NicoleD

Examining E.Coli Cells and Finding Gbr22 through Protein Sampling Series**

**Introduction:** Proteins for experimental work are produced using E.Coli bacteria strains. Proteins are produced by over expressing the target protein in bacteria and purifying the low availability proteins. Protein purification is done by using chromatography and gels to express the purity of a protein sample. Protein purification is obtained by separating the separating the unneeded parts of the protein such as the cell wall, from what is targeted and needed, while also obtaining low contamination. In the series of protein expression, purification, and characterization proteins techniques were used to further analyze the protein at each step. With protein expression a recombinant protein was over expressed in bacteria. From the over expressed bacteria, protein purification was done. For the last step in the series, the gel electrophoresis was used to analyze the samples prepared from the purification and expression labs.

**Materials and Methods:** In protein expression a recombinant protein was over expressed in bacteria. This was done using aseptic techniques with 3 different control plates to ensure the clean technique. Competent bacterial cells were transformed with a DNA plasmid. Then a starter culture of bacteria was grown overnight in LB with ampicillin in the incubator 4degree fridge; this culture was used to create a larger culture and express the recombinant protein in flasks that were placed in the shaking incubator for 16-24 hours. Cells were then harvested and frozen for additional experimental work in the next lab. The next lab was protein purification. The bacterial cells were first lysed, the lysozome added digested the cell walls and the solution was incubated to ensure complete lysis. Then the lysate was clarified using centrifugation and the insoluble cell wall debris was removed by centrifugation, and filtered using a syringe to get rid of large particulate matter. Then the protein was purified using the affinity tag and Ni-NTA resin. The following lab used samples from protein purification and expression to analyze the protein characterization. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins in the samples. The gel was prepared and stained using electrophoresis, and dried in the Biotech lab. Using the results from electrophoresis and spectroscopy the molecular weight of gbr22 protein, the purity, and yield of the final product were estimated. Using UV-Vis spectroscopy measurements from the last lab, the concentration of the protein solution was estimated. **Results:** **TRANSFORMATION PLATES (Protein Purification)**

** BL21(DE3) Amp plate 1 **, with no DNA control showed no change after being placed in the 37C incubator. ** BL21(DE3) Plate two ** was beginning to show many bacteria cultures after about 8hours, but they were not purple, or fully grown, when they were pulled out of the incubator. ** Fun plate with no Amp ** was coughed on, showed no difference after being placed in the 37C incubator for 8 hours; it was placed in the incubator for longer, and still showed no change.

These are the **pellets that formed after centrifugation**. The lighter purple pellet is the PGEM-gbr22 which showed up as yellow in the flask after shake incubation (shown to the right). The dark pellet is BL21(DE3), which showed up as a lighter purple when in the flask did shake incubation. Only the weight of the pellet was taken, which was 48.12mg.

**Elution 1 and 2 in conical tubes—Yield for 280nm is 0.57 mg/ml, Yield for max wavelength 574nm is 0.512mg/ml**



**Nanodrop spectrophotometer 2ul purified protein at absorbance 280nm reading one.** This shows the elution one spectra with its top absorbance and wavelength at 280nm. The absorbance is lower than the reading two. The concentration is 0.16mg/ml.



**Nanodrop spectrophotometer 2ul purified protein at absorbance 280nm reading one.** This shows a repeated measurement of the elution one spectra with its top absorbance and wavelength at 280nm. This spectra looks exactly like reading one but has a smoother curve at the wavelength of 280nm than reading one. The absorbance is higher than that in reading two. The concentration is 0.19mg/ml.



**Nanodrop spectrophotometer UV/VIS mode, Max wavel****ength 574 nm, wavelength two 280nm.** This spectra shows the absorbance for the maximum wavelength 574nm compared to the 280nm absorbance. 574nm has a higher absorbance than 280nm.

**Nanodrop spectrophotometer UV/VIS mode, Max wavelength 574nm, wavelength two 280nm reading two.** This spectra shows the second reading absorbance for the maximum wavelength 574nm compared to the 280nm absorbance. 574nm has a higher absorbance than 280nm. The curve for 574nm appears smoother than the reading one curve.

**BEER’S LAW CALCULATIONS:** **A=b****εc** 574 nm: 0.047= 118300 L/(mol cm )*0.1cm*c c= 3.973e-6 mol/L c*25274.2g/mol*5=0.512mg/ml

280nm: A= εlc 0.015= 39100 L/(mol cm )*1cm*c c= 4.425e-6 c*25274.2g/mol*5 = 0.57mg/ml

**Protein Gel before electrophoresis.** The first five lanes are ladders; the next five are samples 2-6, respectively.

**Gel after electrophoresis**

**Image of Ladder.** This is the molecular weight marker used to estimate the molecular weight of the purified protein, which is about 25kD.

**Discussion:** BL21(DE3) Amp plate 1, with no DNA control showed no change after being placed in the 37C incubator. Plate two was beginning to show many bacteria cultures after about 8hours, but they were not purple, or fully grown, when they were pulled out of the incubator. Fun plate with no Amp was coughed on, showed no difference after being placed in the 37C incubator for 8 hours; it was placed in the incubator for longer, and still showed no change. These are the pellets that formed after centrifugation. The lighter purple pellet is the PGEM-gbr22 which showed up as yellow in the flask after shake incubation (shown to the right). The dark pellet is BL21(DE3), which showed up as a lighter purple when in the flask did shake incubation. Nanodrop 2ul purified protein at absorbance 280nm had a higher absorbance reading for reading two than the initial reading. Reading two also had a smoother curve. This probably means that reading two was more accurate than the first one. Nanodrop UV/VIS mode, Max wavelength 574nm, showed a spectra with the results having the second reading absorbed for the maximum wavelength 574nm compared to the 280nm absorbance. 574nm has a higher absorbance than 280nm. The curve for 574nm appears smoother on the second reading than the reading one curve. It appears that the 25kD lines up to the band from the Gel on sample 5. The purity for the samples is almost 100%, which means the sample was eluted correctly to leave enough gbr22. Five ladders were added to the gel, instead of one. This was done because of inexperience and not knowing how to use the ladder for the gel. The calculations were off for the concentration in the spectrograph results, which means something in the solution created this error that was possibly a contaminant or incorrect measurements.

**Conclusion:** Throughout these series of labs, a protein was expressed, purified, and characterized to obtain a pure sample of gbr22. The concentrations and molecular weight were found to characterize this specific protein, by referring to the ladder. The size of the protein was found in this lab. In a future lab, these techniques can be used to further study and identify proteins.
 * References:**