Zach+R

**Over-expression, purification, and characterization of protein pGEM-gbr22 in non-lethal //E. coli// bacteria**
In order for a foreign protein to be expressed in //Escherichia coli//, used frequently due to its availability and low price, a plasmid containing the protein must transform the cell culture to be resistant against ampicillin[1]. The ampicillin-resistant protein allows all // E. coli // cells not containing the purple gbr22 to die off for better analysis of the target protein. Lysozyme was used to break down cell walls to allow easy transfer of the plasmid, albeit after heat-shocking [2]. Purification through chromatography is effective at isolating the target protein through use of histidine tags. Nanodrop spectroscopy and gel electrophoresis (SDS-PAGE) are used to characterize the protein. This lengthy process is used to determine the purity and molecular weight of the protein, gbr22, while isolating it from all other proteins that could create a false molecular weight or contaminate the protein wanted for isolation.

A tube containing 25 μL of competent bacterial cells (non-lethal //E. coli BL21 (DE3)//, New England Biolabs, Ipswich, Massachusetts, United States) and plasmid were heat-shocked in a water bath at 42 °C for 45 s, iced, and SOC media was added. The cells were placed on a plate and left to incubate overnight at 37 °C. Ten microlitres of ampicillin and 5 mL of LB broth were added to the tube and left to shake and incubate for roughly 8 h at 300 rpm and 37 °C. Ampicillin was later added. Part of that was saved as sample one; the rest was centrifuged and later resuspended in PBS and lysozyme, and placed in the -20 °C freezer. Once thawed, benzonase was added, centrifuged, and the supernatant liquid was drawn off. The supernatant liquid was kept as sample two. The sample was run through a filter column, washed, and eluted twice, collected as different samples. A nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, United States) was then used to analyze the absorbance of the two elutions at 280 nm and 574 nm, from a very small 2 µL sample. Remaining samples were centrifuged once more, and loading buffer was added, mixing the samples well. The samples were then heated at 95 °C for a few minutes and then centrifuged again. These samples were used in comparison against a molecular weight standard used in SDS-PAGE wells, run for 25 min at 200 V. Once the gel was run, it was washed repeatedly, stained, and dried. The gel did not survive the drying process.

__ Results: __

























Error could have stemmed from any one of the many steps in this lab. Lysozyme may have been added twice, doubling its concentration, and potentially affecting the //E. coli//'s uptake of the plasmid, and, eventually, the expression of the purple gbr22 protein. Pipets used during the spectrophotometry phase may have been contaminated with the slightest touch of a foreign object, rendering all data based on the nanodrop useless and inaccurate. The samples run through the Ni-NTA column may have had foreign protein in them, ruining the molecular weight estimate and purity estimate. The purity of the protein, with included errors, is believed to be around 50%.

Sample one contained whole //E. coli// cells from the initial protein expression and harvest, often centrifuged down to a pellet of organic matter. The lysate (collected after the cells were lysed) comprised sample two. Sample three was waste washed through the Ni-NTA column. After being run through the Ni-NTA column and attracted to the histidines (via imidazole), the resulting protein liquid, after being washed, made up sample four. Sample five was the protein liquid (sample four) eluted (protein was washed) once; sample six was sample four eluted twice. The elution buffer had more imidazole (and, thus, histidines) than the wash buffer, better separating the protein from the waste.

During these three labs, over the course of a few weeks, the protein pGEM-gbr22—found in coral off the Great Barrier Reef in Australia—was expressed, purified, and characterized. The molecular weight and concentration of the protein could be determined at various steps in these labs, and was ultimately estimated to be about 25 kDa. Future labs could use these results and products in enzyme assays to better understand the nature of the gbr22 protein.

__ References: __
 * 1) Protein production and purification. //Nat Methods.// **2008**, 5(2), 135-146.
 * 2) European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. (Heidelberg, Germany) []