Team:Grenoble-EMSE-LSU/Documentation/Biobricks

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<h1>Biobricks</h1>
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<h2>KillerRed</h2>
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<p>KillerRed is a key protein in our bacterial density control system. It represents the light-sensitive element that allows the cells to receive signals from the control device.</p>
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<h1>Data Page</h1>
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<p>KillerRed is a red fluorescent protein, meaning that by illuminating it with wavelengths from a certain portion of the visible spectrum, it re-emits light in another portion with longer (less energetic) wavelengths. Below is the absorption and emission spectra for the KillerRed protein:</p>
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<h2> Our parts</h2>
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<p>INSERT ABSORPTION/EMISSION SPECTRA</p>
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<td> <div id='groupparts' style='min-height:100px;width:700px;'><div style='width:300px;margin:2px;padding:20px;color:gray;border:1px solid gray'>Loading.....</div></div><script>$('#groupparts').load('/cgi/api/groupparts.cgi?t=iGEM013\&amp;g=Grenoble-EMSE-LSU',function(){ $('#groupparts .tablesorter').tablesorter();} );</script>
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<p>From the emission and absorption spectra, we can determine that the protein absorbs in the green portion of the spectrum with a peak at 585 nm and emits in the red portion of the spectrum with a peak at 610 nm, hence the name "KillerRed".</p>
 
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<p>Emitted light from bacteria is proportional to the amount of protein in the cells. This allows for measuring protein concentration in a cell culture.</p>
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<h3><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1141001"> BBa_K1141001 </a></h3>
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<p>The second function of the protein is emission of ROS (Reactive Oxygen Species) when fluorescing.</p>
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<img src="https://static.igem.org/mediawiki/2013/7/7b/Grenoble_2013_KR1.png" width="350"></p>
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<p><strong>pLac-RBS-KillerRed</strong>
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This BioBrick was obtained by PCR on an eukaryote vector containing the coding sequence of the <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology#KillerRed">KillerRed </a> protein (generous gift of Stefan Dimitrov and Yohan Roulland, IAB Grenoble). A PCR allowed to flank it with the restriction sites KpnI and BamHI upstream and downstream. It was then ligated into the pQE30 vector(Qiagen), which contains a pLac promoter and RBS.
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The <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology#KillerRed">KillerRed </a> protein fits in our project in 2 ways: its fluorescence makes the transformed cells easily detectable and can be used to quantify the amount of live cells by our device. Its ROS-mediated killing is triggered by light, such that no chemical product has to be added in the medium to make it work and the process can be stopped as soon as the light is switched off.
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<p>ROS are highly unstable and react chemically with many substrates including proteins, lipids and DNA. These reactions are oxidative and damage the affected molecules, making ROS toxic to the cell. With sufficient amounts of ROS, a cell's essential components can be damaged beyond repair, and the cell killed. Thus illuminating KillerRed-expressing cells with light in the green portion of the visible spectrum kills them, a mechanism that we use to control cell density in a culture.</p>
 
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<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1141002"><h3>BBa_K1141002</h3></a>
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<p> <align="center"><img src="https://static.igem.org/mediawiki/2013/7/7b/Grenoble_2013_KR1.png" width="350"></p>
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<p><strong>pLac-RBS-KillerRed</strong>
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<br>This BioBrick was obtained from  BBa_K1141001 using PCR to amplify the part pLac-RBS-KillerRed.  An overlapping PCR was performed to remove an EcoRI restriction site from the sequence in between pLac and RBS. The sequence was then flanked with the iGEM suffix and prefix.</p>
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<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1141000"><h3>BBa_K1141000</h3></a>
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<p> <align="center"><img src="https://static.igem.org/mediawiki/2013/3/33/Grenoble_2013_MCherry1.png" width="350"></p>
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<p><strong>pLac-RBS-mCherry</strong>
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<br>This part contains the BioBricks  <a href="http://parts.igem.org/Part:BBa_R0010">BBa_R0010</a> and  <a href "http://parts.igem.org/Part:BBa_J06702">BBa_J06702</a>.
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It was built with standard assembly.
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<br>In the project this BioBrick was used as a negative control of KillerRed as mCherry has similar excitation and emission peaks without being cytotoxic (very low ROS production). This BioBrick also allowed us to characterize the two BioBricks BBa_R0010 and BBa_J06702 with new data.
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<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1141003"><h3>BBa_K1141003</h3></a>
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<p> <align="center"><img src="https://static.igem.org/mediawiki/2013/a/ab/Grenoble_Gnsns_GFP.png" width="350"></p>
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<p><strong>Green sensor-GFP</strong>
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Built by Gibson Assembly with samples of vectors sent by Pr.Voigt lab.
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This part was used in the <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology/KR#degradation">light controlled module </a> it has been built to characterize the green sensor.
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<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1141004"><h3>BBa_K1141004</h3></a>
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<p> <align="center"><img src="https://static.igem.org/mediawiki/2013/8/80/Grenoble_Redsns_rfp.png" width="350"></p>
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<p><strong>Red sensor-RFP</strong>
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<br>Built by Gibson Assembly with samples of vectors sent by Pr.Voigt lab.
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This part is a negative control that does not produce as much ROS a KillerRed.</p>
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<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1141005"><h3>BBa_K1141005</h3></a>
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<p> <align="center"><img src="https://static.igem.org/mediawiki/2013/7/7b/Grenoble_Redsns_KR.png" width="350"></p>
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<p><strong>Red Sensor-KillerRed</strong>
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<br>Built by Gibson Assembly with samples of vectors sent by Pr.Voigt lab.
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<br>This BioBrick takes part in the <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology/KR">light controlled KillerRed concentration</a> module. It is the enhanced version of <a href="http://parts.igem.org/Part:BBa_K1141001">BBa_K1141001</a> with a light sensitive promoter.
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<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1141006"><h3>BBa_K1141006</h3></a>
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<p> <align="center"><img src="https://static.igem.org/mediawiki/2013/1/15/Grenoble_2013_Sspb.png" width="350"></p>
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<p><strong>pBad-RBS-sspB</strong>
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<br>This part contains the BioBricks <a href="http://parts.igem.org/Part:BBa_K206000"> pBAD K206000</a> and sspB <a href="http://parts.igem.org/Part:BBa_K174000">K174000</a>, the RBS has been added via PCR on sspB, the rest of the BioBrick was built by standard assembly.
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This BioBrick is a part of the <a href="https://2013.igem.org/Team:Grenoble-EMSE-LSU/Project/Biology/KR#degradation">degradation module </a>, it enhances the degradation rate of any protein tagged with an ''ssrA''-tag.</p>
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Latest revision as of 03:35, 5 October 2013

Grenoble-EMSE-LSU, iGEM


Grenoble-EMSE-LSU, iGEM

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