Difference between revisions of "Part:BBa K1604010"

Revision as of 14:34, 6 September 2015

araC-pBAD + Proteorhodopsin

AAA

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, become 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. NEB10β cells transformed with BBa_K1604010 and grown in LB and induced in LB or M9 with 5 mM arabinose and 10 uM of retinal at 30C or 37C. 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. It is a membrane protein that needs time to fold properly into the membrane and requires retinal for the correct folding.

Figure 3. Figure 5. Red pellets: proteorhodopsin is expressed! NEB10β cells transformed with BBa_K1604010 and BBa_K731201 were induced in LB at 37C 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 4C. The supernatant was ultracentrifuged for 100,000 g for 3 hours at 4C. 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 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. 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.

Figura 5. 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 37C until an 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. 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. Sequence and Features