Difference between revisions of "Part:BBa K1604010"

Revision as of 20:09, 19 September 2015

araC-pBAD + Proteorhodopsin

The part is an improvement of the proteorhodopsin part extracted from the registry (BBa_K773002), which was not working, we added a strong RBS, an inducible promoter and we demostrated that the proton pump does work.

Proteorhodopsin (PR) is a light-powered proton pump. It creates a proton motive force across the membrane that can subsequently power cellular processes, such as ATP synthesis.

Usage and Biology

PR is a light-powered proton pump that belongs to the rhodopsin family.

This protein has the property to use light energy to generate an outward proton flux that can subsequently power cellular processes, such as ATP synthesis, chemiosmotic reactions and rotary flagellar motor.[1] It was demonstrated that engineered E. coli with PR, whose cellular respiration is inhibited and undergoes anaerobic conditions, becomes light-powered in presence of light. [2]

FIGURE 1. Proteorhodopsin can drive ATP synthesis. Proposed mechanism of PR associated to ATP-synthase complex. Light-activated proteorhodopsin pumps protons outwards increasing the proton motive force, protons can reenter the cells through ATP-synthase complex powering ATP production.

Figure 2. Optimal conditions for a proper folding. E. coli NEB10β transformed with BBa_K1604010, grown in LB and induced in LB or M9 Minimal Media with 5 mM arabinose and 10 uM of retinal at 30 °C or 37 °C. Negative controls were cells transformed with BBa_K731201 (i.e. araC-pBAD). By the screening of several parameters (media, temperature, time of induction) we discovered that the optimal expression conditions were in LB, at 37 °C overnight in the presence of 10 μM of all-trans retinal. The estimated molecular weight according to the sequence is 27-28 kDa. However, the expected band is approximately 37 kDa due to post-translational modifications, as confirmed in literature. [3] It is a membrane protein that needs time to fold properly into the membrane and requires retinal for the correct folding.

Figure 3. Red pellets: proteorhodopsin is expressed! E. coli NEB10β 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 h at 4 °C. On the left: the three tubes in front contain proteorhodopsin purified fractions and the three tubes in the back are negative controls treated in the same conditions. On the right: crude pellet membrane after ultracentrifugation.

Figure 4. Proteorhodopsin is successfully expressed in M9. Cells transformed with BBa_K1604010 and BBa_K731201 were grown in LB and transferred in M9 Minimal Media at an OD of 0.6 and induced with arabinose with the presence of 10 uM of retinal. 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. 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.

Figure 5. More ATP under anaerobic condition! E. coli NEB10β transformed with BBa_K1604010 (Proteorhodopsin) and BBa_K731201 (araC-pBAD) 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 hours of induction at 37 °C the culture were transferred in sealed bottles in the anaerobic chamber. Sample in the dark (in purple) were kept in aluminum foil. Light-exposed samples (in yellow) were excited with a 160 W halogen light bulb placed outside the incubator. After an overnight incubation an ATP assay was performed. The commercial kit used measures ADP/ATP ratio. Proteorhodopsin-engineered E. coli exposed to light and under anaerobic condition show a much lower ADP/ATP ratio in comparison to control cells (araC-pBAD and dark condition), meaning that the cells are growing. The test confirms that proteorhodpsin supports E. coli viability under anaerobic condition when cells are light-exposed.

Figure 6. more H+ pumping outside! E. coli NEB10β transformed with BBa_K1604010 were grown until an OD600 of 0.7 was reached and induced in M9 Minimal Media with 5mM 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 mimicking apparatus with or without light. pH was measured every 6 h, in a 24 h range. 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 the medium thank to the activation of the proton pump when the bacteria were light exposed.

Figure 7. PR-engineered E. coli survives better anaerobically. E. coli transformed with BBa_K1604010 (blue line) and BBa_K731201 (green line) were grown in LB at 37 °C until an OD of 0.6 and induced in M9 Minimal Media 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 160 W 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. The bacteria expressing proteorhodopsin have an increased lifetime when compared to the negative control. We did not observe significant changes between light and dark with this test. However it seems that there is a basal functionality even in the absence of light, probably due to activation of the proton pump independently from light exposure.

Figure 8. Proteorhodopsin is not genotoxic to cells. Toxicity test by serial dilutions after 24 h of induction. 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.

BBa_K1604010 is an improvement of the proteorhodopsin part that we extracted from the registry (BBa_K773002). We added a strong RBS and placed the gene under an inducible promoter. Furthermore we fully characterized the part 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 under anaerobic conditions. We did experience some difficulties in finding the right conditions of growth, light exposure and to reach anaerobiosis. However we optimized the system and we now have a functioning device that can be used in our pMFC.

Check out our Wiki UNITN-Trento iGEM 2015!