC media was added to each of the tubes and were shaken in a water incubator for 30 minutes at 37 degrees Celsius at 250 rpm. 50ul of bacteria/SOC mixture was pipetted from each tube and added to agar plates containing ampicillin. Overnight incubation followed. A single colony of bacteria was then added to LB/ampicillin media. The two tubes were incubated for 8 hours (37 degrees Celsius 200-350rpm). 0.625mL of starter culture containing pGEM-gbr22 was transferred into an Erlenmeyer flask that contained LB and ampicillin. The flask then shook for twenty-four hours in a dry shaking incubator. A 500uL sample of culture was taken (sample 1). The remaining contents of the bacteria were poured into a 50ml conical tube and were centrifuged into an Allegra X-15 benchtop centrifuge (Beckman Coulter, Inc., Brea, CA) and was set to run for 10 minutes at 5000 rpm and 4 degrees Celsius. The purple pellet at the bottom was saved and the remaining liquid was discarded after getting bleached for approxiamtely 20 minutes. 2.5 mL of 1x PBS solution was mixed with the pellet. Lysozyme was also added to the tube and the tube was stored in the -20 degrees Celsius freezer.

LAB 2 Protein Purification 2uL of Benzonase was added to the conical tube ( refer to figurs 5 & 6) that was stored in the -20 degrees Celsius freezer, in order to facilitate DNA and RNA removal. The lysate was distributed into two Eppendorf tubes and were then centrifuged. 50ul of supernatant was then taken (sample 2). The liquid supernatant from both tubes were transferred into a conical tube and filtered though a PES syringe filter. An Econo column was used to run the supernatant along with the Ni-NTA resin/buffer. The supernatant was mixed with 0.5mL of Ni-NTA resin/buffer mix and run through the column (50uL of this was taken as Sample 3). 5mL of Wash elution that consisted of 1x PBS and 20mM imidazole was run through the column (50uL of this was taken as Sample 4). Then, 5mL of elution buffer that consisted of 1x PBS and 250mM imidazole was run through the column twice (first time: 50uL taken as sample 5, and second time 50uL as sample 6). The absorbance of the protein solution from elution 1 was measured using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE),at 280 and 574 nanometers in order to make sure that the gbr22 protein was purified into the elution solution.

LAB 3 Protein Characterization From expression and purification, six samples [Sample 1 (cell solution) contains E.coli cells that are expressing the purple gbr-22 protein, the lysozyme enzyme, and cell wall fragments. Sample 2 (solution fraction) contains bacterial proteins, gbr-22, and supernatant fluid. Sample 3 (flow through) contains proteins that did not adhere to the nickel; the gbr-22 protein was not collected in this sample because it was bound to the beads. Sample 4 (wash) contains loosely bound proteins. Sample 5 (Elution 1) contains a majority of gbr-22 protein. Sample 6 (Elution 2) contains extra remnants of gbr-22 protein ],were gathered and placed into a heat block at 95 degrees Celsius for 5 minutes, after which they were centrifuged for 2 minutes at 5,000 rpm. loaded into Bio-Rad precast polyacrylamide gel. The SDS electrophoresis was assembled and prepared accordingly so that the samples could be run for twenty-five minutes at 200V in a Mini-PROTEAN electrophoresis tank filled with 750mL of 5x TGS buffer. The samples were resuspended in 10uL 6x sample loading buffer and loaded into the gel along with a Page Ruler Prestained Protein Ladder (ThermoScientific) refer to figure 11. The gel was stained, destained, and dried the following day on Whatman paper and covered with saran wrap at 75 degrees Celsius for approximately 1.5 hours.

Need to improve on caption **Results:**  

<span style="font-family: 'Times New Roman',Times,serif;"> Need to state what is in Elution 1 and 2 <span style="font-family: 'Times New Roman',Times,serif;">

Need to specify what each lane stands for

Lane 1:

Lane 2:

Lane 3:

<span style="font-family: 'Times New Roman',Times,serif;"> Need to talk about the significance of using LB/Amp plate & yield <span style="font-family: 'Times New Roman',Times,serif;">Lysozyme is used to break apart the cell components. With the cell membrane broken down, the DNA is free inside of the solution, able to extract and manipulate. Cyanase <span style="color: #ff0000; font-family: 'Times New Roman',Times,serif;">(used benzonase) <span style="font-family: 'Times New Roman',Times,serif;">reduces the viscosity of the solution by digesting DNA. This allows easier access to the protein itself, and not sticky substances along with it.
 * <span style="font-family: 'Times New Roman',Times,serif;">Discussion: **

<span style="font-family: 'Times New Roman',Times,serif;">The HIS tag is inserted into the protein and attaches to it <span style="color: #ff0000; font-family: 'Times New Roman',Times,serif;">(We did not physically attach the 6X His tag on the protein) (Where is the 6X His tag located on the protein?) <span style="font-family: 'Times New Roman',Times,serif;">. Therefore, when Nickel is added to the solution, the protein binds to it, allowing all else <span style="color: #ff0000; font-family: 'Times New Roman',Times,serif;">(give examples of what flows through the column) <span style="font-family: 'Times New Roman',Times,serif;">in the solution to run through the filter. Then, when large concentrations of imidazole is added to the nickel and protein solution, the HIS tags are replaced by the binding of nickel and imidazole. The protein is then allowed to flow through and be collected <span style="color: #ff0000; font-family: 'Times New Roman',Times,serif;">(in elution 1 and 2) <span style="font-family: 'Times New Roman',Times,serif;">.

<span style="font-family: 'Times New Roman',Times,serif;">Sample 1 is the cell fraction, the bacteria culture after incubation. Sample 2 is the soluble fraction of the bacteria and lysate, breaking down cell components. Sample 3 is the flow through after the lysate and resin and buffer are added to the column and flowed through material. Sample 4 is the wash. This is the waste after the wash buffer is added to Ni-NTA and the protein. Sample 5 is elution 1 which is the isolated protein after elution buffer is run through the column causing nickel to release the HIS tags on protein and bind to imidazole. Sample 6 is elution 2 which is the same and elution 1, just the second round of adding elution buffer to ensure all the protein is extracted.

<span style="font-family: 'Times New Roman',Times,serif;">The wash buffer contains a small concentration of imidazole aiming to remove the loosely bound proteins to resin along with other substances, leaving behind the strong resin-protein solution. In contrast, elution buffer contains a large concentration of imidazole in order to unbind HIS tags on the protein from nickel. The imidazole will bind to the nickel allowing the freed protein to run through for collection.

<span style="font-family: 'Times New Roman',Times,serif;">The known molecular weight of gbr22 is 25.7 kilo Daltons. Comparing to the ladder used in our gel, the protein in elution 1 is in fact gbr22, with the strong band slightly above the 25kDa line.

<span style="font-family: 'Times New Roman',Times,serif;">Sources of error include contaminantion ( non-sterile techniques), human miscalculations and faulty equipment. Other possible errors could have occurred when obtaining purification samples of protein during purification. This is demonstrated in column 7-10 which appear very light. This suggests that not a lot of protein was obtained. This could have also occurred due to the lack of imidazole added to release the protein into sample 5. As seen in figure 9, a dark band appears below the gbr-22 protein band (25 kDa) at around 15 kDa. This is a contamination from another protein; this along with lighter bands indicates that the predicted purity of 80% is now an estimated 30%.


 * <span style="font-family: 'Times New Roman',Times,serif;">Conclusion: **

<span style="font-family: 'Times New Roman',Times,serif;">In this lab a purple protein gbr22 was over expressed in E. Coli cells, harvested, purified using a chromatography column, and characterized with a SDSPAGE electrophoresis gel. The key findings in this lab include the estimated purity of the final protein solution approximately 75%, and therefore the total yield of protein, 1.85 mg.

<span style="font-family: 'Times New Roman',Times,serif;">The techniques used to express target proteins by overexpression in E. Coli followed by the use of Ni-NTA affinity chromatography column purification demonstrated successful production of gbr22 and purification as exhibited by the nanodrop spectroscopy in which the absorbance peak indicated a greater concentration of purple protein (gbr22) and by the electrophoresis where the darkest band after the elution was shown at the molecular weight that corresponded with gbr22. The techniques used demonstrated a relatively high purity purification of protein and could be used in future work to isolate proteins for <span style="color: #ff0000; font-family: 'Times New Roman',Times,serif;">X-ray Diffraction characterization (?) <span style="font-family: 'Times New Roman',Times,serif;"> as well as enzyme-binding assay analyses <span style="color: #ff0000; font-family: 'Times New Roman',Times,serif;">(also inhibition assay) <span style="font-family: 'Times New Roman',Times,serif;">.

<span style="font-family: 'Times New Roman',Times,serif;">[1] Haghi F., Peerayeh S. N., Saidat S.D., Cloning, expression and purification of outer membrane protein PorA of Neisseria meningtidis serogroup B, Journal of Infection in Developing Countries.**2011**, 5(12), p856-862. <span style="font-family: 'Times New Roman',Times,serif;">[2] Zhou, W.; Merrick, B.; Morteza, K.; Tomer, K., Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectroscopy. Journal of the American Society for Mass Spectrometry.**2002**,11(4), 273-282. <span style="font-family: 'Times New Roman',Times,serif;">[3] Halavaty, A. S.; Kim, Y.; Minasov, G.; Shuvalova, L.; Dubrovska, I.; Winsor, J.; Zhou, M.; Onopriyenko, O.; Skarina, T.; Papazisi, L.; Kwon, K.; Peterson, S. N.; Joachimiak, A.; Savchenko, A.; Anderson, W. F., Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. Acta Crystallogr D Biol Crystallogr.**2012**, 68 (Pt 10), 1359-70.
 * <span style="font-family: 'Times New Roman',Times,serif;">References: **