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| | + | <section id="cta" style="background-image:url('https://static.igem.org/mediawiki/2015/1/10/Unitn_pics_cta_proteorhodopsin.jpg'); background-position:center bottom;" > |
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| − | <h2><strong>MFC</strong></h2> | + | <h2><strong>Proteorhodopsin:</strong><br /><span style="font-size:0.8em; text-transform:lowercase; font-weight:300">a light-powered proton pump</span></h2> |
| − | <p>our Microbial Fuel Cell: House of Energy!</p>
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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 class="button small bstyle2" style="line-height:2em;" onclick="javascript:scrollToID('pr_char')"><span class="primary">Characterization</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> |
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| − | <h3 class="wow fadeInDown">The power of the sun</h3>
| + | <h3 class="wow fadeInDown">Proteorhodopsin</h3> |
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| − | <p>We observed that <i>E. coli</i> engineered with proteorhodopsin produced more ATP when exposed to light, due to the activation of the proton pump (see <a href="https://2015.igem.org/Team:UNITN-Trento/Results/Proteorhodopsin" class="i_linker" target="_blank">Proteorhdopsin</a>). We wanted to see if this makes bacteria more MFC friendly (<i>i.e.</i> live happily in the anode chamber) and if that would produce more electricity.</p>
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| − | <p>Cells transformed with <a href="http://parts.igem.org/Part:BBa_K1604010" target="_blank" class="i_linker registry">BBa_K1604010</a> (araC-pBAD + proteorhodopsin) and <a href="http://parts.igem.org/Part:BBa_K731201" target="_blank" class="i_linker registry">BBa_K731201</a> (negative control) were grown in M9 media and induced with arabinose (5 mM) and all-trans retinal (10 μM) for 4 hours in darkness. Preliminary tests showed that the optimal medium to be used was M9 medium supplemented with glucose, which gave a more stable signal (data not shown). </p>
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| − | <p>The bacterial cultures were split and then placed in the anodic chamber of a small Microbial Fuel Cell (borrowed from one of our instructor Martin Hanczyc) and exposed to the light of a blue LED. The experiment was repeated for 3 days, keeping the same experimental conditions.
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| − | In the presence of a blue-light LED, the proteorhodopsin expressing strain showed in all three cases a better electrochemical response than the negative control (<i>i.e.</i> PR-expressing strain shows higher polarization and power curves), with a higher voltage and maximum power (<i>P<sub>max</sub></i>). In a biological scale this means that there is an increased electricity due to the ability of the bacteria to maintain an active metabolism also in the absence of oxygen.<sup><a class="sourced" onclick="javascript:scrollAndHighlight('refs_1')" href="#refs_1">[1]</a></sup> </p>
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| − | <a class="fancybox" rel="group" title="Small Microbial Fuel Cell with bacteria expressing BBa_K1604010 and the negative control BBa_K731201 in the light" href="https://static.igem.org/mediawiki/2015/4/40/Unitn_pics_mfc_ima1.jpg"><img src="https://static.igem.org/mediawiki/2015/2/22/Unitn_pics_mfc_ima1_thumb.jpg" alt="" style="width:100%; "/></a>
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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 <sup><a class="sourced" onclick="javascript:scrollAndHighlight('refs_1')" href="#refs_1">[1]</a></sup>. Furthermore, it was demonstrated that light-activated proton pumping by proteorhodopsin can drive <strong>ATP synthesis</strong> as proton reenter the cell through the H<sup>+</sup>-ATP synthase complex<sup><a class="sourced" onclick="javascript:scrollAndHighlight('refs_2')" href="#refs_2">[2]</a></sup>.</p> |
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| − | <p class="image_caption"><span>Small Microbial Fuel Cell with bacteria expressing BBa_K1604010 and BBa_K731201 in the light.</span> Bacteria were placed in the anode covered with a layer of mineral oil to keep anaerobic conditions. The anode was exposed to blue light LED. Chemical mediators were added in the anode (Methylene blue, 100 μM) and in the cathode (Ferricyanide, 10 mM)</p> | + | <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> |
| − | </div>
| + | <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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| − | <div class="captionbox" style="max-width:965px; width:80%; margin-top:2em;">
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| | | | |
| − | <a class="fancybox" rel="group" title="More electricity with proteorhodopsin!" href="https://static.igem.org/mediawiki/2015/2/2f/Unitn_pics_mfc_graph1.png"><img src="https://static.igem.org/mediawiki/2015/d/d3/Unitn_pics_mfc_graph1_thumb.png" alt="" style="width:100%; "/></a> | + | <p>The sequence of our part belongs to the uncultured marine Gammaproteobacteria of the <strong>SAR86 group</strong>. 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.<sup><a class="sourced" onclick="javascript:scrollAndHighlight('refs_3')" href="#refs_3">[3]</a></sup></p> |
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| − | <p class="image_caption"><span>More electricity with proteorhodopsin!</span> <a href="http://parts.igem.org/Part:BBa_K1604010" target="_blank" class="i_linker registry">BBa_K1604010</a> and <a href="http://parts.igem.org/Part:BBa_K731201" target="_blank" class="i_linker registry">BBa_K731201</a> cells were grown and induced as described before. For each construct one MFC was placed in the light. The cells were connected to a data logging multimeter connected to an external variable resistor to register the voltage parameter of our system. Every hour the resistance was changed starting from 10MΩ to 1 KΩ Panel A: Polarization curve for <a href="http://parts.igem.org/Part:BBa_K1604010" target="_blank" class="i_linker registry">BBa_K1604010</a> and <a href="http://parts.igem.org/Part:BBa_K731201" target="_blank" class="i_linker registry">BBa_K731201</a>; for each data point the voltage was measured, while current and power were calculated with the Ohm law. Panel B: Power curve for <a href="http://parts.igem.org/Part:BBa_K1604010" target="_blank" class="i_linker registry">BBa_K1604010</a> and <a href="http://parts.igem.org/Part:BBa_K731201" target="_blank" class="i_linker registry">BBa_K731201</a>. The calculated power is plotted against the current to estimate the maximum power produced. </p>
| + | <div class="5u 12u(narrower)"> |
| − | </div> | + | <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> |
| − | | + | <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>We wanted to investigate the ability of proteorhodopsin to respond to light. The cells were grown and induced as before. The same sample of cells expressing proteorhodopsin was divided post-induction in two equal samples. One sample was placed in the dark and one in the light. When exposed to light <a href="http://parts.igem.org/Part:BBa_K1604010" target="_blank" class="i_linker registry">BBa_K1604010</a> showed a remarkable response to the external load applied, as shown by the higher values of voltage and current in the light. However, it has to be pointed out that this behavior was not always consistent. A few times we also observed the reverse effect (more electricity in the dark). This data are in agreement with the functional characterization, in which it was shown that a few times there was a basal activation of the proton pump also in the dark.</p>
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| − | <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/ b/ b3/ Unitn_pics_mfc_graph3. png" title=" BBa_K1604010 polarization curve: light versus dark."><img src="https://static.igem.org/mediawiki/2015/ 7/ 70/ Unitn_pics_mfc_graph3_thumb. png" alt="" style="width:100%; "/></a> <br> | + | |
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| − | <p class="image_caption"><span>BBa_K1604010 polarization curve: light versus dark.</span>The experiment was performed with the same experimental details described before. This time MFC with <a href="http://parts.igem.org/Part:BBa_K1604010" target="_blank" class="i_linker registry">BBa_K1604010</a> was placed in the dark and one was exposed to the light of a blue LED.</p> | + | |
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| | + | <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/f/f7/Unitn_pics_prschema.png" title="Parts BBa_K1604010 and BBa_K1604025"><img src="https://static.igem.org/mediawiki/2015/6/64/Unitn_pics_prschema_thumb.png" alt="" style="width:100%; max-width:800px;"/></a> |
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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 <strong>improve this part</strong>!</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"> |
| | + | <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> |
| | + | <li><a class="i_linker registry" href="http://parts.igem.org/Part:BBa_K1604025" target="_blank">BBa_K1604025</a> : Device for the production of Proteorhodopsin and biosynthesis of retinal</li> |
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| − | <h3 class="wow fadeInDown">Electrons thieves and Electrons producers</h3>
| + | <h3 class="wow fadeInDown">Characterization</h3> |
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| − | <p> All previous tests were operated by adding exogenous mediators to the anodic medium (<i>i.e.</i> Methylene blue, Neutral red). However this does not represent a valid method for future applications of the MFC. Related to our main project, we also characterized a < b> mediatorless MFC</ b> by expressing < i> Shewanella oneidensis</ i> electron export system in an engineered < i> E. coli</ i> strain from Ajo- Franklin Lab in Berkeley < sup><a class=" sourced" onclick=" javascript:scrollAndHighlight( 'refs_2')" href=" #refs_2"> [2]</a></ sup>). We characterized this strain in the MFC because we wanted to use it later in our Solar pMFC prototype. It should be noted that the parts used here were not BioBricks. </p> | + | |
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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 <strong>LB at 37 °C overnight</strong> 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 <sup><a class="sourced" onclick="javascript:scrollAndHighlight('refs_4')" href="#refs_4">[4]</a></sup>. 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 <strong>M9 Minimal Media</strong>. This result was not visible by SDS-page, but the expression is demonstrated by the presence of a bright <strong>red colored pellet</strong> 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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| | + | <div class="captionbox" style="max-width:1000px;"> |
| | + | <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-HCl 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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| | + | <h4 class="header4 lateral-icon wow animated fadeInDown delay05"> <span>More ATP in anaerobiosis!</span> <i class="faabig flaticon-lightbulb58"></i></h4> |
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| | + | <p>Proteorhodopsin 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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| | + | <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/3/3a/Unitn_pics_results_pr7.jpg" title="Apparatus for anaerobiosis growth"><img src="https://static.igem.org/mediawiki/2015/7/75/Unitn_pics_results_pr7_thumb.jpg" alt="" style="width:100%; max-width:700px;"/></a> |
| | + | <p class="image_caption"><span>Apparatus for anaerobiosis growth</span>Panel A) sealed sterile bottles. Panel B) |
| | + | Anaerobic chamber.</p> |
| | + | </div> |
| | + | <div class="6u 12u(narrower)"> |
| | + | <p>We tested if light activation with a <strong>white light bulb</strong> (160 W) containing the blue wavelength, activates proteorhodopsin, thus making the bacteria <strong>survive better anaerobically</strong> and produce <strong>more ATP</strong>.</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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| − | <div class=" captionbox" style="max-width:900px; width:80%; margin-top:2em;"> | + | </div> |
| − | <a class="fancybox" rel="group" title="E. coli Mtr electron transport system polarization and power curve." href="https://static.igem.org/mediawiki/2015/c/c9/Unitn_pics_mfc_graph2.png"><img src="https://static.igem.org/mediawiki/2015/7/7e/Unitn_pics_mfc_graph2_thumb.png" alt="" style="width:100%; "/></a>
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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 class="image_caption"> <span><i>E.coli</i> Mtr electron transport system polarization and power curve.</span>C43(DE3) cotransformed with a IPTG inducible plasmid carrying the <i>cymAmtrCAB</i> operon and a plasmid with <i>ccmA-H</i> under pTet constitutive promoter, were grown in LB and induced with IPTG (0.5 mM). The induced cells were placed in a MFC without mediators. The data were acquired as described earlier. </ p> | + | <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 < strong>smaller ADP/ ATP ratio</ strong> means higher ATP levels than ADP: < b>the cells are growing </ b>.</p> |
| − | </div>
| + | |
| − | | + | |
| − | <p>The increased electron flow we saw this time was mediated by the expression of <i>Shewanella</i> electron export complex. Such increase is not related to bacteria’s viability.</p>
| + | |
| − |
| + | |
| − | <p>Last, we wanted to test pncb (BBa_K1604031), our own new device for the production of NAD+, to evaluate any possible improvement in electrons flow. Our characterization data for this part showed an increase in NAD+ intracellular concentration of 13 fold in anaerobiosis. When placed in a MFC the bacteria expressing pncB showed higher values of voltage and current for each resistance applied (data not shown) respect to the negative control. However, this was a preliminary result that we had no time to repeat do to the lack of time.</p>
| + | |
| | | | |
| − | <div class="captionbox" style="max-width:900px; width:80%; margin-top:2em;"> | + | < p><span class=" bacterium"> E. coli</span> engineered with Proteorhodopsin, 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 lower 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 < strong> proteorhodopsin does make more ATP</ strong> in the lack of oxygen. A basal functionality of the pump is observed also in the dark. </p> |
| − | <a class=" fancybox" rel="group" title="" href=" https:// static.igem.org/ mediawiki/2015/e/ec/Unitn_pics_mfc_newgraph.png">< img src=" https:// static.igem.org/ mediawiki/2015/4/4a/Unitn_pics_mfc_newgraph_thumb.png" alt="" style=" width:100%;" /></a> | + | |
| − | | + | |
| − | <p class="image_caption"><span>Polarization curve for BBa_K1604031 and BBa_K731201</span> Bacteria were placed in the anode of a small MFC covered with a layer of mineral oil to keep anaerobic conditions. Chemical mediators were added in the anode (Neutral red, 100 μM) and in the cathode (Ferricyanide, 10 mM). Voltage was measured with an external multimeter changing the external resistance. Current and power were calculated with the Ohm law.</p>
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| | </div> | | </div> |
| − | </section>
| + | </div> |
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| − | <section class="wrapper style4 container" style="margin-top:1em;">
| + | <div class="6u 12u(narrower)"> |
| − | <!-- Content --> | + | <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/7/70/Unitn_pics_results_prATP.png" title="ATP levels of BBa_K1604010"><img src="https://static.igem.org/mediawiki/2015/1/12/Unitn_pics_results_prATP_thumb.jpg" alt="" style="width:100%; max-width:800px;"/></a> |
| − | <div class="content"> | + | </div> |
| − | <header> | + | <div class="6u 12u(narrower)"> |
| − | <h3 class="wow fadeInDown">Applications</h3>
| + | < 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> |
| − | </header>
| + | </div> |
| − | < p> Proteorhodopsin can power the blue-light LED used by UniTN iGEM Trento 2013 to produce ethylene! We used small MFCs filled with proteorhodopsin-expressing bacteria (<a href="http://parts.igem.org/Part:BBa_K1604010" target="_blank" class="i_linker registry"> BBa_K1604010</a> ), connected in series, to light up a few electronic apparatus, including a calculator, a blue-light LED and a lab timer.</p> | + | </div> |
| | + | |
| | + | <div class="row"> |
| | + | <div class="12u 12u(narrower)"> |
| | + | |
| | + | <h4 class="header4 lateral-icon wow animated fadeInDown delay05"> <span>More H<sup>+</sup> pumping outside!</span> <i class="faabig flaticon-science49"></i></h4> |
| | + | |
| | + | |
| | + | <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 <strong>proton pumping experiment</strong> as described in the literature <sup><a class="sourced" onclick="javascript:scrollAndHighlight('refs_2')" href="#refs_2">[2]</a> <a class="sourced" onclick="javascript:scrollAndHighlight('refs_5')" href="#refs_5">[5]</a></sup>.</p> |
| | | | |
| − | <div class="row"> | + | <p>To perform this test we built a solar mimicking apparatus, that would allow us to <strong>directly illuminate</strong> the samples, while growing multiple samples simultaneously and easily measure the pH.</p> |
| − | <div class="6u 12u(narrower) wow animated bounceInLeft">
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| − | <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/2/22/Unitn_pics_mfc_twoexamples1.jpg"><img src="https://static.igem.org/mediawiki/2015/8/81/Unitn_pics_mfc_twoexamples1_thumb.jpg" alt="" style="width:100%; max-width:600px; "/></a>
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| − | <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/c/c0/Unitn_pics_mfc_twoexamples2.jpg"><img src="https://static.igem.org/mediawiki/2015/4/4e/Unitn_pics_mfc_twoexamples2_thumb.jpg" alt="" style="width:100%; max-width:600px; "/></a>
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| − | <div class="row">
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| − | <div class="12u 12u(narrower) wow animated fadeIn" >
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| − | <p class="image_caption"><span><i>E.coli</i> engineered with Proteorhodopsin light activated can power up electrical devices with MFCs connected in series. </span> 3 MFCs can start a lab timer, while 12 MFCs can start a functioning calculator and a blue-light LED. </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| | | | |
| − | <p style="font-weight:500; font-size:1.3em; margin-bottom:2em; margin-top:2em; text-align:center; ">Watch this video to see our MFC in action:</p> | + | <div class="captionbox" style="max-width:1200px;"> |
| − | <div style="height:500px; 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%;"/></a> |
| − | <video controls >
| + | <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> |
| − | <source src="https://static.igem.org/mediawiki/2015/9/9d/Unitn_videomfc.mp4" type="video/mp4"></source>
| + | </div> |
| − | <source src="https://static.igem.org/mediawiki/2015/f/fb/Unitn_videomfc_2.ogg" type="video/ogg">
| + | |
| − | </source>
| + | |
| − | </video> | + | <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> |
| − | </div> | + | |
| | + | <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="Acidification of culture medium by BBa_K1604010"><img src="https://static.igem.org/mediawiki/2015/1/13/Unitn_pics_results_pr10_thumb.jpg" alt="" style="width:100%; max-width:1000px;"/></a> |
| | + | <p class="image_caption"><span>Acidification of culture medium by BBa_K1604010</span> <i> E. coli </i> 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 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> |
| | + | </div> |
| | </div> | | </div> |
| − | </section> | + | |
| | + | <div class="row"> |
| | + | <div class="12u 12u(narrower)"> |
| | + | |
| | + | <h4 class="header4 lateral-icon wow animated fadeInDown delay03 "> <span>Proteorhodopsin is not genotoxic to cells!</span> <i class="faabig flaticon-shield114"></i></h4> |
| | + | |
| | + | <p> We wanted to test if the protein had a <strong>genotoxic effect</strong> on cells in order to confirm the enhanced viability of Proteorhodopsin-expressing bacteria. We performed a Toxicity test by serial dilution as described in Protocols. <span class="bacterium">E. coli</span> NEB10β 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 and induced with arabinose and retinal for 24 hours. The test showed no significant difference between proteorhodopsin expressing bacteria induced and not induced.</p> |
| | + | |
| | + | <div class="captionbox" style="max-width:800px; width:80%; margin:auto;"> |
| | + | <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/6/6d/Unitn_pics_project_pr_toxicitytest.png" title="Proteorhodopsin Toxicity test at 24 h of induction"><img src="https://static.igem.org/mediawiki/2015/6/6d/Unitn_pics_project_pr_toxicitytest.png" alt="" style="width:100%;"/></a> |
| | + | <p class="image_caption"><span>Toxicity test at 24 h of induction.</span> <i>E. coli</i> NEB10β 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 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: proteorhodopsin (PR) induced with 5 mM arabinose and supplemented with 10 μM of all-trans retinal exposed to light. Orange: proteorhodopsin (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 <strong>our proton pump does not have any genotoxic effects</strong> on cells!</p> |
| | + | </div> |
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| | + | </div> |
| | + | </div> |
| | + | </div> |
| | + | </section> |
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| − | <a class="anchor-off" name="pr_conclusion" id="pr_conclusion"></a> | + | <a class="anchor-off" name="pr_conclusion" id="pr_conclusion"></a> |
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| − |
| + | |
| − | <p>We saw an increase of electricity production both with bacteria engineered with proteorhodopsin and bacteria expressing mtrCAB. Although the electrochemical effects are comparable, the biological causes are different. We saw an increase in the viability of the bacteria in the anode chamber, thank to the activity of proteorhodopsin and a more efficient electrons transport with mtrCAB part. Next, we should combine the two biological parts for a better MFC performance.</p> | + | <div class="row"> |
| − |
| + | <div class="12u 12u(narrower) wow animated fadeIn" > |
| | + | |
| | + | <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 <strong>increases the lifespan and the vitality</strong> 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 <strong>we optimized the system</strong> and we now have a <strong>functioning device</strong> that can be used in our MFC.</p> |
| | + | </div> |
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| | + | </div> |
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| | <div class="row"> | | <div class="row"> |
| | <div class="4u 12u(narrower) rotate-box-superhover wow animated fadeIn"> | | <div class="4u 12u(narrower) rotate-box-superhover wow animated fadeIn"> |
| | <h4 class="header4 wow animated flipInX" style="text-align:center;"><div href="#" class="rotate-box square-icon" style="text-align:center;"> | | <h4 class="header4 wow animated flipInX" style="text-align:center;"><div href="#" class="rotate-box square-icon" style="text-align:center;"> |
| − | <span class="rotate-box-icon"><i class="faa flaticon-ecological10"></i></span> | + | <span class="rotate-box-icon"><i class="faa flaticon-award9"></i></span> |
| − | </div><br />Live longer, Live better</h4> | + | </div><br />Part Improvement</h4> |
| | <p> | | <p> |
| − | Proteorhodopsin expressing bacteria are MFC friendly and produce more electricity | + | 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 Proteorhodopsin was expressed in <span class="bacterium">E. coli</span> NEB10β cells and functionally characterized. |
| | </p> | | </p> |
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| − | <span class="rotate-box-icon"><i class="faa flaticon-lightbulb8"></i></span> | + | <span class="rotate-box-icon"><i class="faa flaticon-covered16"></i></span> |
| − | </div><br />Power, Power!</h4> | + | </div><br />More ATP, better survival</h4> |
| | <p> | | <p> |
| − | We powered up different electrical devices: a lab timer, a calculator and a blue-light LED | + | <span class="bacterium">E. coli</span> equipped with proteorhodopsin survive better under anaerobic condition by producing higher levels of ATP |
| | </p> | | </p> |
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| − | <span class="rotate-box-icon"><i class="faa flaticon-sunbeam"></i></span> | + | <span class="rotate-box-icon"><i class="faa flaticon-sun90"></i></span> |
| − | </div><br />Ready for the solar pMFC</h4> | + | </div><br />Towards the pMFC</h4> |
| | <p> | | <p> |
| − | Check out our own designed <a href=" https://2015.igem.org/Team:UNITN-Trento/Design" class="i_linker"> Prototype</a> | + | 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> |
| | </p> | | </p> |
| | </div> | | </div> |
| | </div> | | </div> |
| − | </section> | + | </section> |
| | | | |
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| + | <section class="wrapper style4 container"> |
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| − | <a class="anchor-off" name="refs_1" id="refs_1"></a> | + | <ol type="1" class="sourcebox"> |
| − |
| + | <a class="anchor-off" name="refs_1" id="refs_1"></a> |
| − | <li>Johnson, E. T., D. B. Baron, B. Naranjo, D. R. Bond, C. Schmidt-Dannert, and J. A. Gralnick.<br/> | + | <li>Walter, Jessica M., Derek Greenfield, Carlos Bustamante, and Jan Liphardt.<br/> |
| − | <a href="http://aem.asm.org/content/76/13/4123.short" target="_blank" class="sourcebox-link">"Enhancement of Survival and Electricity Production in an Engineered Bacterium by Light-Driven Proton Pumping."</a><br/> | + | <a href="http://www.pnas.org/content/104/7/2408.abstract" target="_blank" class="sourcebox-link">"Light-powering Escherichia Coli with Proteorhodopsin."</a><br/> |
| − | </li> | + | Proceedings of the National Academy of Sciences 104 (2007): 2408-2412.</li> |
| | + | |
| | + | <a class="anchor-off" name="refs_2" id="refs_2"></a> |
| | + | <li>Martinez, A., A. S. Bradley, J. R. Waldbauer, R. E. Summons, and E. F. Delong.<br/> |
| | + | <a href="http://www.ncbi.nlm.nih.gov/pubmed/17372221" target="_blank" class="sourcebox-link">"Proteorhodopsin Photosystem Gene Expression Enables Photophosphorylation in a Heterologous Host"</a><br/> |
| | + | Proceedings of the National Academy of Sciences 104.13 (2007): 5590-595.</li> |
| | | | |
| − | <a class="anchor-off" name="refs_2" id="refs_2"></a>
| |
| − | <li>Teravest, Michaela A., Tom J. Zajdel, and Caroline M. Ajo-Franklin.<br/>
| |
| − | <a href="" target="_blank" class="sourcebox-link"> "The Mtr Pathway of Shewanella Oneidensis MR-1 Couples Substrate Utilization to Current Production in Escherichia Coli."</a><br/>
| |
| − | <i>ChemElectroChem</i> 1.11 (2014): 1874-879</li>
| |
| − |
| |
| | | | |
| | + | <a class="anchor-off" name="refs_3" id="refs_3"></a> |
| | + | <li>Kim, So Young, Stephen A. Waschuk, Leonid S. Brown, and Kwang-Hwan Jung. <br/> |
| | + | <a href="http://www.ncbi.nlm.nih.gov/pubmed/18433714" target="_blank" class="sourcebox-link">"Screening and Characterization of Proteorhodopsin Color-tuning Mutations in Escherichia Coli with Endogenous Retinal Synthesis."</a><br/> |
| | + | <i>Biochimica Et Biophysica Acta (BBA) - Bioenergetics</i> 1777.6 (2008): 504-13</li> |
| | + | |
| | + | <a class="anchor-off" name="refs_4" id="refs_4"></a> |
| | + | <li>Richard A. Krebs, Ulrike Alexiev, Ranga Partha, Anne Marie DeVita, Mark S.Braiman.<br/> |
| | + | <a href="http://www.biomedcentral.com/1472-6793/2/5" target="_blank" class="sourcebox-link">Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin</a><br/> |
| | + | BMC Physiology (2002), 1472-6793/2/5 |
| | + | </li> |
| | + | |
| | + | <a class="anchor-off" name="refs_5" id="refs_5"></a> |
| | + | <li>Ying Wang, Yan Li, Tuan Xu, Zhenyu Shi, Qiong Wu.<br/> |
| | + | <a href="http://www.ncbi.nlm.nih.gov/pubmed/25421845" target="_blank" class="sourcebox-link">Experimental Evidence for Growth Advantage and Metabolic Shift Stimulated by Photophosphorylation of Proteorhodopsin Expressed in Escherichia Coli at Anaerobic Condition</a><br/> |
| | + | Biotechnology and Bioengineering (2015), 112, 947-956 |
| | + | </li> |
| | </ol> | | </ol> |
| | </section> | | </section> |
