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| | <p style="clear:both;">Our goal was to boost electron production by increasing the concentration of electron carriers (i.e. NADH). To achieve this goal we decided to engineer <span class="bacterium">E. coli</span> with an enzyme that would provide more intracellular NAD<sup>+</sup>, and thus NADH<sup><a class="sourced" onclick="javascript:scrollAndHighlight('refs_1')" href="#refs_1">[1]</a></sup>.</p> | | <p style="clear:both;">Our goal was to boost electron production by increasing the concentration of electron carriers (i.e. NADH). To achieve this goal we decided to engineer <span class="bacterium">E. coli</span> with an enzyme that would provide more intracellular NAD<sup>+</sup>, and thus NADH<sup><a class="sourced" onclick="javascript:scrollAndHighlight('refs_1')" href="#refs_1">[1]</a></sup>.</p> |
| − | <p>PncB catalyzes one of the rate-limiting step in the NAD synthesis pathway. This gene is naturally found in <span class="bacterium">E. coli</span> and encodes for the enzyme <span class="i_enph">NAPRTase</span> (nicotinic acid phosphorbosyl- transferase) that catalyzes the formation of nicotinate mono-nucleotide, a direct precursor of NAD, from NA (nicotinic acid) | + | <p>PncB catalyzes one of the rate-limiting step in the NAD<sup>+</sup> synthesis pathway. This gene is naturally found in <span class="bacterium">E. coli</span> and encodes for the enzyme <span class="i_enph">NAPRTase</span> (nicotinic acid phosphorbosyl- transferase) that catalyzes the formation of nicotinate mono-nucleotide, a direct precursor of NAD<sup>+</sup> , from NA (nicotinic acid) |
| | <sup><a class="sourced" onclick="javascript:scrollAndHighlight('refs_2')" href="#refs_2">[2]</a></sup> | | <sup><a class="sourced" onclick="javascript:scrollAndHighlight('refs_2')" href="#refs_2">[2]</a></sup> |
| | <sup><a class="sourced" onclick="javascript:scrollAndHighlight('refs_3')" href="#refs_3">[3]</a></sup> | | <sup><a class="sourced" onclick="javascript:scrollAndHighlight('refs_3')" href="#refs_3">[3]</a></sup> |
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| | <div class="captionbox" style="width:85%; max-width:950px;"> | | <div class="captionbox" style="width:85%; max-width:950px;"> |
| | <p style="text-align:center;"> | | <p style="text-align:center;"> |
| − | <a class="fancybox" title="Biochemical pathway of NAD synthesis" rel="group" href="https://static.igem.org/mediawiki/2015/0/00/Unitn_pics_pncb_path.jpg"><img src="https://static.igem.org/mediawiki/2015/2/2e/Unitn_pics_pncb_path_thumb.jpg" alt="" style="width:100%;"/></a> | + | <a class="fancybox" title="Biochemical pathway of NAD <sup>+</sup> synthesis" rel="group" href="https://static.igem.org/mediawiki/2015/0/00/Unitn_pics_pncb_path.jpg"><img src="https://static.igem.org/mediawiki/2015/2/2e/Unitn_pics_pncb_path_thumb.jpg" alt="" style="width:100%;"/></a> |
| | </p> | | </p> |
| − | <p class="image_caption"><span>Biochemical pathway of NAD synthesis</span> PncB gene encodes the transcription of the NARPTase which catalyzes the formation of nicotinate mono-nucleotide from nicotinic acid.</p> | + | <p class="image_caption"><span>Biochemical pathway of NAD<sup>+</sup> synthesis</span> PncB gene encodes the transcription of the NARPTase which catalyzes the formation of nicotinate mono-nucleotide from nicotinic acid.</p> |
| | </div> | | </div> |
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| − | <h4 class="header4 lateral-icon wow animated fadeInDown delay05" > <span>PncB enhances NAD production by 13 fold in anaerobic conditions</span><i class="faabig flaticon-bars-graphic"></i></h4> | + | <h4 class="header4 lateral-icon wow animated fadeInDown delay05" > <span>PncB enhances NAD<sup>+</sup> production by 13 fold in anaerobic conditions</span><i class="faabig flaticon-bars-graphic"></i></h4> |
| | </div> | | </div> |
| | </div> | | </div> |
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| − | <p>Our goal was to demonstrate that pncB <strong>increased intracellular levels of NAD+and thus NADH</strong>.We measured NAD+ levels under different growth conditions modifying the culture media conditions and the presence or lack of oxygen, which is the condition of the anodic chamber of a MFC.</p> | + | <p>Our goal was to demonstrate that pncB <strong>increased intracellular levels of NAD<sup>+</sup>and thus NADH</strong>. We measured NAD<sup>+</sup> levels under different growth conditions modifying the culture media conditions and the presence or lack of oxygen, which is the condition of the anodic chamber of a MFC.</p> |
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| − | <p>Colonies expressing pncB (<a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank">BBa_K1604031</a>) and the negative control (<a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker registry" target="_blank">BBa_K731201</a>) were grown in LB broth. When an Optical Density (OD) of 0.6 (600nm) was reached, the cultures were centrifuged and resuspended in fresh medium and induced with 5mM of arabinose. The cultures after 5 hours of induction were transferred in sealed glass bottles with a rubber septum under an anaerobic work station. After additional 20 hours of induction 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.</p> | + | <p>Colonies expressing pncB (<a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank">BBa_K1604031</a>) and the negative control (<a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker registry" target="_blank">BBa_K731201</a>) were grown in LB broth. When an Optical Density (OD) of 0.6 (600nm) was reached, the cultures were centrifuged and resuspended in fresh medium and induced with 5mM of arabinose. The cultures after 5 hours of induction were transferred in sealed glass bottles with a rubber septum under an anaerobic work station. After additional 20 hours of induction we quantified the levels of NAD<sup>+</sup> by a colorimetric test that measures the levels of NAD<sup>+</sup> indirectly by quantifying the concentration of NAD<sup>+</sup> total (NAD<sup>+</sup> + NADH) and NADH only.</p> |
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| − | <p class="image_caption" style="clear:both;"><span>NAD/NADH ratio: pncB and negative control in anaerobic condition</span> NAD/NADH ratio of <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker registry" target="_blank">BBa_K731201</a> (araC-pBAD, blu) and cell expressing <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank">BBa_K1604031</a> (pncB, green), in LB medium after 20 hours post-induction in anaerobic conditions. NAD+ and NADH levels were quantified with Sigma NAD /NADH quantification kit (MAK037). The concentration of NAD and NADH were calculated based on a standard curve.</p> | + | <p class="image_caption" style="clear:both;"><span>NAD/NADH ratio: pncB and negative control in anaerobic condition</span> NAD/NADH ratio of <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker registry" target="_blank">BBa_K731201</a> (araC-pBAD, blu) and cell expressing <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank">BBa_K1604031</a> (pncB, green), in LB medium after 20 hours post-induction in anaerobic conditions. NAD<sup>+</sup> and NADH levels were quantified with Sigma NAD /NADH quantification kit (MAK037). The concentration of NAD<sup>+</sup> and NADH were calculated based on a standard curve.</p> |
| | </div> | | </div> |
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| − | <p>In anaerobic conditions we obtained our best result, however we were able to repeat this measurement only one time due to technical difficulties with the anaerobic chamber. We also tested the performance of pncB in different conditions in LB, in a rich media (Terrific Broth), and with the addition of Nicotinic Acid, the starting molecule used by pncB, in the presence of oxygen. For all these samples we were able to use 3 biological replicates and with 3 technical replicates. Also under the conditions described above pncB enhanced NAD intracellular levels.</p> | + | <p>In anaerobic conditions we obtained our best result, however we were able to repeat this measurement only one time due to technical difficulties with the anaerobic chamber. We also tested the performance of pncB in different conditions in LB, in a rich media (Terrific Broth), with the addition of Nicotinic Acid, the starting molecule used by pncB, in the presence of oxygen. For all these samples we were able to use 3 biological replicates and with 3 technical replicates. Also under the conditions described above pncB enhanced NAD<sup>+</sup> intracellular levels.</p><br> |
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| | <div class="captionbox" style="max-width:860px; width:80%; margin:auto;"> | | <div class="captionbox" style="max-width:860px; width:80%; margin:auto;"> |
| | <a class="fancybox" title="NAD/NADH ratio between negative control (BBa_K731201) and cells expressing BBa_K1604031" rel="group" href="https://static.igem.org/mediawiki/2015/7/71/Unitn_pics_pncb_nadnadhratio.png"><img src="https://static.igem.org/mediawiki/2015/8/8b/Unitn_pics_pncb_nadnadhratio_t.png" alt="" style="width:100%;"/></a> | | <a class="fancybox" title="NAD/NADH ratio between negative control (BBa_K731201) and cells expressing BBa_K1604031" rel="group" href="https://static.igem.org/mediawiki/2015/7/71/Unitn_pics_pncb_nadnadhratio.png"><img src="https://static.igem.org/mediawiki/2015/8/8b/Unitn_pics_pncb_nadnadhratio_t.png" alt="" style="width:100%;"/></a> |
| | <p class="image_caption"> | | <p class="image_caption"> |
| − | <span>NAD/NADH ratio between negative control (BBa_K731201 ) and cells expressing BBa_K1604031.</span>The culture were grown in LB until an OD of 0.5 and induced with 5 mM arabinose in the presence of oxygen. Before inducing the cells, the media were changed with: fresh LB, LB supplemented with nicotinic acid or terrific broth. Panel A: <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker registry" target="_blank">BBa_K731201</a> in LB (blu), <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank">BBa_K1604031</a> in LB (green) and in LB with 10 uM nicotinic acid (yellow). Panel B: <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker registry" target="_blank">BBa_K731201</a> (orange) and BBa_K1604031 (red) both in terrific broth. | + | <span>NAD/NADH ratio between negative control BBa_K731201 and cells expressing BBa_K1604031.</span>The culture were grown in LB until an OD of 0.5 and induced with 5 mM arabinose in the presence of oxygen. Before inducing the cells, the media were changed with: fresh LB, LB supplemented with nicotinic acid or terrific broth. Panel A: <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker registry" target="_blank">BBa_K731201</a> in LB (blu), <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank">BBa_K1604031</a> in LB (green) and in LB with 10 uM nicotinic acid (yellow). Panel B: <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker registry" target="_blank">BBa_K731201</a> (orange) and BBa_K1604031 (red) both in terrific broth. |
| | </p> | | </p> |
| | </div> | | </div> |
| | </div> | | </div> |
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| − | <p> We did not observe significant difference in LB versus terrific broth (2.5 and 2.7 fold, respectively), while nicotinic acid boost the NAD+ levels by 3.7 fold respect to araC-pBAD and 1.5 compared to pncB in LB only.</p> | + | <p> We did not observe significant difference in LB versus terrific broth (2.5 and 2.7 fold, respectively), while nicotinic acid boost the NAD<sup>+</sup> levels by 3.7 fold respect to araC-pBAD and 1.5 compared to pncB in LB only.</p> |
| | </div> | | </div> |
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| − | <p style="clear:both" class="image_caption"><span>Colorimetric Assay for NAD/NADH ratio in aerobic conditions </span><strong>Left:</strong> <strong>Lane B</strong>: samples 2-7 calibration curve (0, 20, 40, 60, 80, 100 pmole/well of NADH). <strong>Lane C</strong>: samples 2-9 NAD total levels; <strong>Lane D</strong>: samples 2-9 NAD total repeated with a 2 fold concentrated sample; <strong>Lane E</strong>: 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. <strong>Right:</strong> Standard curve with 0, 20, 40, 60, 80, 100 pmole/well of NADH</p> | + | <p style="clear:both" class="image_caption"><span>Colorimetric Assay for NAD/NADH ratio in aerobic conditions </span><strong>Left:</strong> <strong>Lane B</strong>: samples 2-7 calibration curve (0, 20, 40, 60, 80, 100 pmole/well of NADH). <strong>Lane C</strong>: samples 2-9 NAD <sup>+</sup> total levels; <strong>Lane D</strong>: samples 2-9 NAD <sup>+</sup> total repeated with a 2 fold concentrated sample; <strong>Lane E</strong>: 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. <strong>Right:</strong> Standard curve with 0, 20, 40, 60, 80, 100 pmole/well of NADH</p> |
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| − | </div> | + | </div> |
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| − | <p style="margin-top:2em;"><a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank">BBa_K1604031</a> does increase NAD levels by <strong>2.5 fold</strong> and NADH levels by <strong>1.4 fold</strong> 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.</p>
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| − | <p>Overexpression of the gene pncB enhances significantly the intracellular level of NAD+. The best data were obtained in anaerobic conditions where the increasing of NAD+ was of 13 fold which are the conditions to be used in a Microbial Fuel Cell (Anode chamber is in anaerobic condition). When nicotinic acid was added in the medium there was a significant enhancement of NAD+ levels (1.5 fold versus pncB and 3.7 fold versus negative control).</p> | + | <p>Overexpression of the gene pncB enhances significantly the intracellular level of NAD+. The best data were obtained in anaerobic conditions where the increasing of NAD<sup>+</sup> was of 13 fold which are the conditions to be used in a Microbial Fuel Cell (Anode chamber is in anaerobic condition). When nicotinic acid was added in the medium there was a significant enhancement of NAD<sup>+</sup> levels (1.5 fold versus pncB and 3.7 fold versus negative control).</p> |
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| − | <p>Also in Terrific broth there was an increase in the NAD level (2.7 fold versus negative control in the same condition) In the future it will be interesting to measures NAD+ in anaerobic conditions with the presence of high levels of Nicotinic Acid. 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.</p> | + | <p>Also in Terrific broth there was an increase in the NAD<sup>+</sup> level (2.7 fold versus negative control in the same condition) In the future it will be interesting to measures NAD<sup>+</sup> in anaerobic conditions with the presence of high levels of Nicotinic Acid. We plan in the future to add a NAD<sup>+</sup> reducing enzyme and to give a medium able to enhance the cell metabolism to further increase NADH intracellular levels.</p> |
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| | <h4 class="header4 wow animated flipInX" style="text-align:center;"><div href="#" class="rotate-box square-icon" style="text-align:center;"> | | <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-speed4"></i></span> | | <span class="rotate-box-icon"><i class="faa flaticon-speed4"></i></span> |
| − | </div><br />More NAD produced</h4> | + | </div><br />More NAD<sup>+</sup> produced</h4> |
| − | <p>NAD production was enhanced in our cells by overexpressing the pncB gene, thanks to our novel part.</p> | + | <p>NAD<sup>+</sup> production was enhanced in our cells by overexpressing the pncB gene, thanks to our novel part.</p> |
| | </div> | | </div> |
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| | <a class="anchor-off" name="refs_2" id="refs_2"></a> | | <a class="anchor-off" name="refs_2" id="refs_2"></a> |
| | <li>Berríos-Rivera, S.<br/> | | <li>Berríos-Rivera, S.<br/> |
| − | <a href="http://www.ncbi.nlm.nih.gov/pubmed/12616693" target="_blank" class="sourcebox-link">"The Effect of NAPRTase Overexpression on the Total Levels of NAD, The NADH/NAD Ratio, and the Distribution of Metabolites in Escherichia Coli."</a><br/> | + | <a href="http://www.ncbi.nlm.nih.gov/pubmed/12616693" target="_blank" class="sourcebox-link">"The Effect of NAPRTase Overexpression on the Total Levels of NAD<sup>+</sup>, The NADH/NAD Ratio, and the Distribution of Metabolites in <i>Escherichia Coli.</i>"</a><br/> |
| | Metabolic Engineering 4.3 (2002): 238-47</li> | | Metabolic Engineering 4.3 (2002): 238-47</li> |
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