ANDREA+F.

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__ Results: __ ABC Figure 1) Agar plates after first overnight incubation period. Plate A contains BL21(DE3) bacteria with pGEM-gbr22 plasmid on an agar plate with ampicillin. Ampicillin-resistant colonies on the experimental plate experienced growth and small colonies are visible as whitish-yellow round dots. Plate B was the control plate. This plate contained BL21(DE3) bacteria with no DNA plasmid on an agar plate with ampicillin. Bacteria did not experience growth on this plate due to ampicillin vulnerability. Plate C was the fun plate and did not contain BL21(DE3), plasmid, or ampicillin. Bacteria on this plate originated from the following sources: Joey's forehead, used gloves from the lab trash bin, the men's room urinals and paper towel dispenser, and coughing (from both lab partners). No growth is visible after the first overnight incubation period. Figure 2) Purple culture in Erlenmeyer flasks after a 24-hour incubation period. Media in flasks appear purple in hue after being shaken in the incubator for 24 hours at approximately 37 degrees Celsius and 200-350 rpm. Media consists of 25 mL of LB broth, 625 microliters of bacterial starter culture (BL21(DE3) bacteria and pGEM-gbr-22 plasmid), and 50 microliters of ampicillin. Figure 3) Wet cell pellet appearing purple in hue due to purple protein expression after centrifuging for 10 minutes at 5000 rpm and 4 degrees Celsius, After being centrifuged, the liquid portion of the culture that separated was drained from the conical tube. The resulting pellet consisted of the bacterial BL21(DE3) cells and the pGEM-gbr22 plasmid coding for the purple protein. Figure 4) Fun plate after a 3-day incubation period at 37 degrees Celsius. Large colonies are visible on the agar plate (no ampicillin). Bacteria colonizing the plate originated from the following sources: Joey's forehead, used gloves from the lab trash bin, the men's room urinals and paper towel dispenser, and coughing (from both lab partners). AB Figure 5) Elution buffers after filtering through column and purifying protein. The solutions contain 1xPBS, 250 mM imidazole, and gbr22 protein. Elution 1 (A) contains the gbr22 protein released in the first rinse of the column using heavy concentration of imidazole. Most of the protein was rinsed into this first elution 15ml conical tube (A) seen with a distinct purple hue. The second 15ml conical tube (B) contains the second rinse, capturing any last protein until the elution buffer ran clear through the column. AB Figure 6) Absorbance spectra for elution 1 at 280nm wavelength. Nanodrop spectrophotometry absorbance of gbr22 protein in Trial 1 (A) and Trial 2 (B) with absorbance of 0.134 and 0.137, respectively, at a 1cm path length. Wavelength (nm) on x-axis and absorbance on y-axis. AB Figure 7) Nanodrop spectrophotometry absorbance of gbr22 protein at 574nm (maximal wavelength) and 280 nm wavelengths. Trial 1 (A) shows absorbance 0.29 and 0.21 absorbance at a 1cm path length, respectively. Trial 2 (B) shows absorbance 0.33 and 0.22 at a 1 cm path length, respectively. Wavelength (nm) on the x-axis and absorbance using a 1mm path length on y-axis. Absorbance readings for elution 1 only. Figure 8) Protein characterization gel electrophoresis results for gbr22 protein after gel was stained with bromophenol blue overnight and dried for approximately 1.5 hours. Lane 1 displays the molecular weight standard. Lane 2 is empty. Remaining lanes are samples 1-8: cell lysate, soluble fraction, flow through, wash, elution 1 (SC), elution 2 (SC), elution 1 (AF), and elution 2 (AF). First six samples are from my lab partner and last two elution samples were mine. Contamination can be seen in all sample lanes because there is more than a single band, which would signal a pure sample of only the gbr22 protein (~25kDa). Figure 9) Pre-stained molecular weight standard ladder used in lane 1 (Fig. 8). Purified protein gbr22 should be in the 25kDa ladder range.

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