User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/09/19

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Inoculation of Expression Culture Media

  • Following the previous lab entry of Sept. 18, the starter culture media inoculated with transformed E. coli were still mixing in the incubator orbital shaker at 237 rpm, 37°C (8:20 AM).
  • Noting the distinct characteristic of broth 1, the appearance had a murky white appearance. In comparison to the other inoculated media, broths 2, 3, and 4, broth 1 is the least turbid. All other media had the same turbidity. Hence, broth 1 will be isolated from the rest.
  • Initiating the inoculation of the expression culture media, the incubator orbital shaker was turned off. Broths 1, 2, 3, and 4 were poured into falcon tubes with corresponding labels and capped tightly. The tubes containing the samples were brought to the balance room for centrifuge at 4500 rpm for 15 min.
  • When the centrifuge was finished, the supernatant from each sample were poured into a biological waste container.
  • Aseptic techniques were still applied during inoculation by conducting the procedure at a close proximity of the Bunsen burner. Each aluminum foil top for the samples were flamed upon opening and after use.
  • Using a graduated 25 mL pipet with fast release, 4 mL of LB broth was taken from the expression culture media (contained in Fernbach flasks). 1 mL was aliquoted to sample 1 and 3 mL was released to sample 2. The 3 mL media in sample 2 was used to resuspend the pellet by the motion of a micropipet.
  • Once all of the pellet was resuspended into the media of sample 2, the media was transferred into sample 3. The same resuspension method was used in sample 3. When all of pellet 3 was resuspended into the media, the contents were transferred to sample 4. Pellet 4 was dissolved into the media. The final 3 mL media is saturated with cells from pellets 2, 3, and 4.
  • Following the same technique, pellet 1 obtained from broth 1 with the unusual turbidity was re-suspended by itself with a separate pipet.
  • From the falcon tube containing the 3 mL media, 1 mL aliquots were transferred to sample 2, 3, and 4 of the expression culture media.
  • The re-suspended pellet 4 was transferred to sample 1 of the expression culture media.
  • The samples were capped with the flamed aluminum foil tops and then placed in the incubator orbital shaker at 160 rpm, 37°C.

Protein Expression

  • While the samples were mixing, 0.4 M of isopropyl β-D-1-thiogalactopyranoside (IPTG, FW 238.31) was prepared by weighing out .4021 g of the white powder and dissolving it into 4 mL of distilled water.
  • After the white powder was completely dissolved, the incubator orbital shaker was paused; followed by 1 mL aliquots of IPTG to each sample.
  • A UV-Vis scan was required as mentioned in the protein expression protocol before the addition of IPTG. This was not executed due to time constraints.
  • There were no observable physical changes upon the addition of IPTG.
  • The expression culture media was allowed to mix for 4 hours.

Preparation of Binding and Elution Buffers

  • In the course of protein expression, the buffers, binding and elution, required for the affinity chromatography were made. One liter of the binding buffer and 500 mL of the elution buffer were needed. The binding buffer consists of .020 M Tris, .5 M NaCl, and .030 M imidazole. In one solution, .020 M of Tris, .5 M NaCl, and .5 M imidazole were combined to make the elution buffer.
  • Calculations for the binding buffer:

.020 mol of Tris × [math]\frac{121.14 g}{1 mol}[/math] of Tris = 2.4228 g of Tris

.5 mol of NaCl × [math]\frac{58.439 g}{1 mol}[/math] of NaCl = 29.2195 g of NaCl

.030 mol of imidazole × [math]\frac{68.077 g}{1 mol}[/math] of imidazole = 2.0243 g of imidazole

  • Calculations for elution buffer:

[math]\frac{.020 mol}{L}[/math] of Tris × .500 L of water = .01 mol of Tris × [math]\frac{121.4g}{1 mol}[/math] of Tris = 1.2114 g of Tris

[math]\frac{.5 mol}{L}[/math] of NaCl × .500 L of water = .25 mol of NaCl × [math]\frac{58.439g}{1 mol}[/math] of NaCl = 14.609 g of NaCl

[math]\frac{.5 mol}{L}[/math] of imidazole × .500 L of water = .25 mol of imidazole × [math]\frac{68.077g}{1 mol}[/math] of imidazole = 17.019 g of imidazole

  • The calculations were followed by weighing the raw materials. The Tris and NaCl appeared as white granular solids while the imidazole had an off-white, crystalline, needle-like structure. The actual weights are listed below in the table.
  • The weighed materials were combined according to their respective buffers. The buffers appeared as clear, colorless liquid solutions. This was followed by adjusting the buffers to pH 7. This was accomplished by the addition of HCl; no physical changes were observed upon the addition of HCl. The binding buffer was adjusted to pH 7.53 and the elution buffer was adjusted to pH 7.44. Next, the buffers were poured into sterilized plastic bottles; capped and placed in the refrigerator.
buffer contents actual weights for the binding buffer (g) actual weights for the elution buffer (g)
Tris 2.4271 1.2164
NaCl 29.2148 14.611
imidazole 2.0245 17.019

Harvesting of Cells

  • Following the 4 h. incubation period of the inoculated expression culture media, the incubator was turned off.
  • The culture media were poured into 4 sterilized plastic containers (holds approximately 500 mL). The containers were brought to the balance room for centrifuge.
  • The centrifuge was set to 25°C at 4500 rpm for 15 min.
  • Once the cycle has terminated, the supernatant from each plastic container were poured into biological waste. The pellets appeared as pastel yellow.
  • 10 mL of the binding buffer was added to each sample to re-suspend and preserve the pellets.
  • The re-suspended pellet of sample 1 was isolated and poured to its own falcon tube. The other 3 re-suspended samples were combined into one falcon tube with a total volume of 30 mL.
  • The falcon tubes were capped and placed in a freezer at -80°C.
  • The binding buffer was returned to the refrigerator and all other materials were cleaned and stored.