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<li><p id="interlab_intro_btn" class="button small bstyle1" style="line-height:2em;" onclick="javascript:scrollToID('results_pncb')"><span class="primary">pncB</span> <span class="secondary">NAD Booster</span></p></li>   
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<li><p class="button small bstyle1" style="line-height:2em;" onclick="javascript:scrollToID('results_pr')"><span class="primary">Proteorhodopsin</span></p></li>
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<li><p class="button small bstyle3" style="line-height:2em;" onclick="javascript:scrollToID('results_pncb')"><span class="primary">PncB</span> <span class="secondary">NAD Booster</span></p></li>   
 
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<h3 class="wow fadeInDown">Proteorhodopsin</h3>
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<p>Proteorhodopsin (PR) is a light-powered proton pump that belongs to the rhodopsin family. It is a 7-transmembrane protein, which uses all-trans-retinal as the chromophore. It uses <span class="i_enph">light energy</span> to generate an <span class="i_enph">outward proton flux</span>. The increased proton motive force across the membrane can power cellular processes, such as ATP synthesis, chemiosmotic reactions and rotary flagellar motor [1]. Furthermore, it was demonstrated that light-activated proton pumping by proteorhodopsin can drive ATP synthesis as proton reenter the cell through the H+-ATP synthase complex[2].</p>
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<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://2015.igem.org/File:Unitn_pics_results_prscheme_thumb.jpg" alt="" style="width:100%; max-width:700px;"/></a>
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<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>The sequence of our part belongs to the uncultured marine Gammaproteobacteria of the  SAR86 group. The original cluster is composed of 6 genes: four are involved in beta-  carotene production; one is implied in beta carotene cleavage into two molecules of  retinal, the other encodes for proteorhodopsin. From the analysis of our part sequence we  found out that our protein belongs to the blue absorbing group. [3]</p>
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<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/1/1b/Unitn_pics_project_cluster_pr.png" title="Schematic representation of the PR gene cluster identified in clone HF10_19P19"><img src="https://static.igem.org/mediawiki/2015/d/db/Unitn_pics_project_cluster_pr_thumb.png" alt="" style="width:100%; max-width:700px;"/></a>
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<p class="image_caption"><span>Schematic representation of the PR gene cluster identified in clone HF10_19P19</span>Predicted transcription terminators are indicated in red. (Four genes are for beta-carotene synthesis, blh for retinal production, and PR itself.</p>
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<p 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 BBa_K1604031. 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 BBa_K1604031. 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 22:09, 5 September 2015

Results

  • Proteorhodopsin

  • PncB NAD Booster

Introduction to the Results

Proteorhodopsin

PncB: nicotinic acid phosphorbosyl-transferase

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.