Protein Extraction from two Deinococcus species

Bacterial Protein Extraction: A Summary

February 11th, 2022

Introduction and Prelab

Proteins play important roles inside organisms, and by better understanding the metabolic pathways that they take part in, one can better study the genes that code for these proteins and alter them to favorably control certain metabolic functions of proteins. This is, of course, only possible if proteins were isolated from the cells. Bacterial protein extraction, a useful procedure necessary for various research applications like measuring the activity of specific enzymes in metabolic pathways that protect bacterial cells against oxidative stress, is the topic of today's blog. Before bacterial cells are ready for protein extraction, they have to be grown in liquid medium and measured for their optical density (OD). 

As a prelab preparation, two biological replicates of Deinococcus aquaticus taken from two separate colonies were inoculated into 50 mL tubes (Labeled A and B) with 20 mL TGY medium and grown over 48 hours. In addition, Deinococcus radiodurans was also inoculated into a 10 mL tube with TGY and grown over the same period. Subsequently, the OD measurement for both Deinococcus species was taken. D. aquaticus A measured 2.17, D. aquaticus B measured 1.93, while D. radiodurans measured 0.53. The immense difference in the OD measurements was taken into account, and it was linked back to the difference in growth conditions for the two species, namely the size of the tubes used for inoculation. This difference was visible in the turbidity of the cultures where both biological replicates of D. aquaticus appeared as opaque, reddish orange broth cultures while D. radiodurans appeared as a transparent, hazy yellow broth culture. 

Procedure and Results 

The procedure involves extracting protein from bacterial cultures of two species of Deinococcus, certain cultures are treated with hydrogen peroxide and others that serve as controls. To begin with, all of the three tubes containing D. aquaticus and D. radiodurans are homogenized by vortexing for 10 seconds. This step insures that the cells are uniform throughout the liquid broth. Next, the tubes containing D. aquaticus A and B are each divided into two subsequent 50 mL tubes containing 10 mL of broth culture to separate treatment and control cultures. This is also done with D. radiodurans, only the two new 10 mL tubes each have 5 mL of broth culture. The resulting six tubes are labeled D. aquaticus A1 (control), D. aquaticus A2 (treatment), D. aquaticus B1 (control), D. aquaticus B2 (treatment), D. radiodurans 1 (control), and D. radiodurans 2 (treatment). In the treatment tubes from D. aquaticus (tubes A2 and B2), 51 microliters of 9.8 M hydrogen peroxide is added to the 10 mL broth cultures while the treatment tube from D. radiodurans (tube 2) receives 26 microliters of the same concentration of hydrogen peroxide into the 5 mL broth culture. The rest of the control tubes for D. aquaticus (tubes A1 and B1) receive 51 microliters of TGY instead of the hydrogen peroxide. Similarly, the D. radiodurans control tube (tube 1) receives 26 microliters of TGY. After, all the tubes are placed in the incubator at 28 degrees Celsius for 30 minutes to allow for the hydrogen peroxide to stress the cells and activate cellular metabolic pathways which may in turn induce growth in the Deinococcus species. 

After the incubation period is over, the OD reading of each tube was measured. Below are the results:

D. aquaticus A1 = 2.77                      D. aquaticus B1 = 2.38                    D. radiodurans 1 = 0.50

D. aquaticus A2 = 3.02                      D. aquaticus B2 = 2.47                    D. radiodurans 2 = 0.63

Further, a 2 mL sample from each of the six tubes is transferred to a clean 2 mL Collection Tube. Following, all tubes are centrifuged for 5 minutes to separate the cells from the supernatant which contains the TGY medium along with hydrogen peroxide in treatment tubes. The supernatant is then discarded and the pellet is preserved. Then, 5 mL of Potassium Phosphate buffer is added to the pellet (mixed by pipetting up and down) to get rid of excess glucose attached to cells from the medium and to keep the pH balanced. Following is another step of 5 minute centrifugation. After disregarding the supernatant, the pellet in each tube is resuspended in 500 microliters of the same buffer (Potassium Phosphate) and the contents are transferred to PowerBead tubes. Next, the PowerBead tubes are taken to a Vortex Adapter and vortexed at maximum speed for 90 seconds. Then the tubes are placed on ice for 60 seconds to prevent protein denaturation as the temperature inside the tubes increases with vortexing. The vortexing for 90 seconds and cooling for 60 seconds is repeated four more times to insure the cell lyses is successfully carried out by the glass beads. Afterwards, the PowerBead tubes are centrifuged for 5 minutes to get cell debris to sink to the bottom and the isolated proteins to remain in the supernatant. Shortly after, the supernatant in each PowerBead tube is transferred to a clean 2 mL tube for a total of six tubes. Finally, the density of the proteins in each tube is measured using a NanoDrop at the wavelength 280 nm, and the tubes are then stored in the freezer at -20 degrees Celsius. The resulting density measurements are as follows:

 D. aquaticus A1 = 14.5                      D. aquaticus B1 = 8.2                    D. radiodurans 1 = 1.9

 D. aquaticus A2 = 14.4                      D. aquaticus B2 = 9.2                    D. radiodurans 2 = 2.8

The end of the procedure gives the basis for future research on the isolated bacterial proteins, especially that concerning the effect of oxidative stress on the Pentose Phosphate Pathway (PPP) in the two Deinococcus species.