Proteorhodopsin (PR) is a light-powered proton pump that belongs to the rhodopsin family. It is a 7-transmembrane protein, which uses all-trans-retinal as the chromophore. It uses light energy to generate an outward proton flux. The increased proton motive force across the membrane can power cellular processes, such as ATP synthesis, chemiosmotic reactions and rotary flagellar motor [1]. 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[2].

Schematic representation of the PR gene cluster identified in clone HF10_19P19Predicted transcription terminators are indicated in red. (Four genes are for beta-carotene synthesis, blh for retinal production, and PR itself.

The sequence of our part belongs to the uncultured marine Gammaproteobacteria of the SAR86 group. The original cluster is composed of 6 genes: four are involved in beta- carotene production; one is implied in beta carotene cleavage into two molecules of retinal, the other encodes for proteorhodopsin. From the analysis of our part sequence we found out that our protein belongs to the blue absorbing group. [3]

Proposed mechanism of PR associated to the ATP-synthase complex Light-activated proteorhodopsin pumps protons outwardly, increasing the proton motive force. Protons can then reenter the cells through ATP-synthase complex, powering the ATP production.

Proteorhodopsin was taken from the Registry (BBa_K773002 ) part of Caltech 2012. From the experience of Caltech 2012 we saw that they were not able to express and functionally characterize the part. We took the challenge to improve this part!

We have built two different devices to produce Proteorhodopsin and added a RBS which was missing:

• BBa_K1604010: Proteorhodopsin producing device under the control of aracpBAD.
• BBa_K16040xx: Device for the production of Proteorhodopsin and biosynthesis of retinal

We have screened several parameters (media, temperature, time of induction) to discover that the optimal expression conditions were in LB at 37°C overnight in the presence of 10 μM of all-trans retinal. Attempts to express the protein in the absence of retinal failed. 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.

The expected molecular size is 28 KDa. The SDS gel shows a band corresponding to around 37 KDa, as it was seen in other studies [4]. This is probably due to post-translational modifications.

Although LB gives the maximum expression as shown in the SDS page, we were able to successfully express Proteorhodopsin also in M9. This result was not visible by SDS page, but it is demonstrated by the presence of a bright red colored pellet typical of retinal bound to Proteorhodopsin.

M9 is the perfect culture media for our MFC, to maintain the correct proton equilibration between the anodic and cathodic chambers, and maintains a more stable signal (see our MFC results). Therefore we decided to use these growth conditions for the functional characterization.

Expression of ProteorhodopsinNEB10β cells transformed with BBa_K1604010 and grown in LB and induced in LB or M9 with 5 mM arabinose and 10 μM of retinal at 30°C or 37°C. Negative control were cells transformed with BBa_K731201 (i.e. araC-pBAD).

Expression of ProteorhodopsinNEB10β cells transformed with BBa_K1604010 and grown in LB and induced in LB or M9 with 5 mM arabinose and 10 μM of retinal at 30°C or 37°C. Negative control were cells transformed with BBa_K731201 (i.e. araC-pBAD).

We attempted also to purify the protein from the bacterial culture by sonication followed by ultracentrifugation and we were happy to see that the purified protein was also RED, while the negative control was not.

Expression of ProteorhodopsinNEB10β cells transformed with BBa_K1604010 and grown in LB and induced in LB or M9 with 5 mM arabinose and 10 μM of retinal at 30°C or 37°C. Negative control were cells transformed with BBa_K731201 (i.e. araC-pBAD).

Anaerobioc growth of BBa_K1604010 E. coli transformed with BBa_K1604010 (blue line) and BBa_K731201 (green line) were grown in LB at 37°C untilan OD of 0.6 and induced in M9 minimal medium with 5 mM arabinose and 10 uM retinal in the dark. After 5 hours of induction the culture were transferred in sealed bottles in the anaerobic chamber and placed again in the thermoshaker. Sample in the dark were kept in aluminum foil. Light exposed samples were excited with a 160W halogen light bulb placed outside the incubator. The blue line (proteorhodopsin) is the result of the average of 6 different samples (3 in the dark and 3 in the light) while the green line (araC-pBAD) is the average of 1 sample in the dark and 1 in the light.