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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+-ATP synthase complex[2].</p> | + | <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> | | <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> |
| <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> | | <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: four are involved in beta- carotene production; one is implied in beta carotene cleavage into two molecules of retinal, the other encodes for proteorhodopsin. From the analysis of our part sequence we found out that our protein belongs to the blue absorbing group. [3]</p> | + | <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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| <p style="margin-bottom:0">We have built two different devices to produce Proteorhodopsin and added a RBS which was missing:</p> | | <p style="margin-bottom:0">We have built two different devices to produce Proteorhodopsin and added a RBS which was missing:</p> |
| <ul class="customlist arrowed" style="margin-top:0.5em"> | | <ul class="customlist arrowed" style="margin-top:0.5em"> |
− | <li><a href="" class="i_linker registry">BBa_K1604010</a>: Proteorhodopsin producing device under the control of aracpBAD.</li> | + | <li><a href="" class="i_linker registry">BBa_K1604010</a>: Proteorhodopsin producing device under the control of araC-pBAD.</li> |
| <li>BBa_K16040xx: Device for the production of Proteorhodopsin and biosynthesis of retinal</li> | | <li>BBa_K16040xx: Device for the production of Proteorhodopsin and biosynthesis of retinal</li> |
| </ul> | | </ul> |
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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> | + | <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> |
− | <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> | + | <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> |
− | <p> 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.<br /><br /> | + | <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.<br /><br /> |
− | M9 is the perfect culture media for our MFC, to maintain the correct proton equilibration between the anodic and cathodic chambers, and maintains a more stable signal (see our MFC results). Therefore we decided to use these growth conditions for the functional characterization.</p> | + | 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). Therefore we decided to use these growth conditions for the functional characterization.</p> |
| </div> | | </div> |
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| <div style="margin:0; text-align:center;"><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%; max-width:1000px;"/></a></div> | | <div style="margin:0; text-align:center;"><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%; max-width:1000px;"/></a></div> |
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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> | + | <p class="image_caption"><span>Expression of Proteorhodopsin</span><i> E. coli </i> NEB10β 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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− | <p>Proteorhodpsin is a light activated proton pump that exploits the conformational change of all trans-retinal to all cis-retinal. The different absorption properties are due to a single amino acid, at position 105 in the retinal binding pocket. The presence of a highly conserved Gln at position 105 in <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604010" target="_blank">BBa_K1604010</a> indicates that it belongs to the blue absorbing family. [3]</p> | + | <p>Proteorhodpsin is a light activated proton pump that exploits the conformational change of all trans-retinal to 13-cis retinal. The different absorption properties are due to a single amino acid, at position 105 in the retinal binding pocket. The presence of a highly conserved Gln at position 105 in <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604010" target="_blank">BBa_K1604010</a> indicates that it belongs to the blue absorbing family. [3]</p> |
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− | <p>We tested if light activation with a white light bulb (160W) containing the blue wavelength, activates proteorhodopsin, thus making the bacteria survive better anaerobically.</p> | + | <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.</p> |
| <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> | | <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 class="image_caption" style="margin-top:0;"><span>Anaerobioc growth of BBa_K1604010</span> E. coli transformed with <a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604010" class="i_linker" target="_blank">BBa_K1604010</a> (blue line) and <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker registry" target="_blank">BBa_K731201</a> (green line) were grown in LB at 37°C untilan 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.</p> | + | <p class="image_caption" style="margin-top:0;"><span>Anaerobioc growth 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> (blue line) and <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker registry" target="_blank">BBa_K731201</a> (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 h 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.</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. <br /> The OD<sub>600</sub> was constantly monitored because E. coli’s growth is slowed down in stressful conditions such as the lack of oxygen.</p> | + | <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. <br /> The OD<sub>600</sub> was constantly monitored because <i>E. coli</i> growth is slowed down in stressful conditions such as the lack of oxygen.</p> |
| <p>The bacteria expressing proteorhodopsin have an increased lifetime when compared to a negative control with araC-pBAD (<a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K731201" target="_blank">BBa_K731201</a>). However we did not observe significant changes between light and dark with this test. The explanations could be several. Most likely we were not exciting properly the system. 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.</p> | | <p>The bacteria expressing proteorhodopsin have an increased lifetime when compared to a negative control with araC-pBAD (<a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K731201" target="_blank">BBa_K731201</a>). However we did not observe significant changes between light and dark with this test. The explanations could be several. Most likely we were not exciting properly the system. 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.</p> |
| <p>While we decided to explore different light sources, we built a solar mimicking apparatus, that would allow us to directly illuminate the samples without the glass of the thermoshaker.</p> | | <p>While we decided to explore different light sources, we built a solar mimicking apparatus, that would allow us to directly illuminate the samples without the glass of the thermoshaker.</p> |
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− | <div style="text-align:center;"><a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/f/f8/Unitn_pics_results_pr9.jpg" title="Our solar mimicking apparatus"><img src="https://static.igem.org/mediawiki/2015/2/2f/Unitn_pics_results_pr9_thumb.jpg" alt="" style="width:100%; max-width:1200px;"/></a><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 Watt 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> </div> | + | <div style="text-align:center;"><a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/f/f8/Unitn_pics_results_pr9.jpg" title="Our solar mimicking apparatus"><img src="https://static.igem.org/mediawiki/2015/2/2f/Unitn_pics_results_pr9_thumb.jpg" alt="" style="width:100%; max-width:1200px;"/></a><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> </div> |
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| <div style="text-align:center; margin:0;"><a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/0/0b/Unitn_pics_results_pr10.png" title=""><img src="https://static.igem.org/mediawiki/2015/1/13/Unitn_pics_results_pr10_thumb.jpg" alt="" style="width:100%; max-width:900px;"/></a> | | <div style="text-align:center; margin:0;"><a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/0/0b/Unitn_pics_results_pr10.png" title=""><img src="https://static.igem.org/mediawiki/2015/1/13/Unitn_pics_results_pr10_thumb.jpg" alt="" style="width:100%; max-width:900px;"/></a> |
− | <p class="image_caption"><span>Acidification of culture medium by BBa_K1604010</span> A NEB10β cells transformed with BBa_K1604010 were grown until an OD600 of 0.7 was reached and induced in M9 Minimal Medium 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” apparatus with or without light. pH was measured every 6h, in a 24h range.</p> | + | <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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