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: 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. 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 araC-pBAD.
• BBa_K1604025 : 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 Minimal Media. 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. 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). The functional characterization in vivo were done in LB which gives the maximum expression, except for a few tests done in M9.

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 proteorhodopsin in M9 Minimal Media Cells transformed with BBa_K1604010 and BBa_K731201 were grown in LB and transferred in M9 at an OD of 0.6 and induced with arabinose with the presence of 10 μM of retinal at 37 °C. After 6 hours of induction the cells were centrifuged and the supernatant was discarded. From left to right: araC-pBAD induced with retinal (A), proteorhodopsin induced with retinal (B), proteorhodopsin induced (C) and not induced (D) both without retinal.

ATP levels of BBa_K1604010 E. coli transformed with BBa_K1604010 and BBa_K731201 were grown in LB at 37 °C until an OD of 0.6 and induced in LB with 5 mM arabinose and 10 uM retinal in the dark. After 5 h of induction the cultures were transferred in sealed bottles in the anaerobic chamber and placed again in the thermoshaker. Sample in the dark were kept in aluminum foil (purple). Light exposed samples were excited with a 160 W halogen light bulb placed outside the incubator (yellow). After an overnight exposure 10⁵ cells were aliquoted and used to measure ADP/ATP ratio with a commercial kit (Sigma MAK135). For this test were used two biological and three technical replicates of each construct.