SHAWN+O

Intro should be more background info on theory or on the technique than restating methods Missing images - Fun plate? Captions could be improved, for example state the absorbance readings for nanodrop Well done overall, good job on being clear and concise

__Protein Isolation of gbr22 Fluorescent Protein__ __Introduction__:

 In recent years, the use of recombinant proteins and the process of protein purification has increased greatly and yielded a large source of protein and biochemical information. [1] In use, throughout the world, this process of cloning, purification and expression has become one of the most successful methods to protein understanding.[2] Proteins are vital to functions in cell processes, thus an understanding of these experimental procedures proves advantageous to our society.[1] Within this lab, the objective involves a 3-part process in which the gene for the gbr-22 protein is placed in a plasmid and allowed to transform and replicate in a bacteria vector. This protein will then extracted and isolated using centrifugation, enzymes and affinity purification. Lastly, gel electrophoresis will be used in the protein characterization step. It is expected that the gbr-22 protein will be successfully replicated, isolated and extracted from the cellular material by the end of the protein isolation process.

__Materials__:

Several materials are needed to proceed with each process.Within the protein expression step, a competent bacteria (i.e BL21(DE3)), plasmid vector( pGEM-gbr22), Agar plates, coilrollers, LB Media, and gas burner are needed. Within the protein purification step, a Bio-Rad Econo chromatography column, syringe filter, centrifuge/centrifuge tubes, Imidazole, 10X PBS, 1X PBS, NI-NTA resin, and Cyanase(or Benzonase) are needed. Lastly, within the protein characterization lab, a heat block, Mini Protein electrophoresis tank, TGS running buffer, Bio-Rad polyacrylamide gel, 6x gel-loading buffer, protein samples, imperial protein stain, and molecular weight standards are needed. Moreover, micropipettes micropipette tips, ice and tubes were needed for all three experiments. PPE (gloves, goggles, footwear, lab apron) are necessary for each experiment

__Methods __:

The first day involved expression and replication of the protein within Ecoli. Initially, 25 ul of bacteria(BL21(DE3)) and 1-2 ul of plasmid DNA were placed in a transformation tube, heat shocked then placed in ice. This allowed for transformation of the plasmid(pGEM-gbr22). 50 ul of this mixture were than pipetted into an agar plate of which coil rollers were added in order to evenly spread the mixture. Nextly, these plates were incubated over night and used to create a starter culture. .625 ml of this starter culture, 25 ml of LB media, and .05 ml of the Ampicillin were transferred to an Erlenmeyer flask and incubated for 16-24 hours. Lastly, bacteria was placed in a conical tube for centrifugation. The pellet was then saved, suspended in PBS and lysosome was added to it. The next step involved the protein purification process. Firstly, the 50 ml were thawed and 2 ul of Cyanase was added to the tubes. This solution was mixed, centrifuged for 20 minutes and the supernatant was saved. Column chromatography and NI-NTA beads were used to further purify the protein. The protein was first washed with 20mM of Imidazole and then eluted with 250 mM. A nanodrop spectrophotometer reading was then performed on Elution 1 at an absorbance of 280 nm and 574 nm. Lastly, the six samples collected were run on gel electrophoresis with PageRuler Prestained Protein Ladder (ThermoScientific, #26616) as molecular weight standard. Gel was then dryed and analyzed.

__Results__:   Figure 1: Image of Agar Plate: BL21(DE3) bacteria with LB+Amp media. Approx. 300 colonies

 Figure 2: Image of Agar Plate: BL21(DE3) bacteria with LB media(Control Plate) Approx. 10 colonies

 Figure 3: Image of Erlenmeyer flask of post-incubated culture. Bacteria(BL21-DE3) transformed with pGEM-gbr22 plamid.  Purple color indicates extensive growth and reproduction.

Figure 4: Image of cell pellet. Pellet yielded from centrifugation of BL21(DE3) bacteria transformed with pGEM-gbr22 plasmid. Mass=.37 g

Figure 5:Image of Elution 1 buffer with protein gbr22. (5 mL) Figure 6: Elution 1 buffer with protein gbr22. 5 mL obtained.

Figure 7: Absorbance vs Wavelength Nanodrop spectrophotometer reading of Elution 1. Measured at wavelength of 280nm. Trial 1

Figure 8: Absorbance vs Wavelength Nanodrop spectrophotometer reading of Elution 1. Measured at wavelength of 280nm. Trial 2

Figure 9: Electrophoresis gel of Samples 1-6. Molecular Marker gel shown Molecular weight standard: PageRuler Prestained Protein Ladder.

Figure 10: Gel Electrophoresis Molecular weight standard: PageRuler Prestained Protein Ladder (Product #26616)

Concentration at 280 nm: Average A: (0.239+0.194)/2= 0.2165 <span style="font-family: Arial,Helvetica,sans-serif;">c= 0.2165/[(38850 M-1 cm-1)(1 cm)]= 5.57*10^-6 mol/L (25,794 g/mol) = 0.144 mg/mL

<span style="font-family: Arial,Helvetica,sans-serif;">Concentration at 574 nm: <span style="font-family: Arial,Helvetica,sans-serif;">Average A: (0.190+0.230)/2= 0.21 <span style="font-family: Arial,Helvetica,sans-serif;">c= (0.21)/[(118300 M-1 cm-1)(1 cm)]= 1.775*10^-7 mol/L (25,794 g/mol)= 0.0458 mg/mL

<span style="font-family: Arial,Helvetica,sans-serif;">__Discussion__:

<span style="font-family: 'Times New Roman',Times,serif;"> This process involved the isolation of the gbr-22 protein from a bacterial cell. As predicted, through the hypothesis, the protein was successfully extracted and isolated from the rest of the cellular material. Initially in the first step, protein expression, ampicillin was used as a marker gene in order to isolate the cells that were successfully transformed with the plasmid. Heat shock is the technique that makes the membrane permeable to plasmid and allows for movement inside the bacterial cell. Moreover, within the protein purification step, the lysozyme was used to digest the cell wall, while the Cyanase/Benzonase was used to digest the nucleic material (RNA/DNA). After centrifuged, the supernatant contained the protein of interest, while the pellet held the heavier cell debris and nucleic material. Also, within this step, HIS tag system involved the binding of nickel to the histidine of the target protein. This solution was then placed through column chromatography in which, the first flow through removed proteins not bound to the nickel resin, the Wash step removed protein loosely bound to the nickel resin, and the Elution step used Imidazole to compete with the nickel and dislodge the nickel-bound gbr-22 protein from the resin. The last step allowed for SDS-PAGE to separate proteins in sample. SDS (Sodium dododecyl Sulfate) coats these proteins in a negative charge and denatures them to allow for all the proteins to move towards the positive charged side. Movement across the gel was based on size. Smaller proteins moved faster, while larger proteins moved slower.

<span style="font-family: 'Times New Roman',Times,serif;"> Sample 1 contained all cellular material including the DNA, RNA and cell wall. Sample 2 contained only the cellular protein. Sample 3 contained the protein that was not bound to the protein. Sample 4 contained protein that was loosely bound to the resin (Wash Step). Sample 5 contained the gbr-22 target protein that was dislodged from the resin with Imidazole (Elution 1). Lastly, Sample 6 contained the leftover gbr-22 protein that was still bound to the nickel. <span style="font-family: 'Times New Roman',Times,serif;"> Based on the Nanodrop spectrophotometry at 280 nm, it was determined that there was .144 mg/ml of protein.According to the reading, gbr22 absorbs at 574 nm. Based on this wavelength, there was .0458 mg/ml protein. Thus, it was expected that there would be other types of proteins present in the sample. Based on the molecular ladder, the size was determined to be ~30kDA/~500 bp. Moreover, the electrophoresis gel contained two bands that indicates 40% purity <span style="font-family: Times New Roman,Times,serif;"> Sources of error within this three-part experiment involved the possible contamination of the bacteria cultures or protein samples. Initially, the presence of colonies on the control plate is evidence of early contamination. Moreover, the handling and ripping of the electrophoresis gel was also a source of error that occurred within the protein characterization step. This, however, did not significantly alter the clarity of our results. Lastly, the drastic misreadings of the spectrophotometer indicate another source of error.

<span style="font-family: Arial,Helvetica,sans-serif;">__Conclusions__:

<span style="font-family: 'Times New Roman',Times,serif;">Within this experiment, gbr-22 was implemented into a plasmid(pGEM-gbr22) and transformed into bacteria (BL21(DE3)). This protein was expressed then isolated in the purification process. Samples were taken and placed in a gel electrophoresis for seperation. This gel was then analyzed and used to analyze the purity/gel characteristics of the protein. Within VDS, disease-causing proteins are analyzed for probable dugs. This process serves as a method of further isolation and provides us the ability to understand the chemical/physical properties of proteins.

<span style="font-family: Arial,Helvetica,sans-serif;">__References__:

<span style="font-family: 'Times New Roman',Times,serif;">[1] Acton, T.B.; Albeck, S.; Almo, S. C.; Anderson, S.; Arrowsmith, C.; Atwell, S., Protein production and purification. Nat Methods 2008, 5, (2), 135. <span style="font-family: 'Times New Roman',Times,serif;">[2] European Molecular Biology Laboratory. Protein Expression and Purification Core Facility.[] (accessed April 17, 2013).