Proteorhodopsin
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
Characterization
Retinal is the key!
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.
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.
Purification of Proteorhodopsin. NEB10β cells transformed with BBa_K1604010 and BBa_K731201 were induced in LB at 37 °C in the presence of retinal. The cell pellets were resuspended in 50 mM Tris-Cl pH 8 with 5 mM MgCl2 and sonicated. The lysate was centrifuged at 10,000 rpm for 20 min at 4 °C.. The supernatant was ultracentrifuged for 100,000 x g for 3 hours at 4 °C. The three tubes in front contain proteorhodopsin purified fractions and the three tubes in the back are negative controls treated in the same conditions
More ATP in anaerobiosis!
Proteorhodpsin is a light activated proton pump that exploits the conformational change of all trans-retinal to 13-cis retinal. The activation of the pump causes an outward proton gradient that is the motive force for the ATP synthase.
Apparatus for anaerobiosis growthPanel A) sealed sterile bottles. Panel B) Anaerobic chamber.
We tested if light activation with a white light bulb (160 W) containing the blue wavelength, activates proteorhodopsin, thus making the bacteria survive better anaerobically and produce more ATP.
Anaerobiosis was achieved using sealed glass bottles with a rubber septum. We got from the local pharmacy 12 sterile bottles of physiological solution. After removing the liquid, washing them and autoclaving them, the bottles were ready to host our bacteria!
After five hours of induction in the dark (i.e. the samples were wrapped in aluminum foils) the cultures were split in the anaerobic chamber in light and dark conditions. The cultures were placed in the thermoshaker that was illuminated from the outside. Half of the cultures were kept in the dark and the other half were exposed to the light.
After 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. A higher ratio corresponds to higher ADP than ATP levels, meaning that the cells are dying. A smaller ADP/ATP ratio means higher ATP levels than ADP: the cells are growing .
Proteorhodopsin-engineered E. coli 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). 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. A basal functionality of the pump is observed also in the dark.
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.
More H+ pumping outside!
Since we observed that there was a possible activation of the proton pump without light, we decided that our next test would be a proton pumping experiment as described in the literature [2] [5].
To perform this test we built a solar mimicking apparatus, that would allow us to directly illuminate the samples, while growing multiple samples simultaneously and easily measure the pH.
Solar mimicking apparatus NEB10β cells transformed with BBa_K1604010 were grown exposed to light (left side) or in dark condition (right side). The cultures were maintained at ~37 °C with magnetic stirring using a laboratory plate. Light was provided by a 160 W halogen lamp placed 4 cm from each culture (left side). The dark condition was simulated by covering the cultures with aluminum foil (right side).
The ΔpH between the light exposed proteorhodopsin and the two negative controls (proteorhodopsin in the dark and araC-pBAD in the light is 0.22. This result evidenced that although there is a basal acidification of the medium due to the bacteria metabolism, our device acidifies more the medium thank to the activation of the proton pump when the bacteria are light exposed.
Acidification of culture medium by BBa_K1604010 E. coli NEB10β cells transformed with BBa_K1604010 were grown until an OD600 of 0.7 was reached and induced in M9 Minimal Media with 5 mM of arabinose and supplemented with 10 uM of all-trans retinal. The induction was done in the dark. The samples were then placed in the “Solar” apparatus with or without light. pH was measured every 6 h, in a 24 h range.
PR is not genotoxic to cells!
We wanted to test if the protein had a genotoxic effect on cells in order to confirm the enhanced viability of PR-expressing bacteria. We performed a Toxicity test by serial dilution as described in Protocols. E. coli NEB10β transformed with BBa_K1604010 were grown and induced with arabinose and retinal for 24 hours. The test showed no significant difference between proteorhodopsin expressing bacteria induced and not induced.
Toxicity test at 24 h of induction. E. coli NEB10β transformed with BBa_K1604010 were grown up to an OD600 of 0,6. The culture was split in induced and not induced samples. Cell pellet of the positive sample was resuspended in M9 Minimal Media and supplemented with 5 mM arabinose and 10 μM all-trans retinal. Both samples were exposed to light provided by a 160 W halogen lamp. Green: PR induced with 5 mM arabinose and supplemented with 10 μM of all-trans retinal exposed to light. Orange: PR not induced exposed to light. The average and the standard deviation were calculated between the CFU/ml counted for the four dilution factors. The test confirmed that following induction our proton pump does not have any genotoxic effects on cells!
To sum up...
We improved the proteorhodopsin part that we extracted from the registry, placed under an inducible promoter and we fully characterized it to demonstrate that the proton pump does work when the bacteria are light exposed. This membrane protein does require retinal to properly fold and increases the lifespan and the vitality of the engineered bacteria in anaerobic conditions. We did experience some difficulties in finding the right conditions of growth, light exposure and to reach anareobiosis. Also from our experience, this is a delicate system that showed sometime variability in the measurements between different biological samples. However we optimized the system and we now have a functioning device that can be used in our MFC.
Part Improvement
We successfully improved BBa_K773002 and now it works! Our PR was expressed in E. coli NEB10β cells and functionally characterized.
More ATP, better survival
E. coli equipped with proteorhodopsin survive better under anaerobic condition by producing higher levels of ATP
Towards the pMFC
Proteorhodopsin-engineered bacteria are happy to stay under the sun in our Microbial Fuel Cell.
Check out our Solar pMFC results
References
- Walter, Jessica M., Derek Greenfield, Carlos Bustamante, and Jan Liphardt.
"Light-powering Escherichia Coli with Proteorhodopsin."
Proceedings of the National Academy of Sciences 104 (2007): 2408-2412. - Martinez, A., A. S. Bradley, J. R. Waldbauer, R. E. Summons, and E. F. Delong.
"Proteorhodopsin Photosystem Gene Expression Enables Photophosphorylation in a Heterologous Host"
Proceedings of the National Academy of Sciences 104.13 (2007): 5590-595. - Kim, So Young, Stephen A. Waschuk, Leonid S. Brown, and Kwang-Hwan Jung.
"Screening and Characterization of Proteorhodopsin Color-tuning Mutations in Escherichia Coli with Endogenous Retinal Synthesis."
Biochimica Et Biophysica Acta (BBA) - Bioenergetics 1777.6 (2008): 504-13 - Richard A. Krebs, Ulrike Alexiev, Ranga Partha, Anne Marie DeVita, Mark S.Braiman.
Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin
BMC Physiology (2002), 1472-6793/2/5 - Ying Wang, Yan Li, Tuan Xu, Zhenyu Shi, Qiong Wu.
Experimental Evidence for Growth Advantage and Metabolic Shift Stimulated by Photophosphorylation of Proteorhodopsin Expressed in Escherichia Coli at Anaerobic Condition
Biotechnology and Bioengineering (2015), 112, 947-956