Team:Northeastern/Experiments
Project Experiments
Proteorhodopsin was characterized using four assays: a growth assay, a cytotoxicity assay, an ATP/ADP ratio assay, and a 24 hour pH measurement assay . The protein requires retinal to fold correctly and imbed itself into the cellular membrane and also requires light to activate the proton pump. We decided to test 4 experimental groups: an active culture induced with beta-carotene and light, one induced with light without beta-carotene, one induced with beta-carotene without light, and the last exposed to neither light nor beta-carotene . The cultures with beta-carotene were given 20uM of beta-carotene. Groups induced with light were incubated exposed to a 200W equivalent LED bulb, which was measured to output .34mW at 520nm perfectly suitable to power proteorhodopsin. Groups not induced with light were wrapped in aluminum-foil to prevent exposure.
Our NOX expression system was characterized in a dissolved oxygen assay to measure the impact that the H2O forming NOX had on a sample of aerated PBS. Several attempts were also made to his-tag purify the protein and confirm its presence using SDS-PAGE.
General Growth Curve
A growth assay was conducted comparing control BL21 E. coli to E. coli transformed with NOX and E. coli transformed with Proteorhodopsin + Blh expression plasmids, BBa_K2182003 and BBa_K2182002 respectively. The main goal of this assay was to compare the metabolic stress of the plasmids and see whether or not the plasmids caused a significant decrease in the growth rate of the chassis bacteria. Cultures were grown with agitation in 2 mL of LB broth at 37 degrees celsius and diluted to an OD600 of 0.1 at the start of analysis. OD600 was taken at 20 minute intervals for the first 5.5 hours, with an additional time-point taken at 10 hours.
Proteorhodopsin Cytotoxicity Growth Assay
Cultures of BL21 E. coli were heat-shock transformed with the PR+Blh plasmid. 50 ml of LB + Chloramphenicol (25ug/ml) was added to two falcon tubes before inoculating with the transformed bacteria. Liquid cultures were grown overnight at 37C on a shaking plate before being measured for optical density, combined, and normalized to .28 OD 600. Cultures were then divided into 3 replicates containing 8ml aliquots. The cultures were incubated for twelve hours before measuring OD600 . They were then incubated for an additional twelve hours before measuring OD600 a final time. This experiment was performed to characterize what, if any, effect active Proteorhodopsin had on cellular growth.
Proteorhodopsin ATP/ADP Assay
To characterize Proteorhodopsin, we conducted an ATP/ADP ratio assay with the EnzyLight Kit comparing the ATP/ADP ratio of BBa_K2182003 transformed BL21 E. coli. Successful proton pumping would be seen as an increase of ATP, so the expected outcome was that the bacteria induced with beta-carotene and grown in the light would have the highest ratio. Transformed cells were grown overnight in 50ul of LB + Chloramphenicol (25ug/ml) and allowed to grow overnight at 37 degrees celsius. Induced groups were given 20uM beta-carotene and light groups were exposed to a 200W equivalent LED bulb. The liquid culture was then divided into 10ml each of the four experimental groups and allowed to grow at 37 degrees celsius 10 µL of cultured cells were transferred into a 96-well plate, with four replicates in each group. The cells were mixed with 90 µL of ATP reagent per well and the luminescence was read on a plate reader. This value is RLU A. After 10 minutes, the luminescence was read again, to get the RLU B value. 5 µL of ADP reagent were added to each well and mixed, and a final reading was done to obtain the RLU C value. The ATP/ADP ratio was calculated using the formula (RLU C - RLU B) / RLU A.
Proteorhodopsin Culture pH Characterization
BBa_K1282002 transformed BL21 E. coli were grown by shaking overnight at 37C in 30 ml of LB+chloramphenicol. Cultures were divided into 6ml experimental groups, induced cultures were given 20uM beta-carotene, and light cultures were exposed to a 200W equivalent LED bulb. Cultures were allowed to grow at 37 degrees celsius for 29 hours. During the 29 hour period pH measurements were taken every six hours and a final measurement at the 29th.
NOX Dissolved Oxygen Consumption
200ml of PBS solution was agitated on a magnetic stirring tray for 6 hours in order to aerate the solution. BBa_K1282003 transformed BL21 E. coli were inoculated into 30ml of LB +chloramphenicol at the same time that untransformed BL21 E. coli were inoculated into 30ml of LB. These cultures were grown at 37 degrees celsius for four hours. 200ml of PBS solution was agitated on a magnetic stirring tray for 6 hours in order to aerate the solution. Optical density of the cultures was measured and the cultures were normalized to an OD of .018. 20ml of each cell culture were centrifuged at 4000 rpm for 15 minutes to pellet the cells, the pellet was then resuspended in 5ml of airated PBS. The dissolved oxygen content of each solution was measured simultaneously by dissolved oxygen probes connected to an arduino for 29 minutes.
NOX His-Tag Isolation SDS-PAGE
An attempt was made to isolate the poly-histidine tagged NOX protein using His-Spin Trap columns and SDS-PAGE. This proved unsuccessful likely due to low concentration of the target protein as it was not under a high expression promoter.
