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

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<h2><strong>Results</strong></h2>  
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<h2><strong>Results:</strong><br /><span style="font-size:0.8em; text-transform:lowercase; font-weight:300">check out what we have achieved!</span></h2>  
 
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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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<a  class="nostyl" href="https://2015.igem.org/Team:UNITN-Trento/Results/pncB"><li><p id="cd-timeline_PNCB_btn" class="button small bstyle3"><span class="primary">PNCB</span> <span class="secondary">NAD Boost</span></p></li>  
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<a class="nostyl" href="https://2015.igem.org/Team:UNITN-Trento/Results/MFC"><li><p id="cd-timeline_MFC_btn"class="button small bstyle5"><span class="primary">MFC</span> <span class="secondary">Microbial Fuel Cell</span></span></li>
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<h3 class="wow fadeInDown">Introduction to the Results</h3>
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<h3 class="wow fadeInDown">Pnc<span class="bigletter">B</span>: nicotinic acid phosphorbosyl-transferase</h3>
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<h4 class="header4 wow flipInDown      delay05"><span>Increasing the levels of NADH</span> <i class="faabig flaticon-speedometer26"></i></h4>
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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="i_enph italic">E. coli</span> with an enzyme that would provide more intracellular NAD, and thus NADH.</p>
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<p>PncB catalyzes one of the rate-limiting step in the NAD synthesis pathway. This gene is naturally found in <span class="i_enph italic">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>
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<p>Our device is controlled by an inducible arabinose promoter built by the <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker" 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" 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" target="_blank">BBa_K1604030</a>).</p>
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<h4 class="header4 wow fliInDown 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" target="_blank">BBa_K1604030</a> (araC-pBAD-pncB) or <a href="http://parts.igem.org/Part:BBa_K731201" class="i_linker" 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>
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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>
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<p class="image_caption"><span>Growth rate of BBa_K1604031 (aracpbad-pncb) and BBa_K731201 (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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<h4 class="header4 wow flipInDown      delay05"> <span>PncB enhances NAD production by ~2.5 fold</span><i class="faabig flaticon-bars-graphic"></i></h4>
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<p>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</p>
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<p>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 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  BBa_K1604030 (green) and one negative control BBa_K731201 (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 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="margin-top:2em;">BBa_K1604031 does increase NAD levels by <span class="i_enph">126%</span> (2.5 fold) and NADH levels by  <span class="i_enph">44%</span> (1.4 fold)    when expressed in NEB10&beta;. 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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Latest revision as of 00:55, 17 September 2015

Results:
check out what we have achieved!