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

m
m
 
(80 intermediate revisions by 3 users not shown)
Line 2: Line 2:
 
</p></div>
 
</p></div>
 
<meta name="viewport" content="width=device-width, initial-scale=1" />
 
<meta name="viewport" content="width=device-width, initial-scale=1" />
−
+
 
 
</html>{{UNITN-Trento/globalIncluder}}<html>
 
</html>{{UNITN-Trento/globalIncluder}}<html>
−
+
<style>a.nostyl:hover{color:inherit;}</style>
 +
<script src="https://2015.igem.org/Template:UNITN-Trento/JSnotebookFunctions?action=raw&ctype=text/javascript"></script>
 
<div id="super_wrap">  
 
<div id="super_wrap">  
 
<div class="no-sidebar">
 
<div class="no-sidebar">
Line 12: Line 13:
 
<header id="header"></html>
 
<header id="header"></html>
 
{{UNITN-Trento/mainMenu}}
 
{{UNITN-Trento/mainMenu}}
−
<html><script>jQuery('#menulin_project, #menulin_project_description').addClass('current');</script></header>
+
<html><script>jQuery('#menulin_project, #menulin_project_results,  #menulin_project_results_proteorhodopsin').addClass('current');</script></header>
 
<!-- Main -->
 
<!-- Main -->
−
<style>.faabig:before,.faabig:after{font-size:1.8em; margina:0;} .faabig{position:absolute; right:0; top:0.9em;} .header4{position:relative; border:0 !important;} .header4 > span {  border-bottom: 1px dotted #AAA;  display: block;  padding-bottom: 5px;  margin-right: 1.7em;}</style>
+
<style>.faabig:before,.faabig:after{font-size:1.8em; margina:0;} .faabig{position:absolute; right:0; top:0.9em;}</style>
 
 
 
 
−
<section id="cta" style="background-image:url('https://static.igem.org/mediawiki/2015/c/ca/Unitn_pics_cta_projectresults.jpg');"  >
+
<section id="cta" style="background-image:url('https://static.igem.org/mediawiki/2015/1/10/Unitn_pics_cta_proteorhodopsin.jpg'); background-position:center bottom;"  >
 
<header>
 
<header>
−
<h2><strong>Results</strong></h2>  
+
<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>  
 
</header>
 
</header>
−
<footer>
 
−
<ul class="buttons">
 
−
<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> 
 
−
</ul>
 
−
</footer>
 
−
</section>
 
 
 
−
 
+
−
<article id="main">
+
<ul class="buttons">  
−
+
<a class="nostyl" href="https://2015.igem.org/Team:UNITN-Trento/Results/Proteorhodopsin"><li><p id="cd-timeline_PR_btn" class="button small bstyle1"><span class="primary">PR</span> <span class="secondary">Proteorhodopsin</span></p></li></a>
−
<a class="anchor-off" name=" " id=" "></a>
+
<a class="nostyl" href="https://2015.igem.org/Team:UNITN-Trento/Results/BLH"><li><p id="cd-timeline_BLH_btn" class="button small bstyle2"><span class="primary">BLH</span> <span class="secondary">Retinal Production</span></p></li>
−
<section class="wrapper style4 container" style="margin-top:1em;">
+
<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>  
−
<!-- Content -->
+
<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>
−
<div class="content">
+
</ul>  
−
<header>
+
−
<h3 class="wow fadeInDown">Introduction to the Results</h3>
+
</section>  
−
</header>
+
−
</div>
+
−
</section>
+
−
+
−
<a class="anchor-off" name="results_pncb" id="results_pncb"></a>
+
−
<section class="wrapper style4 container">
+
−
<!-- Content -->
+
−
<div class="content">
+
−
<header>
+
−
<h3 class="wow fadeInDown">Pnc<span class="bigletter">B</span>: nicotinic acid phosphorbosyl-transferase</h3>
+
−
</header> 
+
−
+
−
<div class="row" >
+
−
<div class="6u 12u(narrower)  wow bounceInLeft">
+
−
<h4 class="header4 wow flipInX delay05"><span>Increasing the levels of NADH</span> <i class="faabig flaticon-bacteria3"></i></h4>
+
−
+
−
<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>
+
−
<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>
+
−
<p style="text-align:center;">
+
−
<a class="fancybox" 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%; max-width:700px;"/></a>
+
−
</p>
+
−
<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>
+
−
</div>
+
−
+
−
<div class="6u 12u(narrower) wow bounceInRight">
+
−
<h4 class="header4 wow flipInX delay05"> <span>PncB is not toxic if overexpressed in <span style="font-style:italic">E.coli</span></span> <i class="faabig flaticon-atom27"></i></h4>
+
−
<p style="clear:both;">
+
−
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.
+
−
</p>
+
−
<p>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 style="text-align:center;">
+
−
<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). 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."><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>
+
−
+
−
<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>
+
−
</div>
+
−
</div>
+
−
+
−
<div class="row">
+
−
<div class="12u 12u(narrower)">
+
−
<h4 class="header4 wow flipInX delay05"> <span>PncB enhances NAD production by ~2.5 fold</span><i class="faabig flaticon-chemistry33"></i></h4>
+
−
</div>
+
−
</div>
+
−
<div class="row">
+
−
<div class="6u 12u(narrower) wow bounceInLeft">
+
−
<p style="clear:both;">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>
+
−
<p>NAD<sub>total</sub> = Amount of total NAD (NAD+NADH) in unknown sample (pmole) from standard curve.<br />
+
−
NADH = Amount of NADH in unknown sample (pmole) from standard curve.</p>
+
−
</div>
+
−
+
−
<div class="6u 12u(narrower) wow bounceInRight centered">
+
−
<p style="text-align:center;">
+
−
<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/4/45/Unitn_pics_pncb_StarndarRatioPncB.png" title="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."><img src="https://static.igem.org/mediawiki/2015/5/5e/Unitn_pics_pncb_StarndarRatioPncB_thumb.png" alt="" style="width:100%; max-width:700px;"/></a>
+
−
</p>
+
−
</div>
+
−
</div>
+
−
+
−
<div class="row">  
+
−
+
−
<div class="4u 12u(narrower) wow bounceInLeft  centered">
+
−
<p>
+
−
<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2015/2/26/Unitn_pics_pncb_wells.jpg" title="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."><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>
+
−
</div>
+
−
+
−
<div class="8u 12u(narrower) wow bounceInRight">
+
−
<p>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>
+
−
</div>
+
−
</div>  
+
−
</div>
+
−
</section>
+
−
</article>
+
−
 
+
−
 
+
 
<!-- Footer -->
 
<!-- Footer -->
 
</html>{{UNITN-Trento/mainFooter}}<html>  
 
</html>{{UNITN-Trento/mainFooter}}<html>  

Latest revision as of 00:55, 17 September 2015

Results:
check out what we have achieved!