![]() Proceedings of the National Academy of Sciences 104 (2007): 2408-2412. "Light-powering Escherichia Coli with Proteorhodopsin." Walter, Jessica M., Derek Greenfield, Carlos Bustamante, and Jan Liphardt.Proteorhodopsin-engineered bacteria are happy to stay under the sun in our Microbial Fuel Cell.Ĭheck out our Solar pMFC results References A basal functionality of the pump is observed also in the dark. In the light the ADP/ATP ratio of BBa_K1604010 is 3 fold lower than BBa_K731201 levels, indicating that proteorhodopsin does make more ATP in the lack of oxygen. coli engineered with Proteorhodopsin, exposed to light and under anaerobic conditions shows a much lower ADP/ATP ratio in comparison to control cells (araC-pBAD and PR in dark condition). A smaller ADP/ATP ratio means higher ATP levels than ADP: the cells are growing. A higher ratio corresponds to higher ADP than ATP levels, meaning that the cells are dying. Half of the cultures were kept in the dark and the other half were exposed to the light.Īfter an overnight exposure to the light, the ATP levels were measured with a luciferase test assay that gives you the ratio between ADP and ATP. The cultures were placed in the thermoshaker that was illuminated from the outside. the samples were wrapped in aluminum foils) the cultures were split in the anaerobic chamber in light and dark conditions. The functional characterization in vivo were done in LB which gives the maximum expression, except for a few tests done in M9.Īfter five hours of induction in the dark (i.e. M9 Minimal Media is the perfect culture media for our MFC to maintain the correct proton equilibration between the anodic and cathodic chambers, and keeps a more stable signal (see our MFC results). This result was not visible by SDS-page, but the expression is demonstrated by the presence of a bright red colored pellet typical of retinal bound to proteorhodopsin. This is probably due to post-translational modifications.Īlthough LB gives the maximum expression as shown in the SDS-page, we were able to successfully express proteorhodopsin also in M9 Minimal Media. The SDS gel shows a band corresponding to around 37 kDa, as it was seen in other studies. Proteorhodopsin is a membrane protein that needs the time to fold properly into the membrane and requires retinal to bind the pocket and help the formation of the proper folding. Attempts to express the protein in the absence of retinal failed. We have screened several parameters (media, temperature, time of induction) to discover that the optimal expression conditions were in LB at 37 ☌ overnight in the presence of 10 μM of all-trans retinal. From the analysis of our part sequence we found out that our protein belongs to the blue absorbing group. The original cluster is composed of 6 genes: in addition to the one encoding proteorhodopsin itself, four are involved in beta-carotene production and one is implied in beta-carotene cleavage into two molecules of retinal. ![]() The sequence of our part belongs to the uncultured marine Gammaproteobacteria of the SAR86 group. Four genes are for beta-carotene synthesis, blh for retinal production, and PR itself. Schematic representation of the PR gene cluster identified in clone HF10_19P19Predicted transcription terminators are indicated in red. ![]() Furthermore, it was demonstrated that light-activated proton pumping by proteorhodopsin can drive ATP synthesis as proton reenter the cell through the H +-ATP synthase complex. The increased proton motive force across the membrane can power cellular processes, such as ATP synthesis, chemiosmotic reactions and rotary flagellar motor. It uses light energy to generate an outward proton flux. It is a 7-transmembrane protein, which uses all-trans retinal as the chromophore. Proteorhodopsin (PR) is a light-powered proton pump that belongs to the rhodopsin family. ![]()
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