Difference between revisions of "Team:Manchester-Graz/Judging"

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<a class="navi" href="https://2015.igem.org/Team:Manchester-Graz/Description"><img src="https://static.igem.org/mediawiki/2015/9/9a/Manchester-Graz_Description.jpg"></a>
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<a class="navi" href="https://2015.igem.org/Team:Manchester-Graz/Project/Vectordesign"><img src="https://static.igem.org/mediawiki/2015/1/18/Manchester-Graz_Vectordesign.jpg"></a>
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<a class="navi" href="https://2015.igem.org/Team:Manchester-Graz/Project/Experiments"><img src="https://static.igem.org/mediawiki/2015/3/30/Manchester-Graz_Experiments.jpg"></a>
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<a class="navi" href="https://2015.igem.org/Team:Manchester-Graz/Project/Results"><img src="https://static.igem.org/mediawiki/2015/5/5e/Manchester-Graz_Results.jpg"></a>
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<a class="navi" style="margin-left: 20px;" href="https://2015.igem.org/Team:Manchester-Graz/Project/Results#SDS-Page"><img src="https://static.igem.org/mediawiki/2015/0/03/Manchester-Graz_SDS-Page.jpg"></a>
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<a class="navi" style="margin-left: 20px;" href="https://2015.igem.org/Team:Manchester-Graz/Project/Results#Assay"><img src="https://static.igem.org/mediawiki/2015/9/9b/Manchester-Graz_Fluorescence-Assay.jpg"></a>
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<a class="navi" style="margin-left: 20px;" href="https://2015.igem.org/Team:Manchester-Graz/Project/Results#BBa"><img src="https://static.igem.org/mediawiki/2015/8/88/Manchester-Graz_BBa.jpg"></a>
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<a class="navi" href="https://2015.igem.org/Team:Manchester-Graz/Attributions"><img src="https://static.igem.org/mediawiki/2015/d/dc/Manchester-Graz_Attributionssmall.jpg"></a>
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<a class="navi" href="https://2015.igem.org/Team:Manchester-Graz/Safety"><img src="https://static.igem.org/mediawiki/2015/d/df/Manchester-Graz_Safetysmall.jpg"></a>
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<a class="navi" href="https://2015.igem.org/Team:Manchester-Graz/Project/Protocols"><img src="https://static.igem.org/mediawiki/2015/8/80/Manchester-Graz_Protocols.jpg"></a>
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<div id="twitterboxsmall"><a class="twitter-timeline" href="https://twitter.com/iGEMMancGraz" data-widget-id="619465263411499008" height="900" width="180">Tweets by @iGEMMancGraz</a> <script>!function(d,s,id){var js,fjs=d.getElementsByTagName(s)[0],p=/^http:/.test(d.location)?'http':'https';if(!d.getElementById(id)){js=d.createElement(s);js.id=id;js.src=p+"://platform.twitter.com/widgets.js";fjs.parentNode.insertBefore(js,fjs);}}(document,"script","twitter-wjs");</script>
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<h1>Judging</h1>
  
  
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<div class="medal" style="height:600px;">
  
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<h2>Bronze</h2>
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<h1>Results</h1>
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<h2>Validation of pCERI</h2>
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<h3>via SDS-Page</h3><a name=SDS-Page>
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<p>To show how <i>E. coli</i> BL21 and <i>E. coli</i> Nissle 1917 deal with our synthetic vector pCERI we started a fermentation in 300 ml cultures. For each <i>E. coli</i> strain seven shaking flasks were inoculated with an ONC of <i>E. coli</i> BL21 and <i>E. coli</i> Nissle 1917, both transformed with <a href=" https://2015.igem.org/Team:Manchester-Graz/Project/Vectordesign">pCERI</a>, to start the fermentation with an OD600 of 0.05. In addition, negative (wild type <i>E. coli</i> BL21 and <i>E. coli</i> Nissle 1917) and positive controls were set up. The positive controls comprise recombinant <i>E. coli</i> BL 21 <a href="http://parts.igem.org/Part:BBa_J04421">pSB3C5_J04421</a> and <a href="http://parts.igem.org/Part:BBa_J04450">pSB3C5_J04450</a>. The transformed plasmids contain a lac promoter, leading to the expression of mRFP and CFP after the induction with IPTG. All samples were cultivated under the same conditions (37°C, 100 rpm). Every hour one fermentation of <i>E. coli</i> BL21_pCERI and <i>E. coli</i> Nissle 1917_pCERI was stopped on ice. One culture was fermented over night to reach 16 h. The positive, as well as the negative controls were stopped after 6h. After fermentation, all samples were centrifuged and prepared for  <a href="https://2015.igem.org/Team:Manchester-Graz/Project/Protocols">SDS analysis</a>. Regarding to protein concentrations measured by spectrophotometry (Nanodrop™) we loaded 10µg of soluble protein from the <i>E. coli</i> BL21_pCERI samples (1h-6h) as well as from the negative and positve controls on a SDS gel (Fig 1). For the <i>E. coli</i> Nissle 1917_pCERI and the <i>E. coli</i> Nissle 1917 negative control 20 µg of soluble protein were used for SDS-PAGE (Fig 2).</p>
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<p>The SDS-PAGE of the soluble protein fractions of <i>E. coli</i> BL21 samples shows well separated but quite weak bands. In comparison to the negative control, the <i>E. coli</i> BL21_pCERI samples only show one further band at about 22 kDa. None of the proteins of our regulatory system refer to a band of this size. However, one of our proteins could be expressed in a truncated form. The band at about 24 kDa that is visible in the samples from hour 1 to 6 on the gel picture is also slightly observable in the negative control on the gel itself.<br>
 
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The positive controls show, next to some <i>E. coli</i> BL21 specific bands, typical bands for CFP (26.89 kDa) and mRFP (25.42 kDa). For the mRFP positive control two further bands at 40 kDa and 20 kDa are conspicuous. Those again, are observable on the gel itself in all other <i>E. coli</i> BL21 samples as well. </p>
 
  
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<img src="https://static.igem.org/mediawiki/2015/f/f9/Manchester-Graz_Results_Fig1.jpg" alt="Manchester-Graz_Results_Fig1" width="700" height="522" /><br><b>Figure 1</b> SDS-PAGE of soluble protein fractions of recombinant <i>E. coli</i> BL21 fermentations. Std = Standard, 1h-6h = <i>E. coli</i> BL21_pCERI <br>fermentations that were stopped on ice after the different time points, - = negative control: wild type <i>E. coli</i> BL21, +CFP/+mRFP = <br>positive controls: <i>E. coli</i> BL21 cultures with IPTG inducible gen expression of CFP and mRFP.
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<img src="https://static.igem.org/mediawiki/2015/1/13/Manchester-Graz_Check-box.png" alt="check-box" width="30" height="30" />
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<div class="medaltext">
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1. Register for iGEM, have a great summer, and attend the Giant Jamboree.
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<div class="criterion">
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<div class="tickbox">
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<img src="https://static.igem.org/mediawiki/2015/1/13/Manchester-Graz_Check-box.png" alt="check-box" width="30" height="30" />
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</div>
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<div class="medaltext">
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2. Complete the Judging form.
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<p>The SDS-PAGE for the <i>E. coli</i> Nissle 1917 as well showed quite separated but even weaker bands as the <i>E. coli</i> BL21 samples. For this SDS gel we tried to load as much soluble protein as possible into 10µl of SDS-PAGE sample volume. Still, not more than 20 µg, regarding spectrophotometric measurements, were possible to be prepared for the SDS analysis gel. It has to be considered, that protein concentration measurements at 280 nm by Nanodrop™ can differ from the actual yields. As <i>E. coli</i> Nissle 1917 is not an engineered protein expression stain, like <i>E. coli</i> BL21 is, the protein concentration stays lower due to protease activity.</p>
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<img src="https://static.igem.org/mediawiki/2015/1/13/Manchester-Graz_Check-box.png" alt="check-box" width="30" height="30" />
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</div>
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<div class="medaltext">
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3. Create and share a Description of the team's project using the iGEM wiki, and document the team's parts using the Registry of Standard Biological Parts. <br>
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<p style="color:green;">A short description about our project can be found at our  <a href="https://2015.igem.org/Team:Manchester-Graz/Description" target="_blank">Project Description</a> page.
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</p>
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<img src="https://static.igem.org/mediawiki/2015/c/c3/Manchester-Graz_Results_Fig2.jpg" alt="Manchester-Graz_Results_Fig2" width="700" height="636" /><br><b>Figure 2</b> SDS-PAGE of soluble protein fractions of recombinant <i>E. coli</i> Nissle 1917 fermentations. Std = Standard, 1h-6h = <i>E. coli</i> Nissle 1917_pCERI fermentations that were stopped on ice after the different time points, - = negative control: wild type <i>E. coli</i> Nissle 1917.
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<div class="tickbox">
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<img src="https://static.igem.org/mediawiki/2015/1/13/Manchester-Graz_Check-box.png" alt="check-box" width="30" height="30" />
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<div class="medaltext">
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4. Present a poster and a talk at the iGEM Jamboree. See the 2015 poster guidelines for more information.
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<div class="criterion">
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<div class="tickbox">
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<img src="https://static.igem.org/mediawiki/2015/1/13/Manchester-Graz_Check-box.png" alt="check-box" width="30" height="30" />
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</div>
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<div class="medaltext">
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5. Create a page on your team wiki with clear attribution of each aspect of your project. This page must clearly attribute work done by the students and distinguish it from work done by others, including host labs, advisors, instructors, sponsors, professional website designers, artists, and commercial services.
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<p style="color:green;">Work done by students can be found at our <a href="https://2015.igem.org/Team:Manchester-Graz/Team" target="_blank">Team</a> page.<br>
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Under <a href="https://2015.igem.org/Team:Manchester-Graz/Attributions" target="_blank">Attributions</a> we listed people we worked with to cover different aspects of iGEM.</p>
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</div>
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</div>
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<div class="criterion" style="margin-bottom:70px;">
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<div class="tickbox">
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<img src="https://static.igem.org/mediawiki/2015/1/13/Manchester-Graz_Check-box.png" alt="check-box" width="30" height="30" />
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</div>
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<div class="medaltext">
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6. Document at least one new standard BioBrick Part or Device central to your project and submit this part to the iGEM Registry (submissions must adhere to the iGEM Registry guidelines). You may also document a new application of a BioBrick part from a previous iGEM year, adding that documentation to the part's main page.
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<p style="color:green;">Our registered BioBricks can be reviewed at our  <a href="https://2015.igem.org/Team:Manchester-Graz/Parts" target="_blank">Parts</a> page.</p>
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<p>This second try of SDS-PAGE of all <i>E. coli</i> BL21 samples showed much weaker bands as the <a href="https://2015.igem.org/Team:Manchester-Graz/Notebook#Week9">first try</a> even though much more protein was loaded. That might indicate that our soluble protein fractions are not stable in 20 mM sodium phosphate buffer. The samples from the <i>E. coli</i> Nissle 1917 cultures were prepared for SDS-PAGE straight after fermentation. Still the final gel only showed very weak bands. A possible reason for that is that the <i>E. coli</i> Nissle 1917 strain still expresses several proteases, which would degrade our proteins and hence lower the total protein yield.<br>
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Both SDS-PAGE results are thus inconclusive. Hence, for more accurate characterization further experiments are performed.</p>
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<h2>Silver</h2>
  
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<div class="criterion">
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<div class="tickbox">
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<img src="https://static.igem.org/mediawiki/2015/1/13/Manchester-Graz_Check-box.png" alt="check-box" width="30" height="30" />
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</div>
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<div class="medaltext">
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1. Experimentally validate that at least one new BioBrick Part or Device of your own design and construction works as expected. Document the characterization of this part in the Main Page section of the Registry entry  for that Part/Device. This working part must be different from the part you documented in Bronze medal criterion #6.
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<p style="color:green;">We further characterized all of our registered <a href="https://2015.igem.org/Team:Manchester-Graz/Parts" target="_blank">BioBricks</a></p>
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</div>
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<div class="criterion">
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<div class="tickbox">
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<img src="https://static.igem.org/mediawiki/2015/1/13/Manchester-Graz_Check-box.png" alt="check-box" width="30" height="30" />
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<div class="medaltext">
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2. Submit this new part to the iGEM Parts Registry. This part must be different from the part you documented in Bronze medal criterion #6. (Submissions must adhere to the iGEM Registry guidelines.)
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<p style="color:green;">From our registered and characterized BioBricks, we submitted 8 to the registry:<br>
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<a href="http://parts.igem.org/Part:BBa_K1670001" target="_blank"> BBa_K1670001</a><br>
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<a href="http://parts.igem.org/Part:BBa_K1670002" target="_blank"> BBa_K1670002</a><br>
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<a href="http://parts.igem.org/Part:BBa_K1670003" target="_blank"> BBa_K1670003</a><br>
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<a href="http://parts.igem.org/Part:BBa_K1670004" target="_blank"> BBa_K1670004</a><br>
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<a href="http://parts.igem.org/Part:BBa_K1670006" target="_blank"> BBa_K1670006</a><br>
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<a href="http://parts.igem.org/Part:BBa_K1670007" target="_blank"> BBa_K1670007</a><br>
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<a href="http://parts.igem.org/Part:BBa_K1670008" target="_blank"> BBa_K1670008</a><br>
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<a href="http://parts.igem.org/Part:BBa_K1670009" target="_blank"> BBa_K1670009</a>
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<div class="criterion" style="margin-bottom:70px;">
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<img src="https://static.igem.org/mediawiki/2015/1/13/Manchester-Graz_Check-box.png" alt="check-box" width="30" height="30" />
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<div class="medaltext">
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3. iGEM projects involve important questions beyond the bench, for example relating to (but not limited to) ethics, sustainability, social justice, safety, security, and intellectual property rights. We refer to these activities as Human Practices in iGEM. <b>Demonstrate how your team has identified, investigated and addressed one or more of these issues in the context of your project.</b> (See the Human PracticesHub for more information.)
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<p style="color:green;">Questions beyond the bench were asked and answered in our <a href="https://2015.igem.org/Team:Manchester-Graz/Practices" target="_blank">Human Practices</a> page.</p>
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<h3>via Fluorescence Assay</h3>
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<h2>Gold</h2>
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<p>For the characterization of the finished pCERI a <a href="https://2015.igem.org/Team:Manchester-Graz/Project/Experiments">fluorescence-based assay</a>  was conducted. From all measured emission values we subtracted the measured values of non transformed BL21 or Nissle 1917 samples at the respective time to account for possible autofluorescence of the cells.  As a blank for the OD<sub>600</sub>  measurements we used sterile controls. As the positive control for mRFP we used E.coli BL21 <a href="http://parts.igem.org/Part:BBa_J04450">pSB3C5_J04450</a> and for CFP E.coli BL21 <a href="http://parts.igem.org/Part:BBa_J04421">pSB3C5_J04421</a>. Both were induced with a final concentration of 0.1 mM IPTG.</p>
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<p>The measurement for mRFP whose expression should be induced by octanoyl-homoserine lactone (C8-HSL) yielded no substantial fluorescence emission at 607 nm, neither in <i>E. coli</i> BL21 (Fig. 3) nor in <i>E. coli</i> Nissle 1917 (Fig. 4). While the positive control emitted nearly 15000 RFU (relative fluorescence units) after 11 hours, none of the pCERI samples was measured with more than 100 RFU. A reason for these results could be that no or at least not enough CepR is produced. CepR works as an activator of P<sub>aidA</sub> and enables the recruitment of the RNA polymerase at the promoter and thus allows for the transcription of mRFP. This assumption is further supported by the fact, that <a href="http://parts.igem.org/Part:BBa_K1670003">BBa_K1670003</a> showed to be functional and highly active, when characterized individually.</p>
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<img src="https://static.igem.org/mediawiki/2015/f/f7/Manchester-Graz_Results_Fig3.jpg" alt="Manchester-Graz_Results_Fig3" width="757" height="456" /><br><b>Figure 3</b> Results of the emission measurement at 607 nm of <i>E. coli</i> BL21 pCERI samples treated with 0.1 nM and 100 nM of C8-HSL, C6-HSL, both combined, 0 nM HSL and the positive control for mRFP.
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<img src="https://static.igem.org/mediawiki/2015/4/4a/Manchester-Graz_Results_Fig4.jpg" alt="Manchester-Graz_Results_Fig4" width="757" height="456" /><br><b>Figure 4</b> Results of the emission measurement at 607 nm of <i>E. coli</i> Nissle 1917 pCERI samples treated with 0.1 nM and 100 nM of C8-HSL, C6-HSL, both combined, 0 nM HSL and the positive control for mRFP.
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<p> In <i>E. coli</i> BL21 the emission measurement at 476 nm (Fig. 5) showed the most CFP fluorescence in the sample induced with 0.1 nM C8-HSL at nearly 8000 RFU, followed by the CFP positive control and the 100 nM C8-HSL sample at 4500 RFU. The samples with 0.1 nM and 100 nM 3-oxo-hexanoyl-homoserine lactone (3OC6-HSL) reached 3300 RFU and 2500 RFU respectively. The samples containing 0.1 nM and 100 nM of both HSLs showed the same fluorescence at 2000 RFU. The lowest result came from the samples without any HSL, which showed no increase in fluorescence at all and 600 RFU after 11 hours. <br>
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These results indicate that apparently EsaR which controls CFP expression not only binds 3OC6-HSL but is also sensitive to C8-HSL. C8-HSL (0.1 nM with 8000 RFU) even appears to have a stronger effect than 3OC6-HSL (0.1 nM with 3300 RFU). For both it appears that they seem to work better at very low concentrations, but inhibit gene expression at higher concentrations. The reason for the behavior of the samples with the combination of C8- and 3OC6-HSL has yet to be found. The fact that the sample with no added HSL showed no increase in fluorescence leads us to believe that EsaI is not expressed strong enough due to  P<sub>esaS</sub> being quite a weak promoter. This would also be consistent with the thesis, that mRFP expression is weak due to a lack of CepR  which is also under the control of P<sub>esaS</sub>. </p>
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<img src="https://static.igem.org/mediawiki/2015/5/5e/Manchester-Graz_Results_Fig5.jpg" alt="Manchester-Graz_Results_Fig5" width="750" height="451" /><br><b>Figure 5</b> Results of the emission measurement at 476 nm of <i>E. coli</i> BL21 pCERI samples treated with 0.1 nM and 100 nM of C8-HSL, C6-HSL, both combined, 0 nM HSL and the positive control for CFP.
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<p>The OD<sub>600</sub> measurement (Fig. 6) shows that the 0.1 nM C8-HSL sample did not just simply outgrow the others. All of the samples grew in a similar manner. Only the positive control for CFP expression showed slightly weaker growth rates.</p>
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<img src="https://static.igem.org/mediawiki/2015/5/55/Manchester-Graz_Results_Fig6.jpg" alt="Manchester-Graz_Results_Fig6" width="748" height="448" /><br><b>Figure 6</b> Results of the OD<sub>600</sub> measurements of <i>E. coli</i> BL21 pCERI samples treated with 0.1 nM and 100 nM of C8-HSL, C6-HSL, both combined , 0 nM HSL and the positive controls for mRFP and CFP.
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<p>In <i>E. coli</i> Nissle 1917 the results (Fig. 7) were very different. At the start of the measurement the fluorescence was similar to BL21 around 1000 RFU and 2000 RFU. In the course of the measurement however the emission decreased substantially in all samples. The 0 nM sample had the most fluorescence left after 11 h. It showed 1500 RFU, which is still very low compared to the results of the BL21 samples.</p>
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<img src="https://static.igem.org/mediawiki/2015/d/d4/Manchester-Graz_Results_Fig7.jpg" alt="Manchester-Graz_Results_Fig7" width="749" height="449" /><br><b>Figure 7</b> Results of the emission measurement at 476 nm of <i>E. coli</i> Nissle 1917 pCERI samples treated with 0.1 nM and 100 nM of C8-HSL, C6-HSL, both combined, 0 nM HSL and the positive control for CFP in BL21.
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<p>These results for the Nissle 1917 pCERI transformants can be explained by the presence of proteases in <i>E. coli</i> Nissle 1917. It is much closer to a wild type <i>E. coli</i> than laboratory strains like BL21 which are optimized for labwork by the knockout of several proteases and nucleases. Apparently at first there was CFP produced but then the cells reacted to the presence of the foreign protein, putatively increased their protease expression, degraded the already formed CFP and kept CFP from reaching a measurable amount again. Comparing the different speed of CFP degradation in different samples while looking at the BL21 results, we see a possible correlation. The sample induced 0.1 nM C8-HSL in BL 21 rises fast and high to nearly 8000 RFU while in Nissle 1917 it starts at around 2000 RFU and is rapidly degraded until no emission is left after 6 h. The 100 nM 3OC6- and C8-HSL in BL21 rises slowly to about 2000 RFU while in Nissle 1917 the emission also slowly decreases over the course of time. So one could suspect that the stronger the expression of CFP occurs the stronger Nissle 1917’s protease response will be. </p>
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<p>While this data could result from insufficient growth of the cells, this thought can be dismissed when we look at the OD<sub>600</sub> curves (Fig. 8). They all grew perfectly well and also quite uniformly. </p>
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<img src="https://static.igem.org/mediawiki/2015/e/eb/Manchester-Graz_Results_Fig8.jpg" alt="Manchester-Graz_Results_Fig8" width="743" height="448" /><br><b>Figure 8</b> Results of the OD<sub>600</sub> measurements of <i>E. coli</i> Nissle 1917 pCERI samples treated with 0.1 nM and 100 nM of C8-HSL, C6-HSL, both combined, 0 nM HSL and the positive controls for mRFP and CFP in BL21.
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<h2>BioBrick Characterization</h2>
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<p>The characterization of <a href="http://parts.igem.org/Part:BBa_K1670002">BBa_K1670002</a> along with <a href="http://parts.igem.org/Part:BBa_K1670003">BBa_K1670003</a>  was conducted with a fluorescence-based assay as described in the <a href="https://2015.igem.org/Team:Manchester-Graz/Project/Experiments">experiments section</a>.  As a positive control for mRFP expression <i>E.coli</i> BL21 <a href="http://parts.igem.org/Part:BBa_J04450">pSB3C5_J04450</a> and as a positive control for CFP expression  <i>E.coli</i> BL21<a href="http://parts.igem.org/Part:BBa_J04421">pSB3C5_J04421</a> diluted to an OD<sub>600</sub> of 0.02 and induced with a final concentration of 0.1 mM IPTG are used.  From all measured emission values we subtracted the measured values of non transformed BL21 or Nissle 1917 samples at the respective time to account for possible autofluorescence of the cells.  As a blank for the OD<sub>600</sub>  measurements we used sterile controls.</a></p>
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<p> The constitutively expressed cepR is an activator that will bind to its corresponding promoter P<sub>aidA</sub> when a certain C8-HSL concentration is reached, starting the expression of mRFP. </p>
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<img src="../../iGEM%20Manchester-Graz/Wiki/Project/Experiments/Characterization_BBa/Results/Manchester-Graz_Results_Fig9.jpg" alt="Manchester-Graz_Results_Fig9" width="749" height="455" /><br<b>Figure 9</b> Results of the mRFP emission measurement (607 nm) of <i> E.coli </i> BL 21 J61002_BBa_J23100_BBa_ K1670002 pSB3C5_BBa_K1670003 induced with at C8-HSL concentrations from 0.01 nM to 100 nM, 0 nM and the positive control for mRFP.
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<p> As seen in figure  9 the positive control delivered the strongest signal with up to 14900 RFU, followed by the 0.01 nM C8-HSL just above 10000 RFU. The other samples showed a very similar behavior with their emission peaking at around 7000 RFU.<br>
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Closer comparing the samples, induced with 0.01 nM, 100 nM, 0 nM respectively and the positive control (Fig. 10) we see that the 0.01 nM sample shows a higher fluorescence emission than the sample without any C8-HSL. However the 100 nM sample had a notably lower emission than the one with 0.01 nM. When cepR binds C8-HSL it should work as an activator for P<sub>aidA</sub>. Apparently this is only the case at rather low levels of C8-HSL, like 0.01 nM. At higher concentrations like 100 nM the promoter activity seems to be the same as in the sample without any C8-HSL. It looks like cepR is inhibited in its role as an activator at those C8-HSL concentrations. Considering the fluorescence of the 0 nM sample we can say that P<sub>aidA</sub> appears to have a high basal expression without induction by cepR or that cepR is also working as an inducer even when no C8-HSL is present. </p>
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<img src="../../iGEM%20Manchester-Graz/Wiki/Project/Experiments/Characterization_BBa/Results/Manchester-Graz_Results_Fig10.jpg" alt="Manchester-Graz_Results_Fig10" width="749" height="455" /><br><b>Figure 10</b> Results of the mRFP emission measurement (607 nm) of <i> E.coli </i> BL 21 J61002_BBa_J23100_BBa_ K1670002 pSB3C5_ BBa_K1670003 induced with C8-HSL concentrations of 0.01 nM, 100 nM, 0 nM and the positive control for mRFP.  Error bars show the average deviation from the mean of the three measurements.
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<p>Another possible explanation for those results could be that at 100 nM C8-HSL is somehow toxic for the cells. Looking at the measured OD<sub>600</sub> values (Fig. 11) that assumption can be dismissed. All samples carrying the genes for CepR and mRFP grow in a similar manner. After about 10 hours the OD<sub>600</sub> of the 0.01 nM sample decreases, probably due to nutrient depletion.</p>
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<img src="../../iGEM%20Manchester-Graz/Wiki/Project/Experiments/Characterization_BBa/Results/Manchester-Graz_Results_Fig11.jpg" alt="Manchester-Graz_Results_Fig11" width="750" height="450" /><br><b>Figure 11</b> Results of the OD<sub>600</sub> measurement of <i>E.coli</i> BL 21 J61002_BBa_J23100_BBa_ K1670002 pSB3C5_ BBa_K1670003 induced with C8-HSL concentrations of 0.01 nM, 100 nM, 0 nM and the positive control for mRFP.  Error bars show the average deviation from the mean of the three measurements.
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<p> <a href="http://parts.igem.org/Part:BBa_K1670005">BBa_K1670005</a> is characterized along with <a href="http://parts.igem.org/Part:BBa_K1670001"> BBa_K1670001</a> with a <a href="https://2015.igem.org/Team:Manchester-Graz/Project/Experiments"> fluorescence-based assay</a>.</p>
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<p>The constitutively expressed EsaR is expected to act as a repressor on P<sub>esaRC</sub>. In the presence of 3OC6-HSL it should dissociate from the DNA and allow expression of the P<sub>esaRC</sub> controlled CFP.</p>
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<p>As seen in figure 12 the sample with 0.01 nM 3OC6-HSL had the highest emission at 476 nm of nearly 7000 RFU, followed by the 0 nM sample at 5000 RFU, the positive control at 4500 RFU sample, induced with 0.1 nM 3OC6-HSL, at around 3000 RFU. The other samples showed a very low emission at or under 1500 RFU.</p>
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<img src="../../iGEM%20Manchester-Graz/Wiki/Project/Experiments/Characterization_BBa/Results/Manchester-Graz_Results_Fig12.jpg" alt="Manchester-Graz_Results_Fig12" width="749" height="449" /><br><b>Figure 12</b> Results of the CFP emission measurement (476 nm) of <i> E.coli </i> BL 21 J61002_BBa_J23100_BBa_ K1670005 pSB3C5_BBa_K1670001 induced with 3OC6-HSL concentrations from 0.01 nM to 100 nM, 0 nM and the positive control for CFP.
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<p>For a better overview we compare 0.01 nM, 10 nM, 0 nM and the positive control more closely (Fig. 13). Here we can observe that the fluorescence of the 0.01 nM sample is the highest. The sample without any 3OC6-HSL and the positive control are at about the same level and the sample with 10 nM showed no increase in fluorescence at all. Those results suggest that EsaR ceases its repressor function at very low 3OC6-HSL concentrations of 0.01 nM but inhibit the expression at 3OC6-HSL concentrations higher than 10 nM. The relatively high level of basal CFP expression in the 0 nM sample suggests that P<sub>esaR</sub> is not a very tightly regulated promoter. </p>
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<img src="../../iGEM%20Manchester-Graz/Wiki/Project/Experiments/Characterization_BBa/Results/Manchester-Graz_Results_Fig13.jpg" alt="Manchester-Graz_Results_Fig13" width="747" height="449" /><br><b>Figure 13</b> Results of fluorescent emission measurement at 476 nm of <i> E.coli </i> BL 21 J61002_BBa_J23100_BBa_ K1670005 pSB3C5_BBa_K1670001 induced with 3OC6-HSL at concentrations of 0.01 nM, 10 nM, 0 nM and the positive control for CFP.  Error bars show the average deviation from the mean of the three measurements.
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<p>As a reason for the low fluorescence at high 3OC6-HSL concentrations you could also expect a possible toxicity for the cells, as low cell density would also mean low fluorescence. After looking at the OD<sub>600</sub> measurements (figure 14) this thought can be dismissed. The cells inoculated with 10 nM even grew a little better than the ones with 0.01 nM or 0 nM but still showed a less fluorescence.</p>
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<img src="../../iGEM%20Manchester-Graz/Wiki/Project/Experiments/Characterization_BBa/Results/Manchester-Graz_Results_Fig14.jpg" alt="Manchester-Graz_Results_Fig14" width="749" height="440" /><br><b>Figure 14</b> Results of the OD<sub>600</sub> measurement of <i> E.coli </i> BL 21 J61002_BBa_J23100_BBa_ K1670005 pSB3C5_BBa_K1670001 induced with 3OC6-HSL concentrations of 0.01 nM, 10 nM, 0 nM and the positive control for CFP.  Error bars show the average deviation from the mean of the three measurements.
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<p style="color:green;">We participated in multiple <a href="https://2015.igem.org/Team:Manchester-Graz/Collaborations" target="_blank">Collaborations</a> in key areas such as modelling and human practices.</p>
 
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Latest revision as of 03:59, 19 September 2015

iGEM Manchester Header

iGEM Manchester - Judging

Judging

Bronze

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1. Register for iGEM, have a great summer, and attend the Giant Jamboree.
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2. Complete the Judging form.
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3. Create and share a Description of the team's project using the iGEM wiki, and document the team's parts using the Registry of Standard Biological Parts.

A short description about our project can be found at our Project Description page.

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4. Present a poster and a talk at the iGEM Jamboree. See the 2015 poster guidelines for more information.
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5. Create a page on your team wiki with clear attribution of each aspect of your project. This page must clearly attribute work done by the students and distinguish it from work done by others, including host labs, advisors, instructors, sponsors, professional website designers, artists, and commercial services.

Work done by students can be found at our Team page.
Under Attributions we listed people we worked with to cover different aspects of iGEM.

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6. Document at least one new standard BioBrick Part or Device central to your project and submit this part to the iGEM Registry (submissions must adhere to the iGEM Registry guidelines). You may also document a new application of a BioBrick part from a previous iGEM year, adding that documentation to the part's main page.

Our registered BioBricks can be reviewed at our Parts page.

Silver

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1. Experimentally validate that at least one new BioBrick Part or Device of your own design and construction works as expected. Document the characterization of this part in the Main Page section of the Registry entry for that Part/Device. This working part must be different from the part you documented in Bronze medal criterion #6.

We further characterized all of our registered BioBricks

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2. Submit this new part to the iGEM Parts Registry. This part must be different from the part you documented in Bronze medal criterion #6. (Submissions must adhere to the iGEM Registry guidelines.)

From our registered and characterized BioBricks, we submitted 8 to the registry:
BBa_K1670001
BBa_K1670002
BBa_K1670003
BBa_K1670004
BBa_K1670006
BBa_K1670007
BBa_K1670008
BBa_K1670009

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3. iGEM projects involve important questions beyond the bench, for example relating to (but not limited to) ethics, sustainability, social justice, safety, security, and intellectual property rights. We refer to these activities as Human Practices in iGEM. Demonstrate how your team has identified, investigated and addressed one or more of these issues in the context of your project. (See the Human PracticesHub for more information.)

Questions beyond the bench were asked and answered in our Human Practices page.

Gold

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1. Choose one of these two options: (1) Expand on your silver medal Human Practices activity by demonstrating how you have integrated the investigated issues into the design and/or execution of your project. OR (2) Demonstrate an innovative Human Practices activity that relates to your project (this typically involves educational, public engagement, and/or public perception activities; see the Human Practices Hub for information and examples of innovative activities from previous teams).

We strongly believe that our work done on Human Practices, having huge input in the project shaping, exceeds Silver Medal criteria and makes us eligible for Gold Medal.

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2. Help any registered iGEM team from a high-school, different track, another university, or institution in a significant way by, for example, mentoring a new team, characterizing a part, debugging a construct, modeling/simulating their system or helping validate a software/hardware solution to a synbio problem.

We participated in multiple Collaborations in key areas such as modelling and human practices.

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3. Improve the function OR characterization of a previously existing BioBrick Part or Device (created by another team, or by your own team in in a previous year of iGEM), and enter this information in the part's page on the Registry. Please see the Registry Contribution help page for help on documenting a contribution to an existing part. This part must not come from your team's 2015 range of part numbers.

We improved and further characterized the function of BioBricks BBa_E0020 and BBa_K1362461, as seen on our Description and Results page

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4. Demonstrate a functional prototype of your project. Your prototype can derive from a previous project (that was not demonstrated to work) by your team or by another team. Show this system working under real-world conditions that you simulate in the lab. (Remember, biological materials may not be taken outside the lab.)