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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;" > | + | <section id="cta" class="cta-pncb"> |
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| | <h2><strong>PncB:</strong><br /><span style="font-size:0.8em; text-transform:lowercase; font-weight:300">a Nicotinic acid -> NAD producer</span></h2> | | <h2><strong>PncB:</strong><br /><span style="font-size:0.8em; text-transform:lowercase; font-weight:300">a Nicotinic acid -> NAD producer</span></h2> |
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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> | + | <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_K1604031</a>).</p> |
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| − | <h4 class="header4 lateral-icon wow animated 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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| − | NEB10β 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>
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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>
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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 (araC-pBAD - pncb) and BBa_K731201 (i.e. araC-pBAD)"><img src="https://static.igem.org/mediawiki/2015/2/21/Unitn_pics_pncb_ToxicityTestPncB_thumb.jpg" alt="" style="width:100%;"/></a>
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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> (araC-pBAD - pncb) and <a href="http://parts.igem.org/Part:" class="i_linker registry" target="_blank">BBa_K731201</a> (i.e. araC-pBAD).</span> Cells were grown up to an OD of 0.6 and splitted before induction with arabinose. <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank"> BBa_K1604031</a> (Orange line) and <a href="http://parts.igem.org/Part:" class="i_linker registry" target="_blank">BBa_K731201</a> (green line) induced with 5 mM arabinose. <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank"> BBa_K1604031</a> (yellow line) and <a href="http://parts.igem.org/Part:" class="i_linker registry" target="_blank">BBa_K731201</a> (blue line) not induced.</p>
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| − | <h4 class="header4 lateral-icon wow animated fadeInDown delay05" > <span>PncB enhances NAD production by ~2.5 fold</span><i class="faabig flaticon-bars-graphic"></i></h4> | + | <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> |
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| − | <p>Our goal was to demonstrate that pncB <strong>increased intracellular levels of NAD+and thus NADH</strong> . 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:</p> | + | <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> |
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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> |
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| − | <p style="text-align:center;">$\frac{NAD}{NADH} = \frac{NAD_{TOTAL} - NADH}{NADH}$<br />
| + | <a class="fancybox" title=" NAD/NADH ratio: pncB and negative control in anaerobic condition (LB-Broth)" rel="group" href="https://static.igem.org/mediawiki/2015/ d/ d6/ Unitn_pics_results_pncb_ratiograph_e.png"><img src="https://static.igem.org/mediawiki/2015/ d/ d6/ Unitn_pics_results_pncb_ratiograph_e.png" alt="" style="width:100%; max-width: 500px;"/></a> |
| − | NAD<sub>total</sub> = Amount of total NAD (NAD+NADH) in unknown sample (pmole) from standard curve.<br />
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| − | NADH = Amount of NADH in unknown sample (pmole) from standard curve.</p>
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| − | <p>We started by looking at NAD+ after 6 hours of induction in LB. Colonies expressing pncB (BBa_K1604031) and the negative control (<a href="http://parts.igem.org/Part:" class="i_linker registry" target="_blank">BBa_K731201</a>) were grown in LB broth with chloramphenicol (CF). When an Optical Density (OD) of 0.6 (600nm) was reached, the cultures were centrifuged and resuspended in fresh LB medium, CF and 5mM of arabinose. After 6 hours of induction the OD (600nM) was measured and an aliquot of 1 mL of culture corresponding to 10^8 cells was centrifuged at 2000 rpm for 5 minutes and washed with cold PBS. The NAD and NADH levels were calculated with the Sigma NAD and NADH Quantification Kit (MAK037) following the technical bulletin.</p>
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| − | <a class="fancybox" title=" Standard curve of NADH" rel="group" href="https://static.igem.org/mediawiki/2015/ 9/ 9b/ Unitn_pics_results_pncb_standardcurve.png"><img src="https://static.igem.org/mediawiki/2015/ 9/ 9b/ Unitn_pics_results_pncb_standardcurve.png" alt="" style="width:100%; max-width: 600px;"/></a > | + | |
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| − | <p style="text-align:center;"> | + | <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> |
| − | <a class="fancybox" title="NAD/NADH ratio in LB-Broth" rel="group" href="https://static.igem.org/mediawiki/2015/9/97/Unitn_pics_results_pncb_standardcurve_b.png"><img src="https://static.igem.org/mediawiki/2015/9/97/Unitn_pics_results_pncb_standardcurve_b.png" alt="" style="width:100%; max-width:600px;"/></a>
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| − | <p class="image_caption" style="clear:both;"> <span>Standard curve of NADH and NAD/NADH ratio in LB -Broth </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 BBa_K1604030 (green) and one negative control BBa_K731201 (blue).</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 ), 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> |
| | </div> | | </div> |
| | + | <div class="captionbox" style=" max-width:860px; width:80%;"> |
| | + | <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"> |
| | + | <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> |
| | + | </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> |
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| − | <p style="clear:both" class="image_caption"><span>Colorimetric Assay for NAD/NADH ratio</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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| − | <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/2/26/Unitn_pics_pncb_wells.jpg" title=""><img src="https://static.igem.org/mediawiki/2015/1/1b/Unitn_pics_pncb_wells_thumb.jpg" alt="" style="width:100%; max-width:500px;"/></a> | + | <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/2/26/Unitn_pics_pncb_wells.jpg" title=""><img src="https://static.igem.org/mediawiki/2015/1/1b/Unitn_pics_pncb_wells_thumb.jpg" alt="" style="width:100%; max-width:500px;"/></a> |
| | </p> | | </p> |
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| | + | <a class="fancybox" title="Standard curve of NADH" rel="group" href="https://static.igem.org/mediawiki/2015/9/9b/Unitn_pics_results_pncb_standardcurve.png"><img src="https://static.igem.org/mediawiki/2015/9/9b/Unitn_pics_results_pncb_standardcurve.png" alt="" style="width:100%; max-width:600px;"/></a> |
| | + | </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> 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. <strong>Right:</strong> Standard curve with 0, 20, 40, 60, 80, 100 pmole/well of NADH</p> |
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| − | <p class="image_caption" style="clear:both;"><span>NAD/NADH ratio: aerobic condition, pncB and negative control in LB and Terrific Broth</span> Panel A. NAD/NADH ratio between negative control and cells expressing BBa_K1604031, both samples were grown in LB medium. pncB does increase NAD+ levels <strong>2,5 fold</strong>. (p-value= 0,0078) Panel B. NAD/NADH ratio between negative control and cells expressing BBa_K1604031, both samples were grown in Terrific Broth medium. pncB does increase NAD+ levels by <strong>2,7 fold</strong>. (p-value=0,0024)</p> | + | <p class="image_caption" style="clear:both;"><span>NAD/NADH ratio: aerobic condition, pncB and negative control in LB and Terrific Broth</span> Panel A. NAD/NADH ratio between negative control and cells expressing <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank">BBa_K1604031</a> , both samples were grown in LB medium. pncB does increase NAD+ levels <strong>2,5 fold</strong>. (p-value= 0,0078) Panel B. NAD/NADH ratio between negative control and cells expressing <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank">BBa_K1604031</a>, both samples were grown in Terrific Broth medium. pncB does increase NAD+ levels by <strong>2,7 fold</strong>. (p-value=0,0024)</p> |
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| − | <p class="image_caption" style="clear:both;"><span>NAD/NADH ratio: aerobic condition, pncB + NA 10μM and negative control</span> Panel C. NAD/NADH ratio between negative control and cells expressing BBa_K1604031 10 μM of Nicotinic Acid, both samples were grown in LB medium. pncB+NA does increase NAD+ levels by <strong>~3,7 fold</strong>. (p-value=0,0001) Panel D. NAD/NADH ratio between pncB in LB + 10 μM NA and cells expressing BBa_K1604031 in LB broth. pncB+NA does increase NAD+ levels by <strong>~1,5 fold</strong>. (p-value=0,0231)</p> | + | <p class="image_caption" style="clear:both;"><span>NAD/NADH ratio: aerobic condition, pncB + NA 10μM and negative control</span> Panel C. NAD/NADH ratio between negative control and cells expressing <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank">BBa_K1604031</a> 10 μM of Nicotinic Acid, both samples were grown in LB medium. pncB+NA does increase NAD+ levels by <strong>~3,7 fold</strong>. (p-value=0,0001) Panel D. NAD/NADH ratio between pncB in LB + 10 μM NA and cells expressing <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank">BBa_K1604031</a> in LB broth. pncB+NA does increase NAD+ levels by <strong>~1,5 fold</strong>. (p-value=0,0231)</p> |
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| − | < a class=" fancybox" title=" NAD/ NADH ratio: pncB and negative control in anaerobic condition ( LB- Broth)" rel="group" href="https://static.igem.org/mediawiki/2015/ d/ d6/ Unitn_pics_results_pncb_ratiograph_e.png"><img src="https://static.igem.org/mediawiki/2015/ d/ d6/ Unitn_pics_results_pncb_ratiograph_e. png" alt="" style="width:100% ; max-width:500px;"/></a> | + | <div class="row" > |
| − | </div>
| + | <div class="12u 12u(narrower) wow animated bounceInRight" > |
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| + | <h4 class="header4 lateral-icon wow animated 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> |
| − | <div class="6u 12u(narrower)">
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| − | <p class="image_caption" style="clear:both;"><span>NAD/NADH ratio: pncB and negative control in anaerobic condition (LB-Broth) </span> NAD/NADH ratio between negative control and cell expressing BBa_K1604031, both samples were growth in LB medium. Both samples after 6 hours of induction in Thermoshaker (37° C, 190 rpm) were transferred in sealed glass bottles with a rubber septum under anaerobic work station to keep samples without oxygen. PncB does</p>
| + | NEB10β 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. |
| | + | </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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| | + | <div class="captionbox" style="width:80%; max-width:700px;"> |
| | + | <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 (araC-pBAD - pncb) and BBa_K731201 (i.e. araC-pBAD)"><img src="https://static.igem.org/mediawiki/2015/ 2/ 21/ Unitn_pics_pncb_ToxicityTestPncB_thumb. jpg" alt="" style="width:100%;"/></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> (araC-pBAD - pncb) and <a href="http://parts.igem.org/Part:" class="i_linker registry" target="_blank">BBa_K731201</a> (i.e. araC-pBAD).</span> Cells were grown up to an OD of 0.6 and splitted before induction with arabinose. <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank"> BBa_K1604031</a> (Orange line) and <a href="http://parts.igem.org/Part:" class="i_linker registry" target="_blank">BBa_K731201</a> (green line) induced with 5 mM arabinose. <a href="http://parts.igem.org/Part:BBa_K1604031" class="i_linker registry" target="_blank"> BBa_K1604031</a> (yellow line) and <a href="http://parts.igem.org/Part:" class="i_linker registry" target="_blank">BBa_K731201</a> (blue line) not induced.</p> |
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| | <div class="12u 12u(narrower)"> | | <div class="12u 12u(narrower)"> |
| − | <p>Overexpression of the gene pncB <span class="i_enph">enhances significantly</span> the intracellular level of NAD+. The best data were obtained in anaerobic conditions where the increasing of NAD+ was of <strong>13 fold</strong>. which are the conditions to be used in a Microbial Fuel Cell (Anode chamber is in anaerobic condition).</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+ 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> |
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| − | <p>When Nicotinic acid was added in the medium there was a significant enhancement of NAD+ levels (<strong>1,5 fold</strong> versus pncB and <strong>3,7 fold</strong> versus negative control). Also in Terrific broth condition there was an increasing in the NAD level (<strong>2,7 fold</strong> 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.</p> | + | <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> |
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| | </div><br />Towards the pMFC</h4> | | </div><br />Towards the pMFC</h4> |
| | <p> | | <p> |
| − | Bacteria equipped with pncB produce more electrons and are thus ideal for our Microbial Fuel Cell environment.<br />Check out our <a href="#" class="i_linker">Solar pMFC results to know more!</a> | + | Bacteria equipped with pncB produce more electrons and are thus ideal for our Microbial Fuel Cell environment.<br />Check out our <a href="https://2015.igem.org/Team:UNITN-Trento/Results/MFC" class="i_linker">Solar pMFC</a> results to know more! |
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