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| − | <h2><strong>Proteorhodopsin:</strong><br /><span style="font-size:0.8em; text-transform:lowercase; font-weight:300">a light-powered proton pump</span></h2> | + | <h2><strong>Results:</strong><br /><span style="font-size:0.8em; text-transform:lowercase; font-weight:300">check out what we have achieved!</span></h2> |
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| − | <li><p class="button small bstyle1" style=" line-height: 2em;" onclick=" javascript:scrollToID('pr_intro')"><span class=" primary"> Introduction</span></p></li> | + | <li><p id="cd-timeline_PR_btn" class="button small bstyle1" href=" https: //2015.igem.org/Team:UNITN-Trento/Results/Proteorhodopsin" ><span class=" primary" >PR</span> <span class=" secondary"> Proteorhodopsin</span></p></li> |
| − | <li><p class="button small bstyle2" style=" line-height: 2em;" onclick=" javascript:scrollToID('pr_char')"><span class=" primary"> Characterization</span></p></li> | + | <li><p id="cd-timeline_BLH_btn" class="button small bstyle2" href=" https: //2015.igem.org/Team:UNITN-Trento/Results/pncB" ><span class=" primary" >BLH</span> <span class=" secondary"> Retinal Production</span></p></li> |
| − | <li><p class="button small bstyle3" style=" line-height: 2em;" onclick=" javascript: scrollToID('pr_conclusion')"><span class="primary"> Conclusions</span></ p></li> | + | <li><p id="cd-timeline_PNCB_btn" class="button small bstyle3" href=" https: //2015.igem.org/Team:UNITN-Trento/Results/BLH" ><span class=" primary">PNCB</span> <span class="secondary">NAD Boost</span></p></li> |
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| + | <li><p id="cd-timeline_MFC_btn"class="button small bstyle5" href="https://2015.igem.org/Team:UNITN-Trento/Results/BLH"><span class="primary">MFC</span> <span class="secondary">Microbial Fuel Cell</span></span></li> |
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| − | <p>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 <span class="i_enph">light energy</span> to generate an <span class="i_enph">outward proton flux</span>. 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<sup>+</sup>-ATP synthase complex[2].</p>
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| − | <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/1/1b/Unitn_pics_project_cluster_pr.png" title="Schematic representation of the PR gene cluster identified in clone HF10_19P19"><img src="https://static.igem.org/mediawiki/2015/d/db/Unitn_pics_project_cluster_pr_thumb.png" alt="" style="width:100%; max-width:700px;"/></a>
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| − | <p class="image_caption"><span>Schematic representation of the PR gene cluster identified in clone HF10_19P19</span>Predicted transcription terminators are indicated in red. Four genes are for beta-carotene synthesis, blh for retinal production, and PR itself.</p>
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| − | <p>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]</p>
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| − | <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/5/50/Unitn_pics_results_prscheme.jpg" title="Proposed mechanism of PR associated to the ATP-synthase complex"><img src="https://static.igem.org/mediawiki/2015/2/2b/Unitn_pics_results_prscheme_thumb.jpg" alt="" style="width:100%; max-width:700px;"/></a>
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| − | <p class="image_caption"><span>Proposed mechanism of PR associated to the ATP-synthase complex</span> 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.</p>
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| − | <p>Proteorhodopsin was taken from the Registry (<a href="http://parts.igem.org/Part:BBa_K773002" class="i_linker registry" target="_blank">BBa_K773002</a> ) part of <a href="https://2012.igem.org/Team:Caltech" target="_blank" class="authorCite">Caltech 2012</a>. 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!</p>
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| − | <p style="margin-bottom:0">We have built two different devices to produce Proteorhodopsin and added a RBS which was missing:</p>
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| − | <li><a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604010" target="_blank">BBa_K1604010</a>: Proteorhodopsin producing device under the control of araC-pBAD.</li>
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| − | <li>BBa_K1604025: Device for the production of Proteorhodopsin and biosynthesis of retinal</li>
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| − | <h4 class="header4 lateral-icon wow animated fadeInDown delay05"> <span>Retinal is the key!</span> <i class="faabig flaticon-ask3"></i></h4>
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| − | <p style="clear:both;">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.</p>
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| − | <p>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.</p>
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| − | <p> 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 <i>in vivo</i> were done in LB which gives the maximum expression, except for a few tests done in M9.</p>
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| − | <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/e/ee/Unitn_pics_results_prsds.jpg" title="Expression of Proteorhodopsin in NEB10β cells"><img src="https://static.igem.org/mediawiki/2015/7/7c/Unitn_pics_results_prsds_thumb.jpg" alt="" style="width:100%; max-width:700px;"/></a>
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| − | <p class="image_caption"><span>Expression of Proteorhodopsin</span>NEB10β cells transformed with <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604010" target="_blank">BBa_K1604010</a> 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 <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker registry" target="_blank">BBa_K731201</a> (i.e. araC-pBAD).</p>
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| − | <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/0/01/Unitn_pics_results_prfalcons2_thumb.jpg" title="Expression of Proteorhodopsin in NEB10β cells"><img src="https://static.igem.org/mediawiki/2015/0/01/Unitn_pics_results_prfalcons2_thumb.jpg" alt="" style="width:100%; max-width:700px;"/></a>
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| − | <p class="image_caption"><span>Expression of proteorhodopsin in M9 Minimal Media</span> Cells transformed with <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604010" target="_blank">BBa_K1604010</a> and <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K731201" target="_blank">BBa_K731201</a> 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.</p>
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| − | <p style="margin-bottom:1em;">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.</p>
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| − | <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/1/12/Unitn_pics_results_prfalcons.jpg" title="Expression of proteorhodopsin"><img src="https://static.igem.org/mediawiki/2015/8/84/Unitn_pics_results_prfalcons_thumbs.jpg" alt="" style="width:100%;"/></a>
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| − | <p class="image_caption"><span> Purification of Proteorhodopsin.</span> NEB10β cells transformed with <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604010" target="_blank">BBa_K1604010</a> and <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K731201" target="_blank">BBa_K731201</a> 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 </p>
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| − | <p>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.</p>
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| − | <p class="image_caption"><span>Apparatus for anaerobiosis growth</span>Panel A) sealed sterile bottles. Panel B)
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| − | <p>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.</p>
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| − | <p>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!</p>
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| − | <p>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. </p>
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| − | <p>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: <b>the cells are growing </b>.</p>
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| − | <p>Proteorhodopsin-engineered <span class="bacterium">E. coli</span> 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 <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604010" class="i_linker" target="_blank">BBa_K1604010</a> is 3 fold more than <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K731201" class="i_linker" target="_blank">BBa_K731201</a> 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. </p>
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| − | <p class="image_caption"><span>ATP levels of BBa_K1604010</span> <i>E. coli</i> transformed with <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604010" class="i_linker" target="_blank">BBa_K1604010</a> and <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker registry" target="_blank">BBa_K731201</a> 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 10<sup>5</sup> 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.</p>
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| − | <p>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].</p>
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| − | <p class="image_caption"><span>Solar mimicking apparatus</span> NEB10β cells transformed with <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604010" class="i_linker" target="_blank">BBa_K1604010</a> 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).</p>
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| − | <p>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.</p>
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| − | <p class="image_caption"><span>Acidification of culture medium by BBa_K1604010</span> <i> E. coli </i> 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.</p>
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| − | <p>We <strong>improved the proteorhodopsin</strong> 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.</p>
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| − | We successfully improved <a href="http://parts.igem.org/Part:BBa_K773002" target="_blank" class="i_linker registry">BBa_K773002</a> and now <strong>it works</strong>! Our PR was expressed in <span class="bacterium">E. coli</span> NEB10β cells and functionally characterized.
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| − | </p>
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| − | <span class="rotate-box-icon"><i class="faa flaticon-covered16"></i></span>
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| − | </div><br />More ATP, better survival</h4>
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| − | <p>
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| − | <span class="bacterium">E. coli</span> equipped with proteorhodopsin survive better under anaerobic condition by producing higher levels of ATP
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| − | <h4 class="header4 wow animated flipInX" style="text-align:center;"><div href="#" class="rotate-box square-icon" style="text-align:center;">
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| − | <span class="rotate-box-icon"><i class="faa flaticon-sun90"></i></span>
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| − | </div><br />Towards the pMFC</h4>
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| − | <p>
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| − | Proteorhodopsin-engineered bacteria are happy to stay under the sun in our Microbial Fuel Cell.<br />Check out our <a href="#" class="i_linker">Solar pMFC results</a>
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| − | </p>
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| − | </section>
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| − | </article>
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