Difference between revisions of "Team:UNITN-Trento/Results"

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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>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>
 
<p>We have built two different devices to produce Proteorhodopsin and added a RBS which was missing:</p>
 
<p>We have built two different devices to produce Proteorhodopsin and added a RBS which was missing:</p>
 
<ul class="customlist arrowed">
 
<ul class="customlist arrowed">
<li><a href="" class="i_linker_registry">BBa_K1604010</a>: Proteorhodopsin producing device under the control of aracpBAD.</li>
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<li><a href="" class="i_linker registry">BBa_K1604010</a>: Proteorhodopsin producing device under the control of aracpBAD.</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>  
 
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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 NEB10-Beta 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>
 
<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/e/ee/Unitn_pics_results_prsds.jpg" title="Expression of Proteorhodopsin in NEB10-Beta 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>
 
 
<p class="image_caption"><span>Expression of Proteorhodopsin</span>NEB10&beta; 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 &mu;M of retinal at 30&deg;C or 37&deg;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 class="image_caption"><span>Expression of Proteorhodopsin</span>NEB10&beta; 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 &mu;M of retinal at 30&deg;C or 37&deg;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 NEB10-Beta 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>
 
<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 NEB10-Beta 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>
 
 
<p class="image_caption"><span>Expression of Proteorhodopsin</span>NEB10&beta; 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 &mu;M of retinal at 30&deg;C or 37&deg;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 class="image_caption"><span>Expression of Proteorhodopsin</span>NEB10&beta; 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 &mu;M of retinal at 30&deg;C or 37&deg;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/e/ee/Unitn_pics_results_prsds.jpg" title="Expression of Proteorhodopsin in NEB10-Beta cells"><img src="https://static.igem.org/mediawiki/2015/7/7c/Unitn_pics_results_prsds_thumb.jpg" alt="" style="width:100%; max-width:650px;"/></a>
 
<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/e/ee/Unitn_pics_results_prsds.jpg" title="Expression of Proteorhodopsin in NEB10-Beta cells"><img src="https://static.igem.org/mediawiki/2015/7/7c/Unitn_pics_results_prsds_thumb.jpg" alt="" style="width:100%; max-width:650px;"/></a>
 
 
<p class="image_caption"><span>Expression of Proteorhodopsin</span>NEB10&beta; 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 &mu;M of retinal at 30&deg;C or 37&deg;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 class="image_caption"><span>Expression of Proteorhodopsin</span>NEB10&beta; 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 &mu;M of retinal at 30&deg;C or 37&deg;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/2015/3/37/Unitn_pics_project_prpellets.png" title="Purification of Proteorhodopsin. "><img src="https://static.igem.org/mediawiki/2015/3/3f/Unitn_pics_project_prpellets_thumb.jpg" alt="" style="width:100%; max-width:1500px;"/></a>
 
<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/3/37/Unitn_pics_project_prpellets.png" title="Purification of Proteorhodopsin. "><img src="https://static.igem.org/mediawiki/2015/3/3f/Unitn_pics_project_prpellets_thumb.jpg" alt="" style="width:100%; max-width:1500px;"/></a>
<p class="image_caption"><span>Purification of Proteorhodopsin. </span>NEB10&beta; cells transformed with    <a href="http://parts.igem.org/Part:BBa_K1604010" class="i_linker_registry" 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 induced in LB at 37&deg;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 4C. The supernatant was    ultracentrifuged for 100,000 g for 3 hours at 4C. 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 class="image_caption"><span>Purification of Proteorhodopsin. </span>NEB10&beta; cells transformed with    <a href="http://parts.igem.org/Part:BBa_K1604010" class="i_linker registry" 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 induced in LB at 37&deg;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 4C. The supernatant was    ultracentrifuged for 100,000 g for 3 hours at 4C. 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>
 
 
<p class="image_caption" style="margin-top:1.5em !important;"><span>Proteorhodopsin expression in M9</span>Cells transformed with <a href="http://parts.igem.org/Part:BBa_K1604010" class="i_linker_registry" 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 and transferred in M9 at an OD of 0.6 and induced    with arabinose with the presence of 10 &micro;M 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.</p>
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<p class="image_caption" style="margin-top:1.5em !important;"><span>Proteorhodopsin expression in M9</span>Cells transformed with <a href="http://parts.igem.org/Part:BBa_K1604010" class="i_linker registry" 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 and transferred in M9 at an OD of 0.6 and induced    with arabinose with the presence of 10 &micro;M 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.</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>
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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>
 
 
 
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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&deg;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>
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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&deg;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>
 
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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 E. coli’s 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>
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     <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>
  
<div style="text-align:center;"><a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/2/2f/Unitn_pics_results_pr9_thumb.jpg" title=""><img src="https://static.igem.org/mediawiki/2015/2/2f/Unitn_pics_results_pr9_thumb.jpg" alt="" style="width:100%; max-width:900px;"/></a><p class="image_caption"><span>Solar mimicking apparatus</span> NEB10&beta; 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&deg;C with magnetic stirring using a laboratory plate. The light was provided by a 160 Watt halogen lamp placed 4 cm from each culture. The dark condition was simulated covering the cultures with aluminum foil (not shown).</p> </div>  
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<div style="text-align:center;"><a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/2/2f/Unitn_pics_results_pr9_thumb.jpg" title=""><img src="https://static.igem.org/mediawiki/2015/2/2f/Unitn_pics_results_pr9_thumb.jpg" alt="" style="width:100%; max-width:900px;"/></a><p class="image_caption"><span>Solar mimicking apparatus</span> NEB10&beta; 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&deg;C with magnetic stirring using a laboratory plate. The light was provided by a 160 Watt halogen lamp placed 4 cm from each culture. The dark condition was simulated covering the cultures with aluminum foil (not shown).</p> </div>  
 
 
 
 
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<p>Our device is controlled by an inducible arabinose promoter built by the <a class="i_linker_registry"  href="http://parts.igem.org/Part:BBa_K731201" target="_blank">Unitn iGEM team in 2012</a>. PncB was extracted by <span class="i_enph italic">E. coli</span> genome, the illegal site PstI was removed, and it was placed in pSB1C3 (<a href="http://parts.igem.org/Part:BBa_K1604030" class="i_linker_registry"  target="_blank">BBa_K1604030</a>). Subsequently it was placed under the araC-pBAD promoter (<a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker_registry"  target="_blank">BBa_K1604030</a>).</p>
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<p>Our device is controlled by an inducible arabinose promoter built by the <a class="i_linker registry"  href="http://parts.igem.org/Part:BBa_K731201" target="_blank">Unitn iGEM team in 2012</a>. PncB was extracted by <span class="i_enph italic">E. coli</span> genome, the illegal site PstI was removed, and it was placed in pSB1C3 (<a href="http://parts.igem.org/Part:BBa_K1604030" class="i_linker registry"  target="_blank">BBa_K1604030</a>). Subsequently it was placed under the araC-pBAD promoter (<a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry"  target="_blank">BBa_K1604030</a>).</p>
 
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<h4 class="header4 wow fadeInDown delay05"> <span>PncB is not toxic if overexpressed in <span style="font-style:italic">E.coli</span></span> <i class="faabig flaticon-shield114"></i></h4>
 
<h4 class="header4 wow fadeInDown delay05"> <span>PncB is not toxic if overexpressed in <span style="font-style:italic">E.coli</span></span> <i class="faabig flaticon-shield114"></i></h4>
 
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NEB10&beta; transformed with <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker_registry" target="_blank">BBa_K1604030</a> (araC-pBAD-pncB) or <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker_registry"  target="_blank">BBa_K731201</a>  (i.e. araC-pBAD) were grown up to an OD of 0.6, splitted in two tubes of 23 mL each and induced  with 5 mM of arabinose. Negative controls were not induced. <br />The OD (600 nm) was measured  every 45 minutes for 5 hours.  All measurements were done for 3 different biological samples and 3 technical measures.
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NEB10&beta; transformed with <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank">BBa_K1604030</a> (araC-pBAD-pncB) or <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker registry"  target="_blank">BBa_K731201</a>  (i.e. araC-pBAD) were grown up to an OD of 0.6, splitted in two tubes of 23 mL each and induced  with 5 mM of arabinose. Negative controls were not induced. <br />The OD (600 nm) was measured  every 45 minutes for 5 hours.  All measurements were done for 3 different biological samples and 3 technical measures.
 
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<p>Although the growth rate is slightly decreased, due to the cell stress when expressing pncB, the data indicate that this enzyme does not have toxicity effect on the cells.</p>  
 
<p>Although the growth rate is slightly decreased, due to the cell stress when expressing pncB, the data indicate that this enzyme does not have toxicity effect on the cells.</p>  
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<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/c/ce/Unitn_pics_pncb_ToxicityTestPncB.png" title="Growth rate of BBa_K1604031 (aracpbad-pncb) and BBa_K731201 (i.e. aracpBad)"><img src="https://static.igem.org/mediawiki/2015/2/21/Unitn_pics_pncb_ToxicityTestPncB_thumb.jpg" alt="" style="width:100%; max-width:700px;"/></a>
 
<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/c/ce/Unitn_pics_pncb_ToxicityTestPncB.png" title="Growth rate of BBa_K1604031 (aracpbad-pncb) and BBa_K731201 (i.e. aracpBad)"><img src="https://static.igem.org/mediawiki/2015/2/21/Unitn_pics_pncb_ToxicityTestPncB_thumb.jpg" alt="" style="width:100%; max-width:700px;"/></a>
<p class="image_caption"><span>Growth rate of <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker_registry" target="_blank"> BBa_K1604031</a> (aracpbad-pncb) and <a href="http://parts.igem.org/Part:" class="i_linker_registry" target="_blank">BBa_K731201</a> (i.e. aracpBad).</span> Cells were grown up to an OD of 0.6 and splitted before induction with arabinose. BBa_K1604031 (Orange line) and BBa_K731201 (green line) induced with 5 mM arabinose. BBa_K1604031 (yellow line) and BBa_K731201 (blue line) not induced.</p>
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<p class="image_caption"><span>Growth rate of <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank"> BBa_K1604031</a> (aracpbad-pncb) and <a href="http://parts.igem.org/Part:" class="i_linker registry" target="_blank">BBa_K731201</a> (i.e. aracpBad).</span> Cells were grown up to an OD of 0.6 and splitted before induction with arabinose. BBa_K1604031 (Orange line) and BBa_K731201 (green line) induced with 5 mM arabinose. BBa_K1604031 (yellow line) and BBa_K731201 (blue line) not induced.</p>
 
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<p class="image_caption" style="clear:both;"><span>__Title of the Figure__</span> NAD and NADH levels were quantified with Sigma NAD /NADH quantification kit (MAK037)  following the instructions described in the technical bulletin. Panel A: Standard curve (0, 20, 40, 60,  80, 100 pmole/well of NADH)). Panel B: NAD/NADH levels for three biological samples of  <a href="http://parts.igem.org/Part:BBa_K1604030" class="i_linker_registry" target="_blank">BBa_K1604030</a> (green) and one negative control <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker_registry" target="_blank">BBa_K731201</a> (blue). The cells were grown as described previously.</p>
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<p class="image_caption" style="clear:both;"><span>__Title of the Figure__</span> NAD and NADH levels were quantified with Sigma NAD /NADH quantification kit (MAK037)  following the instructions described in the technical bulletin. Panel A: Standard curve (0, 20, 40, 60,  80, 100 pmole/well of NADH)). Panel B: NAD/NADH levels for three biological samples of  <a href="http://parts.igem.org/Part:BBa_K1604030" class="i_linker registry" target="_blank">BBa_K1604030</a> (green) and one negative control <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker registry" target="_blank">BBa_K731201</a> (blue). The cells were grown as described previously.</p>
 
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<p style="clear:both" class="image_caption"><span>__Title of the Figure__</span> Lane B samples 2-7 calibration curve (0, 20, 40, 60, 80, 100 pmole/well of NADH). Lane C    samples 2-9 NAD total levels; Lane D samples 2-9 NAD total repeated with a 2 fold concentrated    sample; Lane E NADH only; Lane F NADH only, repeated with a 2 fold concentrated sample. In    lanes C-F the order of the samples is: 2 technical replicates of the negative control, and 2 technical    replicates of each of the 3 biological samples of  <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker_registry" target="_blank">BBa_K1604031</a>. The plate was read with a Tecan    Infinite M-200 pro instrument at 450 nm. The measurements were taken after 0.5, 1, 2, 3, 4 hours    to allow color development. The data shown are representative of the best measurement at 2 hours.</p>
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<p style="clear:both" class="image_caption"><span>__Title of the Figure__</span> Lane B samples 2-7 calibration curve (0, 20, 40, 60, 80, 100 pmole/well of NADH). Lane C    samples 2-9 NAD total levels; Lane D samples 2-9 NAD total repeated with a 2 fold concentrated    sample; Lane E NADH only; Lane F NADH only, repeated with a 2 fold concentrated sample. In    lanes C-F the order of the samples is: 2 technical replicates of the negative control, and 2 technical    replicates of each of the 3 biological samples of  <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank">BBa_K1604031</a>. The plate was read with a Tecan    Infinite M-200 pro instrument at 450 nm. The measurements were taken after 0.5, 1, 2, 3, 4 hours    to allow color development. The data shown are representative of the best measurement at 2 hours.</p>
 
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Revision as of 08:50, 9 September 2015

Results

  • Proteorhodopsin

  • PncB NAD Booster

Introduction to the Results

Proteorhodopsin

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 BBa_K1604010 indicates that it belongs to the blue absorbing family. [3]

Apparatus for anaerobiosis growthPanel A) sealed sterile bottles. Panel B) Anaerobic chamber.

We tested if light activation with a white light bulb (160W) containing the blue wavelength, activates proteorhodopsin, thus making the bacteria survive better anaerobically.

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!

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.
The OD600 was constantly monitored because E. coli’s growth is slowed down in stressful conditions such as the lack of oxygen.

The bacteria expressing proteorhodopsin have an increased lifetime when compared to a negative control with araC-pBAD (BBa_K731201). 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.

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.

Solar mimicking apparatus NEB10β cells transformed with BBa_K1604010 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. The light was provided by a 160 Watt halogen lamp placed 4 cm from each culture. The dark condition was simulated covering the cultures with aluminum foil (not shown).

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].

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.

cidification of culture medium by BBa_K1604010 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.

We improved the proteorhodopsin part that we extracted from the registry, placed under an inducible promoter and we fully characterized it to demonstrate that the proton pump doeswork 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.

Proteorhodopsin are happy to stay in our Microbial fuel Cell under the sun. Check out our results. [link alla pag della MFC]

PncB: nicotinic acid phosphorbosyl-transferase

Our device is controlled by an inducible arabinose promoter built by the Unitn iGEM team in 2012. PncB was extracted by E. coli genome, the illegal site PstI was removed, and it was placed in pSB1C3 (BBa_K1604030). Subsequently it was placed under the araC-pBAD promoter (BBa_K1604030).

Our goal was to demonstrate that pncB increased intracellular levels of NAD and thus NADH. We quantified the levels of NAD by a colorimetric test that measures the levels of NAD indirectly by quantifying the concentration of NAD total (NAD + NADH) and NADH only. To make precise quantitation a standard curve with NADH was built. The test provides the ratio of NAD/NADH

NADtotal = Amount of total NAD (NAD+NADH) in unknown sample (pmole) from standard curve.
NADH = Amount of NADH in unknown sample (pmole) from standard curve.

BBa_K1604031 does increase NAD levels by 126% (2.5 fold) and NADH levels by 44% (1.4 fold) when expressed in NEB10β. Although we did see an enhancement in NAD levels, this did not correlate to a proportional boost in NADH levels. We plan in the future to add a NAD reducing enzyme and to give a medium able to enhance the cell metabolism to further increase NADH intracellular levels.