http://2011.igem.org/wiki/index.php?title=Special:Contributions/Pana&feed=atom&limit=50&target=Pana&year=&month=2011.igem.org - User contributions [en]2024-03-29T10:17:21ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/Team:Bielefeld-Germany/StudentsTeam:Bielefeld-Germany/Students2011-11-20T16:34:31Z<p>Pana: </p>
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<area shape="rect" coords="50,177,130,265" href="http://www.youtube.com/watch?v=k-wdsM-7W_g" alt="Simon Schäper" title="Simon Schäper" /><br />
<area shape="rect" coords="160,182,240,270" href="" alt="Panagiotis Papavasiliou" title="Panagiotis Papavasiliou" /><br />
<area shape="rect" coords="252,155,310,229" href="http://www.youtube.com/watch?v=hC1Jm-V90ys&feature=fvwrel" alt="Michael Limberg" title="Michael Limberg" /><br />
<area shape="rect" coords="298,231,364,300" href="http://www.youtube.com/watch?v=lidFipyLG8k" alt="Katharina Thiedig" title="Katharina Thiedig" /><br />
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<area shape="rect" coords="430,233,476,314" href="http://www.dailymotion.com/video/x2bc5b_freundeskreis-a-n-n-a_music" alt="Anna Drong" title="Anna Drong" /><br />
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<area shape="rect" coords="596,187,662,273" href="http://www.youtube.com/watch?v=YXRos2bEi7o" alt="Jonas Aretz" title="Jonas Aretz" /><br />
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<area shape="rect" coords="801,179,853,254" href="http://www.youtube.com/watch?v=2yRIV078puA" alt="Jan Schwarzhans" title="Jan Schwarzhans" /><br />
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In 2011 a student command unit locked themselves in the Bielefeld University for the construction of cell-free biosensors. They promptly identified S-layer proteins as nanobiotechnological building blocks. Today still characterizing their Bisphenol A Biosensor they are the iGEM team Bielefeld. If your cells are a problem, if no one else can help and if you can find them, maybe you can hire the Bisphenol A-Team. <br />
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<img src="https://static.igem.org/mediawiki/2011/e/ed/Bielefeld-Germany-2011-Jonas.jpg" alt="Jonas Aretz" /><br />
<h3>Jonas Aretz<br/></h3><br />
Studies: Molecular Biotechnology<br />
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<img src="https://static.igem.org/mediawiki/2011/0/04/Bielefeld-Germany-2011-Robert.jpg" alt="Robert Braun" /><br />
<h3>Robert Braun<br/></h3><br />
Studies: Molecular Biotechnology<br />
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<img src="https://static.igem.org/mediawiki/2011/e/eb/Bielefeld-Germany-2011-Anna.jpg" alt="Anna Drong" /><br />
<h3>Anna Drong<br/></h3><br />
Studies: Molecular Biotechnology<br />
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<img src="https://static.igem.org/mediawiki/2011/f/f1/Bielefeld-Germany-2011-Matthias.jpg" alt="Matthias Eder" /><br />
<h3>Matthias Eder<br/></h3><br />
Studies: Molecular Biotechnology<br />
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<img src="https://static.igem.org/mediawiki/2011/a/ae/Bielefeld-Germany-2011-Michael.jpg" alt="Michael Limberg" /><br />
<h3>Michael Limberg<br/></h3><br />
Studies: Molecular Biotechnology<br />
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<img src="https://static.igem.org/mediawiki/2011/4/4d/Bielefeld-Germany-2011-Pana.jpg" alt="Panagiotis Papavasiliou" /><br />
<h3>Panagiotis Papavasiliou<br/></h3><br />
Studies: Genome Based Systems Biology<br />
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<img src="https://static.igem.org/mediawiki/2011/a/a2/Bielefeld-Germany-2011-Simon.jpg" alt="Simon Sch&auml;per" /><br />
<h3>Simon Sch&auml;per<br/></h3><br />
Studies: Genome Based Systems Biology<br />
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<img src="https://static.igem.org/mediawiki/2011/9/99/Bielefeld-Germany-2011-Jan.jpg" alt="Jan Schwarzhans" /><br />
<h3>Jan Schwarzhans<br/></h3><br />
Studies: Molecular Biotechnology<br />
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<img src="https://static.igem.org/mediawiki/2011/2/2c/Bielefeld-Germany-2011-Kathi.jpg" alt="Katharina Thiedig" /><br />
<h3>Katharina Thiedig<br/></h3><br />
Studies: Genome Based Systems Biology<br />
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<img src="https://static.igem.org/mediawiki/2011/a/ac/Bielefeld-Germany-2011-Manuel.jpg" alt="Manuel Wittchen" /><br />
<h3>Manuel Wittchen<br/></h3><br />
Studies: Molecular Biotechnology<br />
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<img src="https://static.igem.org/mediawiki/2011/a/a7/Bielefeld-Germany-2011-Timo.jpg" alt="Timo Wolf" /><br />
<h3>Timo Wolf<br/></h3><br />
Studies: Molecular Biotechnology<br />
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[[File:Bielefeld-Germany-team4.jpg|600px|center]]</div>Panahttp://2011.igem.org/File:Bielefeld-header-achiefments.pngFile:Bielefeld-header-achiefments.png2011-10-29T03:30:13Z<p>Pana: uploaded a new version of &quot;File:Bielefeld-header-achiefments.png&quot;: Reverted to version as of 03:28, 29 October 2011</p>
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<div></div>Panahttp://2011.igem.org/File:Bielefeld-header-achiefments.pngFile:Bielefeld-header-achiefments.png2011-10-29T03:28:48Z<p>Pana: uploaded a new version of &quot;File:Bielefeld-header-achiefments.png&quot;: Reverted to version as of 02:34, 29 October 2011</p>
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<div></div>Panahttp://2011.igem.org/File:Bielefeld-header-achiefments.pngFile:Bielefeld-header-achiefments.png2011-10-29T03:28:30Z<p>Pana: uploaded a new version of &quot;File:Bielefeld-header-achiefments.png&quot;</p>
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<div></div>Panahttp://2011.igem.org/Team:Bielefeld-Germany/NutshellTeam:Bielefeld-Germany/Nutshell2011-10-29T02:52:39Z<p>Pana: </p>
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<h1>The project</h1><br />
<p>The development of sensitive and selective biosensors is an important research field in synthetic biology. Unfortunately, cellular biosensors often possess undesirable features that complicate any practical application. Common problems are the limited applicability outside of a laboratory due to the use of genetically engineered cells, the low durability because of the utilization of living cells and the appearance of undesired signals induced by endogenous metabolic pathways.</p><br />
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<p>To solve these problems, the iGEM-Team Bielefeld 2011 aims at developing a cell-free bisphenol A (BPA) biosensor based on a coupled enzyme reaction fused to S-layer proteins for everyday use. Bisphenol A is a harmful substance which is used in the production of polycarbonates. To detect BPA, it is degraded by a fusion protein under formation of NAD<sup>+</sup> which is detected by an NAD<sup>+</sup>-dependent enzymatic reaction with a molecular beacon. Both enzymes are fused to S-layer proteins which build up well-defined nanosurfaces and are attached to the surface of beads. By providing these nanobiotechnological building blocks the system is expandable to other applications.</p><br />
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<h1>Subprojects</h1><br />
Our project is composed of three subprojects which are joined together in the project overview image below. <br />
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<img id="Image-Maps_6201110280540057" src="https://static.igem.org/mediawiki/2011/d/d7/IGEM_Bielefeld_Project.jpg" usemap="#Image-Maps_6201110280540057" border="0" width="930" alt="" /><br />
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<h2>S-layer</h2><br />
<p>S-layers (crystalline bacterial surface layer) are crystal-like layers consisting of multiple protein monomers and can be found in various (archae-)bacteria. Especially their ability for self-assembly into distinct geometries is of scientific interest. At phase boundaries, in solutions and on a variety of surfaces they form different lattice structures. By modifying the characteristics of the S-layer through combination with functional groups and protein domains it is possible to realize various practical applications. In particular for the production of cell-free biosensors, functional fusion proteins are of great importance. Enzymes fused to immobilized S-layers exhibited a significantly longer durability and were more stable against physical and chemical treatment. </p><br />
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[[Image:Bielefeld-Germany2011-405_100-fractions.jpg|400px|rigth|thumb| '''Measured fluorescence of collected fractions after immobilization of purified S-layer proteins fused with fluorescent protein on silica dioxide beads.''']]<br />
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<p>We aim to assemble, produce and immobilize S-layer fusion proteins for the detection of BPA by a coupled enzymatic reaction. The provision of various S-layers with different geometries offers the possibility for the scientific community to create functional nanobiotechnological surfaces with simple and standardized methods. More information on the background can be found <a href=Project/Background/S-Layer>here</a>. </p><br />
<p><b><a href=/Team:Bielefeld-Germany/Results/S-Layer>Results</a></b>: Four different S-layer BioBricks with different lattice structures were created and sent to the Partsregistry. The performance of these genes when expressed in <i>E. coli</i> was characterized and purification strategies for the expressed proteins were developed. Two purified fluorescent S-layer fusion proteins from different organisms were immobilized on beads, leading to a highly significant fluorescence enhancement of these beads (p < 10<sup>-14</sup>). Furthermore, regarding the other two S-layers (CspB from <i>Corynebacterium glutamicum</i> and <i>Corynebacterium halotolerans</i>), we discovered that while expression with a lipid anchor resulted in an integration into the cell membrane, the expression with a TAT-sequence resulted in a secretion into the medium. We also detected that those S-layers seem to stabilize the biologically active conformation of mRFP. Furthermore, we expressed and purified a fluorescent CspB fusion protein from <i>C. halotolerans</i> which had not been expressed in <i>E. coli</i> before. </p><br />
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<h2>Bisphenol A</h2><br />
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[[Image:Bielefeld-Germany2011_K123000K123001char.jpg |350px|thumb|right| '''BPA degradation by ''E. coli'' KRX carrying genes for BisdA and BisdB (only ''bisdA'' (black), polycistronic ''bisdAB'' (red) and fusion protein between BisdA and BisdB (green)).''']]<br />
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<p>The organic compound bisphenol A is a key monomer in the production of polycarbonate plastics and epoxy resins. BPA monomers can leak in small doses from BPA-based plastics into aqueous solutions. This leads to a daily exposure to BPA. As BPA is an endocrine disruptor that mimics the natural hormone estrogen, the exposure may induce negative health effects.</p><br />
<p>In 2005 a soil bacterium was isolated which is able to degrade the environmental poison bisphenol A with a unique rate and efficiency compared to other BPA degrading organisms. Three genes which are responsible for the first step of this effective BPA degradation were identified: a cytochrome P450 (<i>bisdB</i>), a ferredoxin (<i>bisdA</i>) and a ferredoxin-NADP+ oxidoreductase (<i>FNR</i>). More information on the background can be found <a href=Project/Background/BPA>here</a>.</p><br />
<p><b><a href=/Team:Bielefeld-Germany/Results/BPA>Results</a></b>: We enabled <i>E. coli</i> to degrade BPA <i>in vivo</i> and improved the specific BPA degradation rate by creating a Fd<sub>bisd</sub>:CYP<sub>bisd</sub> fusion protein (BisdA | BisdB), changing the cytochrome P450 electron transport system from a putida-like bacterial class I type to a class V type. Furthermore we could show that the fusion protein has a high specificity for BPA when compared to similar bisphenols. Lastly we were impressed by the fact that the fusion protein of FNR, BisdA and BisdB wasn't only capable to degrade BPA but also showed the highest maximal specific BPA degradation rate of all constructs. <br />
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<h2>NAD<sup>+</sup> detection</h2><br />
<p>Our selected NAD<sup>+</sup> detection method utilizes a molecular beacon based approach. The ends of a single-stranded DNA molecule are labeled with a fluorophore as well as with an appropriate quencher. Due to a formed stem-loop both are in such a close proximity to each other that no fluorescence signal is detected. Using two complementary targets hybridizing side-by-side with the hairpin enables NAD<sup>+</sup>-dependent DNA ligation by <i>E.coli</i> DNA ligase. Only after closing the gap between both hybridized targets the stem opens and the secondary structure gets broken down to a linearized probe-target hybrid. The immediate consequence is a disruption of the close proximity of the fluorophore and the quencher, so that an excitation with light is converted into a visible fluorescence signal. Therefore, NAD<sup>+</sup> concentration directly correlates with the emerging fluorescence signal. More information on the background can be found <a href=Project/Background/NAD>here</a>. </p><br />
<p><b><a href=Team:Bielefeld-Germany/Results/NAD>Results</a></b>: We were able to utilize NAD<sup>+</sup>-dependent DNA ligase from <i>E. coli</i> (LigA) for a highly sensitive and selective molecular beacon based bioassay detecting NAD<sup>+</sup> in nano molarity scale (limit of detection: 2 nM). The deadenylated form of LigA could ligate nicked DNA (a split target hybridized to a molecular beacon) resulting in an increase of fluorescence intensity. In relation to this, the initial velocity displayed a linear dependence on the employed NAD<sup>+</sup> concentrations as long as these remained the limiting factor for DNA ligation. Additionally, the NAD+ bioassay has been successfully coupled to the NADH-dependent conversion of pyruvate to L-lactate by lactic acid dehydrogenase. </p><br />
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[[Image:Bielefeld-Germany-2011_NAD+_bioassay_1.png|475px|thumb|left| '''NAD<sup>+</sup> can be detected utilizing the NAD<sup>+</sup>-dependence of LigA for a molecular beacon based bioassay.''']]<br />
[[Image:Bielefeld-Germany-2011_NAD+_bioassay_imaging.png|407px|thumb|right| '''Visualization of NAD<sup>+</sup> presence with the help of the NAD<sup>+</sup> bioassay.]]<br />
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<h1>Human Practice</h1><br />
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While developing our project idea, we were already integrating biosafety aspects in our blueprint and came up with the idea of cell-free biosensors. Besides higher durability and more specific signals, cell-free biosensors have the significant advantage that for the production all GMOs stay in the controlled environment of the lab and are not taken outside the lab for the application of the biosensor. All cells are grown under controlled conditions and only qualified personnel have access to them. This minimizes the risk of releasing GMOs into the environment and therefore the possibility of horizontal gene transfer. <br />
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We got an expert view on cell-free biosensors concerning technology assessment, biosafety and the embedment in the current discussions about synthetic biology by <a href="http://www.tab-beim-bundestag.de/en/about-tab/staff.html#Sauter-Arnold">Dr. Arnold Sauter</a> from the <a href="http://www.tab-beim-bundestag.de/en/index.html">Office of Technology Assessment at the German Bundestag (TAB)</a>.<br />
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The goals of our outreach are to rise public awareness, start public discussions and participate in the discussion about iGEM. Additonally, we want to promote the open source idea behind iGEM, arouse interest and hopefully prevented fear when facing the principles of synthetic biology. Therefore, we organized and participated in various events. Check out our <a href=Public_relations_Overview>Human Practices</a> section for more information.</p><br />
<p>Furthermore we provided a <a href="https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/Guide">guide to do it yourself nanobiotechnology</a> for fellow scientists, with detailed step by step instructions.</p><br />
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<h1>Achievements</h1><br />
<p>With the BioBricks submitted by our team, we enable a fast and selective BPA degradation in <i>E.coli</i>, a highly sensitive and selective NAD<sup>+</sup> detection that provides a versatile NAD<sup>+</sup> bioassay for future iGEM teams and the immobilization of S-layer fusion proteins, which implies the use of our S-layer proteins as nanobiotechnological building blocks.</p><br />
<p>Since our approach is cell-free, we can guarantee a high biosafety of our biosensor and were able to create a rather simple <a href=Modell> model</a> for BPA detection. </p><br />
<p>Check out our <a href=Achievements>Achievements</a> page, if you want to know about further achievements of our team. </p><br />
<html></div>Panahttp://2011.igem.org/File:Bielefeld-header-briefing.pngFile:Bielefeld-header-briefing.png2011-10-29T02:52:03Z<p>Pana: </p>
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<div></div>Panahttp://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/Guide/4aTeam:Bielefeld-Germany/Results/S-Layer/Guide/4a2011-10-29T02:48:11Z<p>Pana: </p>
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=Small scale cell disruption=<br />
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[[Image:IGEM-Bielefeld2011-Sonifier.JPG|430px|thumb|left|A Branson Sonifier 450 which we used to disrupt our cells in small scale.]] [[Image:IGEM-Bielefeld2011-Centrifuge.JPG|430px|thumb|left|A Sigma 6K15 centrifuge which we used in our project.]]<br />
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The S-layer fusion proteins form inclusion bodies in the ''E. coli'' cells (at least most of them). Inclusion bodies have the advantage that they are relatively easy to clean-up and are resistant to proteases. But inclusion bodies are unsoluble so they have to be solubilized by urea or guanidin hydrochloride. In addition, these chemicals suppress the self-assembly ability of the S-layer proteins which leeds to monomeric S-layer proteins. The cell disruption is carried out in a buffer containing 6 M urea, 50 mM phosphate buffer (pH 7.4), 300 mM sodium chloride and 10 mM imidazol. The cells are lyzed by sonification (5 times 2 minutes on ice, max. 20 W, cooling on ice between these steps). Afterwards the lysate is incubated for 1 h at 4 °C on a shaker or vertical rotator to solubilize the inclusion bodies. The cell debris is removed by centrifugation and the supernatant is used for further purification.<br />
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Only purified S-layer proteins will self-assemble - [[Team:Bielefeld-Germany/Results/S-Layer/Guide/5 | click here for further purification steps]].<br />
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</html></div>Panahttp://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/Guide/3aTeam:Bielefeld-Germany/Results/S-Layer/Guide/3a2011-10-29T02:48:00Z<p>Pana: </p>
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=<html><div style="line-height:1.2em;">Easy and small scale expression of S-layer protein under control of T7 promoter</div></html>=<br />
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[[Image:IGEM-Bielefeld2011-Shaker.JPG|400px|thumb|right|Shaking flasks on a Kuhner Lab-Shaker LS-X]]<br />
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The expression of S-layer proteins is stressful for ''Escherichia coli''. So using ''E. coli'' [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ KRX] to express genes under the control of a T7 promoter is an easy way to overexpress your proteins and seperate growth and production phase. This strain carries a T7 polymerase gene under the control of a rhamnose promoter in its genome and is also optimized for cloning so you do not have to transform your plasmids after assembly in e.g. TOP10, isolate them and bring them in an expression strain like BL21(DE3) - you can go with a single transformation step from assembly to production. The rhamnose promoter is a tightly controlled promoter (compared to arabinose or lactose promoter) and is inhibited by glucose. Using glucose and L-rhamnose supplemented LB medium leads to an autoinduction of the rhamnose promoter when glucose is completely metabolized by the cells. L-rhamnose can not be metabolized by the cells but has the same effect like D-rhamnose on the rhamnose promoter. <br />
<br />
Summarized: Use glucose, L-rhamnose and antibiotic supplemented LB medium, put it in a shaking flask, add your ''E. coli'' KRX cells carrying your S-layer fusion protein under the control of a T7 promoter, put the shaking flask on a shaker at 37 °C and then just wait! The cells will grow until the glucose is depleted (OD<sub>600</sub> ~ 1 when inoculating with OD<sub>600</sub> ~ 0.1) and the expression of the S-layer will start. Stop the cultivation after about 8 - 10 h and harvest your cells by centrifugation. <br />
<br />
Want to know how to continue? Then read [[Team:Bielefeld-Germany/Results/S-Layer/Guide/4a | how to disrupt your cells here]].<br />
<br />
To show that this really works that easy: Look at the following figures displaying the expression of the fluorescent S-layer fusion protein SgsE | mCitrine using the autoinduction protocol. <br />
<br />
[[Image:Bielefeld_2011_305_Growthcurve.png|430px|left|thumb| '''Figure 1: Growth curve of ''E. coli'' KRX expressing the fusion protein of SgsE and mCitrine with and without induction, cultivated at 37 °C in autoinduction medium with and without inductor, respectively. A curve depicting KRX wildtype is shown for comparison. After induction at approximately 4 h the OD<sub>600</sub> of the induced <partinfo>K525305</partinfo> visibly drops when compared to the uninduced culture. Both cultures grow significantly slower than KRX wildtype probably due to a leaky promoter and metabolic stress by the high copy plasmid.''']]<br />
<br />
[[Image:Bielefeld_2011_305_RFU_OD.png|430px|left|thumb| '''Figure 2: RFU to OD<sub>600</sub> ratio of ''E. coli'' KRX expressing the fusion protein of SgsE and mCitrine with and without induction. A curve depicting KRX wildtype is shown for comparison. After induction at approximately 4 h the RFU to OD<sub>600</sub> ratio starts to rise in the induced culture. Compared to the uninduced culture the ratio is roughly four times higher. The KRX wildtype shows no variation in the RFU to OD<sub>600</sub> ratio.''']]<br />
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__NOTOC__<br />
<br />
=Immobilization and recrystallisation of S-layer proteins=<br />
<br />
You have your S-layer protein monomer solution? Then you are no longer than five hours away from your nanobiotechnological device - and this only because you have to wait 4 hours for the recrystallization and immobilization of your S-layer proteins. But before you begin...<br />
<br />
<br />
<br />
==Choose your surface==<br />
<br />
[[Image:IGEM-Bielefeld2011-Silicabeads.JPG|400px|thumb|left|Silicon dioxide beads.]]<br />
[[Image:IGEM-Bielefeld2011-Waver.JPG|400px|thumb|left|A silicon wafer.]]<br />
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<br />
S-layers can reassemble on various different surfaces like gold, nylon, functionalized glass or silicon dioxide. We used silicon dioxide because it is low-priced and easy to handle. For microscopy we used silicon wafers with a silicon dioxide surface. Then...<br />
<br />
==Let the immobilization begin==<br />
<br />
[[Image:IGEM-Bielefeld2011-Rotator.JPG|330px|thumb|left|A vertical rotator which we used for coating silica beads with S-layers.]]<br />
[[Image:IGEM-Bielefeld2011-HBSS.JPG|180px|thumb|left|HBSS and recrystallization buffer for recrystallization.]]<br />
[[Image:IGEM-Bielefeld2011-HTimmo.JPG|330px|thumb|left|High-throughput coating of silica beads.]]<br />
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<br />
'''Using silica beads'''<br />
<br />
Weigh in 100 mg silica beads in a 2 mL reaction tube, add 900 µL HBSS buffer (pH 7.4, for SgsE) or recrystallization solution (pH 9.0, for SbpA) and 100 µL of your monomeric solution. You need these buffers because the S-layers we provided only self-assemble in the presence of ions e.g. calcium. Incubate the mix 4 h at room temperature on a vertical rotator. Bring down the beads afterwards by centrifugation, wash them with ddH<sub>2</sub>O and store them in ddH<sub>2</sub>O at 4 °C - finished. Now you have silica beads coated with your own functionalized nanobiotechnological surface. This is what we call '''do it yourself nanobiotechnology'''. The methods for recrystallisation were developed at and published by the BoKu Wien ([[Team:Bielefeld-Germany/Results/S-Layer/Guide/7#References |view references]]).<br />
<br />
<br />
'''Silicon wavers'''<br />
<br />
Silicon wavers are not that cheap and not that applicable as silica beads but they represent a very plane surface. Using silicon wavers with a silicon dioxide surface: Cut or break the waver into small pieces, place it in a 1.5 mL reaction tube, add 900 µL HBSS buffer (pH 7.4, for SgsE) or recrystallization solution (pH 9.0, for SbpA), add 100 µL of your monomeric solution and wait for 4 h (room temperature). Rinse the waver with ddH<sub>2</sub>O and store it in ddH<sub>2</sub>O at 4 °C until use. The methods for recrystallisation were developed at and published by the BoKu Wien ([[Team:Bielefeld-Germany/Results/S-Layer/Guide/7#References |view references]]).<br />
<br />
==References==<br />
<br />
Badelt-Lichtblau H, Kainz B, V&ouml;llenkle C, Egelseer EM, Sleytr UB, Pum D, Ilk N (2009) Genetic engineering of the S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 for the generation of functionalized nanoarrays, [http://pubs.acs.org/doi/abs/10.1021/bc800445r Bioconjug Chem. 20(5):895-903].<br />
<br />
<br />
Kainz B, Steiner K, Möller M, Pum D, Schäffer C, Sleytr UB, Toca-Herrera JL (2010) Absorption, Steady-State Fluorescence, Fluorescence Lifetime, and 2D Self-Assembly Properties of Engineered Fluorescent S-Layer Fusion Proteins of ''Geobacillus stearothermophilus'' NRS 2004/3a, [http://pubs.acs.org/doi/abs/10.1021/bm901071b ''Biomacromolecules'' 11(1):207-214].<br />
<br />
<br />
Schäffer C, Novotny R, Küpcü R, Zayni S, Scheberl A, Friedmann J, Sleytr UB, Messner P (2007) Novel Biocatalysts Based on S-Layer Self-Assembly of ''Geobacillus Stearothermophilus'' NRS 2004/3a: A Nanobiotechnological Approach, [http://onlinelibrary.wiley.com/doi/10.1002/smll.200700200/pdf ''Small'' 3(9):1549-1559].<br />
<br />
<br />
Tschiggerl H, Breitwieser A, de Roo G, Verwoerd T, Schäffer C, Sleytr U B (2008) Exploitation of the S-layer self-assembly system for site directed immobilization of enzymes demonstrated for an extremophilic laminarinase from ''Pyrococcus furiosus'', [http://www.sciencedirect.com/science/article/pii/S0168165607016070 ''Journal of Biotechnology'' 133:403-411].<br />
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=His-tag affinity chromatography=<br />
<br />
[[Image:IGEM-Bielefeld2011-Histraps.JPG|280px|thumb|left|We used HisTrap FF crude columns by GE Healthcare.]]<br />
[[Image:IGEM-Bielefeld2011-AEKTA.JPG|280px|thumb|left|GE Healthcare ÄKTAprime™ plus chromatography.]]<br />
[[Image:IGEM-Bielefeld2011-Saeulen.JPG|280px|thumb|left|Some chromatography columns.]]<br />
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<br />
[http://lmgtfy.com/?q=his-tag Polyhistidine tags] are used for easy purification. They bind to cobalt or nickel ions which are normally immobilized on chromatography beads or in spin columns. We used a 1&nbsp;mL ''HisTrap FF crude'' by [http://www.gehealthcare.com/ GE Healthcare] in combination with a [http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/aktadesign_platform~akta_primeplus GE Healthcare ÄKTAprime™ plus] FPLC device. Alternatively, you can just use a syringe which fits to [http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/Products?OpenDocument&moduleid=165904 these columns]. We did not test commercial spin column kits for his-tag purification but perhaps it even works with these (offered e.g. by Qiagen). After binding of the proteins to the column and removal from the filtered cell lysate, they were eluted with 50 mM imidazol. This fraction was collected and dialysed against ddH<sub>2</sub>O at 4 °C for 18 h using dialysis tubes obtained from [http://www.carlroth.com/catalogue/catalogue.do;jsessionid=36FBA09A4B8B2D86BA4321073514B5FA?favOid=00000009000256d300020023&act=showBookmark&lang=de-de&market=DE Roth] with a 6 kDa cut-off. <br />
<br />
<br />
[[Image:Bielefeld Germany_322_Histrap.png|280px|thumb|left|SDS-PAGE from purification.]]<br />
[[Image:IGEM-Bielefeld2011-Nanodropitlikeithot.JPG|280px|thumb|left|A NanoDrop spectrophotometer.]]<br />
[[Image:IGEM-Bielefeld2011-BCA.JPG|280px|thumb|left|A BCA assay.]]<br />
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<br />
After dialysis, you have purified S-layer fusion protein monomers which are ready to assemble (the so-called monomer solution). Check the protein concentration for example via BCA assay or NanoDrop and dilute to 1 mg mL<sup>-1</sup>. Check the purity of your monomer solution by SDS-PAGE as well.<br />
<br />
Now you have your S-layer monomer solution and can start [[Team:Bielefeld-Germany/Results/S-Layer/Guide/7 | immobilization and recrystallization to build functionalized nanobiotechnological surfaces]]. <br />
<br />
We have tested a lot of purification strategies - look them up in our [[Team:Bielefeld-Germany/Protocols/Downstream-processing | upstream and downstream protocols]].<br />
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=Filtration of the cell lysate=<br />
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[[Image:IGEM-Bielefeld2011-Pump.JPG|400px|thumb|right|A Milipore Pellicon XL 300 membrane actuated with a SciLog TANDEM 1081 peristaltic pump.]]<br />
<br />
Centrifugation does not remove all of the cell debris, therefore a tangential flow ultrafiltration step with a 300 kDa membrane is performed. The retentate is collected. Instead of this device you can also use a simple dead-end sterile filter (0.22 µm poresize) but it is easier the way described above (the dead-end filter gets clogged after some time...). <br />
<br />
With the cleared lysate the histidine affinity tag comes into play - [[Team:Bielefeld-Germany/Results/S-Layer/Guide/6 | want to know how]]?<br />
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=Cell disruption with a high-pressure homogenizer=<br />
<br />
[[Image:IGEM-Bielefeld2011-Homo.JPG|430px|thumb|left|Rannie high-pressure homogenizer was used for cell disruption.]]<br />
[[Image:IGEM-Bielefeld2011-Centrifuge.JPG|430px|thumb|left|A Sigma 6K15 centrifuge was used in our project.]]<br />
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<br />
The S-layer fusion proteins form inclusion bodies in the ''E. coli'' cells (at least most of them). Inclusion bodies have the advantage that they are relatively easy to clean-up and are resistant to proteases. But inclusion bodies are unsoluble so they have to be solubilized by urea or guanidin hydrochloride. In addition, these chemicals suppress the self-assembly ability of the S-layer proteins which leads to monomeric S-layer proteins. The cell disruption is carried out in a buffer containing 6 M urea, 50 mM phosphate buffer (pH 7.4), 300 mM sodium chloride and 10 mM imidazol.<br />
<br />
Commonly used lab methods like sonification or enzymes for cell disruption are unpracticable when you have to disrupt bigger amounts of biomass. In this case, mechanical methods like pebble mills or high-pressure homogenizers are the procedures of choice. But mechanical application of energy always leads to a heat input. Since heat can damage your proteins, you have to ensure a sufficient cooling of the solution during cell disruption. We achieved this by placing our high-pressure homogenizer in the cooling chamber of our lab and not running it continuously, but in cycles (3 cycles with cooling phases between the cycles, p = 800 bar). <br />
<br />
The cell debris is removed with a centrifugation step where the supernatant is decanted and used for further purification.<br />
<br />
Only purified S-layer proteins will self-assemble - [[Team:Bielefeld-Germany/Results/S-Layer/Guide/5 | click here for further purification steps]].<br />
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=<html><div style="line-height:1.2em;">Expression of S-layer protein under control of T7 promoter in a bioreactor</div></html>=<br />
[[Image:IGEM-Bielefeld2011-Bioreaktor.JPG|400px|thumb|right|Inoculating a [http://www.bioengineering-inc.com/standard-reactors.php?id=2.1 Bioengineering NLF22 7 L] with Bioengineering DCU.]]<br />
<br />
The expression of S-layer proteins is stressful for ''Escherichia coli''. So using ''E. coli'' [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ KRX] to express genes under the control of a T7 promoter is an easy way to overexpress your proteins and seperate growth and production phase. This strain carries a T7 polymerase gene under the control of a rhamnose promoter in its genome and it is also optimized for cloning so you do not have to transform your plasmids after assembly in e.g. TOP10, isolate them and bring them in an expression strain like BL21(DE3) - you can go with a single transformation step from assembly to production.<br />
<br />
The advantage of a bioreactor over shaking flasks is the possibility to control cultivation parameters like temperature, pH or dissolved oxygen (DO). When cultivating ''E. coli'' in shaking flasks the cells are often growing oxygen-limited because of a oxygen transmission rate (normally k<sub>L</sub>a&nbsp;= 40 - 80&nbsp;h<sup>-1</sup> in shaking flasks but up to 600 h<sup>-1</sup> in a bioreactor). The production of acetate and lactate leads to a pH shift shaking flasks. In a bioreactor pH and DO is steady during the whole cultivation leading to higher growth rates and yields of biomass. <br />
<br />
For our cultivations we used a [http://www.bioengineering-inc.com/standard-reactors.php?id=2.1 Bioengineering NLF22 7&nbsp;L] or a [http://www.gmi-inc.com/BioEngineering-KLF-Small-Laboratory-Fermenter.html#product_desc Bioengineering KLF] with Bioengineering DCU and software. We implemented a sequencer which automatically pumped an inducer solution after 4 h cultivation time to start protein expression. Other parameters were: <br />
<br />
* Medium: [[Team:Bielefeld-Germany/Protocols/Materials#HSG_medium | HSG medium]] with 20 mg L<sup>-1</sup> chloramphenicol or 100 mg L<sup>-1</sup> ampicillin<br />
* Culture volume: 2.5 - 5 L<br />
* Starting OD<sub>600</sub>: 0.1 - 0.4<br />
* DO: 60 % air saturation (controlled with stirrer cascade starting with 200 rpm)<br />
* pH: 7.0 (controlled with 20 % phosphoric acid and 4 M NaOH)<br />
* Antifoam: BASF Pluronic PE-8100<br />
* Induction solution: 0.2 % L-rhamnose and 1 mM IPTG<br />
<br />
The use of the autoinduction sequencer enables a cultivation over night. The next morning the cells have to be harvested by centrifugation. <br />
<br />
Now you have a lot of biomass which you have to disrupt - [[Team:Bielefeld-Germany/Results/S-Layer/Guide/4b | learn more about it]]!<br />
<br />
The following figure shows the expression of an SgsE | luciferase S-layer fusion protein <partinfo>K525311</partinfo> in ''E. coli'' KRX in HSG medium with autoinduction sequencer as described above. Optical density, activity of the fused luciferase, dissolved oxygen and agitator speed are plotted against the cultivation time. <br />
<br />
[[Image:IGEM-Bielefeld2011-Cultivation311.jpg|650px|thumb|center|'''Fig. 1: Bioreactor cultivation of ''E. coli'' KRX expressing the fusion protein SgsE :: luciferase <partinfo>K525311</partinfo> under the control of a T7 / lac promoter. Induction with 1 mM IPTG and 0.2 % L-rhamnose after 4 h controlled by sequencer. Cultivation in [http://www.gmi-inc.com/BioEngineering-KLF-Small-Laboratory-Fermenter.html#product_desc Bioengineering KLF] bioreactor with 2.5 L [[Team:Bielefeld-Germany/Protocols/Materials#HSG_medium | HSG medium]] with 20 mg L<sup>-1</sup> chloramphenicol starting with OD<sub>600</sub> = 0.4. Dissolved oxygen was regulated to 60 % air saturation (controlled with stirrer cascade starting with 200 rpm) and the pH to 7.0 (controlled with 20 % phosphoric acid and 4 M NaOH). The dissolved oxygen sensor had to be recalibrated after about 140 min. ''']]<br />
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=Assembly of functionalized S-layer proteins=<br />
<br />
[[Image:IGEM-Bielefeld2011-Wirbeimklonen.JPG|200px|thumb|left|iGEM assembly line]] <br />
[[Image:IGEM-Bielefeld2011-Vieleplatten.JPG|300px|thumb|right|Clone wars]]<br />
[[Image:Bielefeld-Germany-2011-Simple-cloning-example.png|400px|thumb|right|Freiburg Assembly example]]<br />
To create fusion proteins you have to use a BioBrick assembly which assembles in frame so your fusion protein is functional. The standard BioBrick assembly '''does not assemble in frame''' so you have to use other standards - like the Freiburg or the Gibson assembly. Our S-layer gene is already provided in the [http://partsregistry.org/Assembly_standard_25 Freiburg assembly standard] so after ordering it you can directly start assembling your fusion proteins of choice to it. In addition, we provided these genes not only in this easy to use standard, but they also exist under the control of a T7 / lac promoter (<partinfo>K525303</partinfo> and <partinfo>K525403</partinfo>). So after cloning you can easily express them in an ''E. coli'' strain which carries a T7 polymerase in its genome like BL21(DE3) or KRX. Do not forget to add a '''his-tag''' (<partinfo>K157011</partinfo>) to your fusion protein so you can easily purify it and follow the next steps in this guide!<br />
<br />
<br />
Want to know how to express S-layer fusion proteins based on <partinfo>K525303</partinfo> the simplest way? Then [[Team:Bielefeld-Germany/Results/S-Layer/Guide/3a | click here]].<br />
<br />
Or do you want to produce bigger amounts of your S-layer to coat all the things? Then [[Team:Bielefeld-Germany/Results/S-Layer/Guide/3b | get the big one here]].<br />
<br />
Have problems with your assemblies? Check out our [[Team:Bielefeld-Germany/Protocols/Genetics | genetic engineering protocols]].<br />
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=Planning a nanobiotechnological device=<br />
<br />
[[Image:IGEM_Bielefeld2011_Planinggruppe.JPG|270px|thumb|left|Only a well-planned project is a good project!]]<br />
<br />
The nanobiotechnological approach we present you here is based on so called S-layer proteins. S-layers (crystalline bacterial surface layer) are crystal-like layers consisting of multiple protein monomers and can be found in various (archae-)bacteria. They constitute the outermost part of the cell wall. Especially their ability for self-assembly into distinct geometries is of scientific interest. At phase boundaries, in solutions and on a variety of surfaces they form different lattice structures. The geometry and arrangement is determined by the C-terminal self-assembly domain, which is specific for each S-layer protein. The most common lattice geometries are oblique, square and hexagonal. By modifying the characteristics of the S-layer through combination with functional groups and protein domains as well as their defined position and orientation to each other (determined by the S-layer geometry), it is possible to realize various practical applications ([http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2006.00573.x/full Sleytr ''et al.'', 2007]). The usability of such well-defined nano-lattice structures is far-reaching from ultrafiltration membranes to the development of immobilized biosensors.<br />
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<br />
[[Image:Bielefeld-Germany2011-S-Layer-Geometrien.jpg|270px|thumb|left|The most common S-layer lattice geometries.]]<br />
[[Image:Bielefeld-Germany-AFM.jpg|260px|thumb|left|AFM microscopy of PS2 with hexagonal lattice structure.]]<br />
<br />
So before you can build your device you have to plan it - on genetic level. We provide you two different fully BioBrick-compatible S-layer genes (<partinfo>K525301</partinfo> and <partinfo>K525401</partinfo>) which cover the lattice geometries oblique and square. Choose the one you like to use for your application, choose proteins to functionalize the nanobiotechnological surface you are going to build, model your final device (it's easy because it's cell-free so no endogenous signals and 20+ different differential equations are needed) and then: <br />
<br />
[[Team:Bielefeld-Germany/Results/S-Layer/Guide/2 | Let the assembly begin!]]<br />
<br />
Want to know more about S-layers in general? <br />
<br />
Check our [[Team:Bielefeld-Germany/Project/Background/S-Layer | S-layer theory page]] or Sleytr UB, Huber C, Ilk N, Pum D, Schuster B, Egelseer EM (2007) S-layers as a tool kit for nanobiotechnological applications, [http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2006.00573.x/full ''FEMS Microbiol Lett'' 267(2):131-144].<br />
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=Guide to do it yourself nanobiotechnology=<br />
<br />
Have you ever considered to improve the biosafety of your BioBrick-based device? <br />
<br />
Have you ever tried to use cells for an application but they had negative side effects like metabolism or dying?<br />
<br />
Does your country have laws that prohibit the use of genetically engineered organisms outside of your lab because of safety reasons?<br />
<br />
Have you ever wanted to do nanobiotechnology by yourself, but it was too difficult?<br />
<br />
If you answer one or more of the questions above with "yes", you should definitely take a look at our '''guide to do it yourself nanobiotechnology''' to solve your problems! Click below to...<br />
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[[Team:Bielefeld-Germany/Results/S-Layer/Guide/1 | Start the guide to do it yourself nanobiotechnology]]<br />
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<div></div>Panahttp://2011.igem.org/Team:Bielefeld-Germany/SinceAmsterdamTeam:Bielefeld-Germany/SinceAmsterdam2011-10-29T02:39:24Z<p>Pana: </p>
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<br />
=What we improved since the European Regional Jamboree=<br />
<br />
==BPA degradation==<br />
<br />
===Specificity of bisphenol A degradation with ''E. coli''===<br />
In order to access the specificity of the bisphenol A degradation by the bisdA | bisdB fusion protein, we tested how well two similar bisphenols, bisphenol F (BPF) and bisphenol S (BPS), were utilized. The structure of those bisphenols differs only in the chemical group linking the two phenols from that of bisphenol A (see Figure 1).<br />
<br />
[[Image:Bielefeld_2011_Bisphenols.png|500px|center|thumb| '''Figure 1: Chemical structure of BPA, BPF and BPS showing the different chemical groups linking the two phenols.''']]<br />
<br />
BPF and BPS are used in a broad range of applications that involve the use of polycarbonates or epoxy resins and thus can often be found were BPA is also present. Accordingly their presence is a potential disruptive factor that could lead to a false positive signal with our biosensor. This is especially true for BPS that in some cases is used as a substitute for BPA in baby bottles [http://www.nytimes.com/2011/04/18/business/global/18iht-rbog-plastic-18.html]. Studies concerning the environmental pollution with BPF ([http://www.sciencedirect.com/science/article/pii/S0043135401003670 Fromme ''et al.'' (2002)]) and the acute toxicity, mutagenicity and estrogenicity of BPF and BPS ([http://onlinelibrary.wiley.com/doi/10.1002/tox.10035/abstract Chen ''et al.'' (2001)] and [http://toxsci.oxfordjournals.org/content/84/2/249 Kitamura ''et al.'' (2005)]) indicate their potential harmfulness but further research is needed to fully assess their impact on human health.<br />
<br />
''E. coli'' KRX carrying genes for the bisdA | bisdB fusion protein behind the medium strong constitutive promoter <partinfo>J23110</partinfo> with RBS <partinfo>B0034</partinfo> was cultivated at 30 °C for 36 h with LB-Medium containing 120 mg L<sup>-1</sup> BPA, BPF respectively BPS. The BPF and BPS concentration were determined by HPLC using the same method as with BPA. Figure 2 shows the degradation of the respective bisphenol after 24 h of cultivation in percent.<br />
<br />
[[Image:Bielefeld_2011_517_BPA-BPF-BPS_Degradation_24h_2.png|500px|center|thumb| '''Figure 2: Degradation of BPA, BPF and BPS after 24 h cultivation with ''E.coli'' KRX carrying genes for the bisdA <html>&#x007C;</html> bisdB fusion protein behind the medium strong constitutive promoter <partinfo>J23110</partinfo> with RBS <partinfo>B0034</partinfo>. [[Team:Bielefeld-Germany/Protocols/Downstream-processing#Expression_of_bisphenol_A_degrading_BioBricks_in_E._coli | Cultivations]] were carried out at different temperatures in LB + Amp + bisphenol medium (starting concentration 120 mg L<sup>-1</sup> BPA, BPF or BPS respectively) for 24 h in 300 mL shaking flasks without baffles with silicon plugs. Samples were taken at the end of the cultivation. Two biological replicates were analyzed. While BPA is fully degraded, only a small fraction of BPF (~7%) and BPS (~3%) was degraded.''']]<br />
<br />
The results of the experiment indicate that the bisdA | bisdB fusion protein has a '''high specificity for the degradation of BPA'''. In addition it is possible that the decrease in BPF and BPS concentration is due to internalization of those bisphenols or a endogenous enzyme of ''E. coli'' KRX and not the bisdA | bisdB fusion protein was responsible. It can be assumed that false positive signals because of BPF or BPS present in a sample are unlikely.<br />
<br />
<br />
===BPA degradation with constructs containing FNR===<br />
Constructs containing FNR, BisdA and BisdB polycistronic (<partinfo>K525551</partinfo>), FNR and a fusion protein of BisdA and BisdB (<partinfo>K525582</partinfo>) and a fusion protein of FNR, BisdA and BisdB (<partinfo>K525560</partinfo>) were tested for their ability to degrade BPA. [[Team:Bielefeld-Germany/Protocols/Downstream-processing#Expression_of_bisphenol_A_degrading_BioBricks_in_E._coli | Cultivations]] were done using the same conditions as with previous constructs. The results are shown in Figure 3 below.<br />
<br />
[[Image:Bielefeld_2011_552_562_582_BPA_Degradation_24h.png|500px|center|thumb| '''Figure 3: Degradation of BPA after 24 h cultivation with ''E.coli'' KRX carrying genes for FNR, BisdA and BisdB polycistronic <partinfo>K525551</partinfo>, FNR and a fusion protein of BisdA and BisdB <partinfo>K525582</partinfo> and a fusion protein of FNR, BisdA and BisdB <partinfo>K525560</partinfo> behind the medium strong constitutive promoter <partinfo>J23110</partinfo> with RBS <partinfo>B0034</partinfo>. [[Team:Bielefeld-Germany/Protocols/Downstream-processing#Expression_of_bisphenol_A_degrading_BioBricks_in_E._coli | Cultivations]] were carried out at 30 °C in LB + Amp + 120 mg L<sup>-1</sup> BPA for 24 h in 300 mL shaking flasks without baffles with silicon plugs. Samples were taken every 3 hours. Two to four biological replicates were analyzed. All constructs were able to degrade BPA, with the constructs containing fusion proteins outperforming the construct where all proteins were expressed polycistronic.''']]<br />
<br />
The results indicate that the BPA degradation was improved using fusion proteins compared to the completely polycistronic construct. Also the '''fusion protein of all three enzymes''' (<partinfo>K525560</partinfo>) involved in the degradation of BPA worked as intended which is quite impressive.<br />
<br />
===Comparison of all constructs used for BPA degradation===<br />
Figure 4 gives an overview of the constructs employed for the degradation of BPA.<br />
<br />
[[Image:BPA-Konstrukte-fusion-polycistronisch.png|700px|center|thumb| '''Figure 4: Schematic depiction of all constructs used for BPA degradation: [http://partsregistry.org/wiki/index.php/Part:BBa_K525512 BBa_K525511], <partinfo>K525515</partinfo>, <partinfo>K525551</partinfo>, [http://partsregistry.org/wiki/index.php/Part:BBa_K525582 BBa_K525580] and <partinfo>K525560</partinfo>. Constructs are divided into those without FNR (left) and with FNR (right). <br />
''']]<br />
<br />
All constructs were cultivated under the same conditions as described under [[Team:Bielefeld-Germany/Protocols/Downstream-processing#Expression_of_bisphenol_A_degrading_BioBricks_in_E._coli | Cultivations]]. BPA concentrations were measured using [[Team:Bielefeld-Germany/Protocols/Analytics#Bisphenol_A_analysis | HPLC]]. Figure 5 shows the percentage of BPA degraded in 24 h of cultivation and the specific BPA degradation rate calculated with the [[Team:Bielefeld-Germany/Modell#Regular_model | regular model]] and in the case of <partinfo>K525560</partinfo> with [[Team:Bielefeld-Germany/Modell#Model_for_the_fusion_protein_between_FNR.2C_BisdA_and_BisdB | the model for the fusion protein between FNR, BisdA and BisdB]].<br />
<br />
[[Image:Vergleich_aller_Konstrukte.png|700px|center|thumb| '''Figure 5: Results of the BPA degradation experiments with [http://partsregistry.org/wiki/index.php/Part:BBa_K525512 BBa_K525511], <partinfo>K525515</partinfo>, <partinfo>K525551</partinfo>, [http://partsregistry.org/wiki/index.php/Part:BBa_K525582 BBa_K525580] and <partinfo>K525560</partinfo> and the corresponding specific BPA degradation rates calculated with the model. The construct consisting only of BisdA shows almost no degradation activity, which was expected since BisdA alone can't facilitate the degradation of BPA. In comparsion the fusion protein of BisdA and BisdB degraded BPA considerbly more efficent than the polycistronic construct, indicating that the fusing of the two proteins improved its activity. Regarding the constructs containing FNR the completely polycistronic construct lags distinctively behind the other two constructs. Again, the fusing of the BisdA and BisdB proteins improved the BPA degradation activity. Using the fusion protein between FNR, BisdA and BisdB we measured the highest specific BPA degradation rate of all constructs. Although it has to be taken into account that a different model had to be used for this particular construct.<br />
''']]<br />
<br />
We are especially impressed that the '''fusion protein of FNR, BisdA and BisdB''' (<partinfo>K525560</partinfo>) not only was '''capable to degrade BPA''' but also was '''the fastest construct''' we employed. This also contributes to the feasibility of our approach for a cell free BPA biosensor since in a cell free environment fusion proteins are beneficial when compared to their non-fused counter parts.<br />
<br />
===Model for the fusion protein between FNR, BisdA and BisdB===<br />
The cultivations and BPA degradation of ''E. coli'' KRX carrying a fusion protein consisting of ferredoxin-NADP<sup>+</sup> oxidoreductase, ferredoxin<sub>bisd</sub> and cytochrome P450<sub>bisd</sub> differ from the cultivations with the other BPA degrading BioBricks. Thus, the model for these cultivations has to be adjusted to this behaviour. First of all, no diauxic growth is observed so the growth can be modeled more easily like<br />
<br />
<br />
[[Image:IGEM-Bielefeld2011-Ecoligrowthsimple.jpg|center|220px]] <div align="right">(6)</div><br />
<br />
<br />
The BPA degradation starts when the imaginary substrate is depleted, like observed in the other cultivations with BPA degrading BioBricks. But it seems that the BPA degradation is getting slower with longer cultivation time. So it is modeled with a Monod-like term in which the specific BPA degradation rate is dependent on the BPA concentration:<br />
<br />
<br />
[[Image:IGEM-Bielefeld2011-BPAdegradcomp.jpg|center|220px]] <div align="right">(7)</div><br />
<br />
<br />
with the maximal specific BPA degradation rate q<sub>D,max</sub> and the constant K<sub>D</sub>. <br />
<br />
Figures 6-10 show a comparison between modeled and measured data for cultivations with BPA degrading fusion protein ''E. coli''. In Table 1 the parameters for the model are given, obtained by curve fitting the model to the data.<br />
<br />
===Comparison between modeled and measured data===<br />
[[Image:IGEM-Bielefeld2011-Modell_K525512.png|left|430px|thumb|'''Figure 6: Comparison between modeled (lines) and measured (dots) data for [[Team:Bielefeld-Germany/Protocols/Downstream-processing#Expression_of_bisphenol_A_degrading_BioBricks_in_E._coli | cultivations]] of ''E. coli'' KRX carrying BPA degrading BioBrick <partinfo>K525512</partinfo>. The BioBrick <partinfo>K525512</partinfo> (polycistronic ''bisdAB'' genes behind medium strong promoter) was cultivated seven times in ''E. coli'' KRX in LB + Amp + BPA medium at 30 °C, using 300 mL shaking flasks without baffles with silicon plugs. The BPA concentration (closed dots) and the cell density (open dots) is plotted against the cultivation time. ''']]<br />
<br />
[[Image:IGEM-Bielefeld2011-ModellK525517.png|left|430px|thumb|'''Figure 7: Comparison between modeled (lines) and measured (dots) data for [[Team:Bielefeld-Germany/Protocols/Downstream-processing#Expression_of_bisphenol_A_degrading_BioBricks_in_E._coli | cultivations]] of ''E. coli'' KRX carrying BPA degrading BioBrick <partinfo>K525517</partinfo>. The BioBrick <partinfo>K525517</partinfo> (fusion protein between BisdA and BisdB behind medium strong promoter) was cultivated five times in ''E. coli'' KRX in LB + Amp + BPA medium at 30 °C, using 300 mL shaking flasks without baffles with silicon plugs. The BPA concentration (closed dots) and the cell density (open dots) is plotted against the cultivation time. ''']]<br />
<br />
<br style="clear: both" /><br />
<br />
[[Image:IGEM-Bielefeld2011-ModellK525552.png|left|430px|thumb|'''Figure 8: Comparison between modeled (lines) and measured (dots) data for [[Team:Bielefeld-Germany/Protocols/Downstream-processing#Expression_of_bisphenol_A_degrading_BioBricks_in_E._coli | cultivations]] of ''E. coli'' KRX carrying BPA degrading BioBrick <partinfo>K525552</partinfo>. The BioBrick <partinfo>K525552</partinfo> (polycistronic ''fnr'' : ''bisdA'' : ''bisdB'' genes behind medium strong promoter) was cultivated four times in ''E. coli'' KRX in LB + Amp + BPA medium at 30 °C, using 300 mL shaking flasks without baffles with silicon plugs. The BPA concentration (closed dots) and the cell density (open dots) is plotted against the cultivation time. ''']]<br />
<br />
[[Image:IGEM-Bielefeld2011-ModellK525582.png|left|430px|thumb|'''Figure 9: Comparison between modeled (lines) and measured (dots) data for [[Team:Bielefeld-Germany/Protocols/Downstream-processing#Expression_of_bisphenol_A_degrading_BioBricks_in_E._coli | cultivations]] of ''E. coli'' KRX carrying BPA degrading BioBrick <partinfo>K525582</partinfo>. The BioBrick <partinfo>K525582</partinfo> (polycistronic ''fnr'' : BisdAB (fusion protein) genes behind medium strong promoter) was cultivated four times in ''E. coli'' KRX in LB + Amp + BPA medium at 30 °C, using 300 mL shaking flasks without baffles with silicon plugs. The BPA concentration (closed dots) and the cell density (open dots) is plotted against the cultivation time. ''']]<br />
<br />
<br style="clear: both" /><br />
<br />
[[Image:IGEM-Bielefeld2011-ModellK525562.png|center|430px|thumb|'''Figure 10: Comparison between modeled (lines) and measured (dots) data for [[Team:Bielefeld-Germany/Protocols/Downstream-processing#Expression_of_bisphenol_A_degrading_BioBricks_in_E._coli | cultivations]] of ''E. coli'' KRX carrying BPA degrading BioBrick <partinfo>K525562</partinfo>. The BioBrick <partinfo>K525562</partinfo> (fusion protein between FNR, BisdA and BisdB behind medium strong promoter) was cultivated four times in ''E. coli'' KRX in LB + Amp + BPA medium at 30 °C, using 300 mL shaking flasks without baffles with silicon plugs. The BPA concentration (closed dots) and the cell density (open dots) is plotted against the cultivation time. ''']]<br />
<br />
<br style="clear: both" /><br />
<br />
<center><br />
<br />
'''Table 1: Parameters of the model. '''<br />
{| cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Parameter<br />
!style="border-style: solid; border-width: 1px"| <partinfo>K525512</partinfo> <br />
!style="border-style: solid; border-width: 1px"| <partinfo>K525517</partinfo><br />
!style="border-style: solid; border-width: 1px"| <partinfo>K525552</partinfo><br />
!style="border-style: solid; border-width: 1px"| <partinfo>K525562</partinfo><br />
!style="border-style: solid; border-width: 1px"| <partinfo>K525582</partinfo><br />
|-<br />
!style="border-style: solid; border-width: 1px"| X<sub>0</sub> <br />
|style="border-style: solid; border-width: 1px"|0.112 10<sup>8</sup> mL<sup>-1</sup> <br />
|style="border-style: solid; border-width: 1px"|0.138 10<sup>8</sup> mL<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|0.109 10<sup>8</sup> mL<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|0.115 10<sup>8</sup> mL<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|0.139 10<sup>8</sup> mL<sup>-1</sup><br />
|-<br />
!style="border-style: solid; border-width: 1px"| µ<sub>max</sub> <br />
|style="border-style: solid; border-width: 1px"|1.253 h<sup>-1</sup> <br />
|style="border-style: solid; border-width: 1px"|1.357 h<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|0.963 h<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|1.730 h<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|0.858 h<sup>-1</sup><br />
|-<br />
!style="border-style: solid; border-width: 1px"| K<sub>S,1</sub> <br />
|style="border-style: solid; border-width: 1px"|2.646 AU<sup>-1</sup> <br />
|style="border-style: solid; border-width: 1px"|1.92 AU<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|5.35 AU<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|13.87 AU<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|3.05 AU<sup>-1</sup><br />
|-<br />
!style="border-style: solid; border-width: 1px"| K<sub>S,2</sub> <br />
|style="border-style: solid; border-width: 1px"|265.1 AU<sup>-1</sup> <br />
|style="border-style: solid; border-width: 1px"|103.1 AU<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|82.6 AU<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|- <br />
|style="border-style: solid; border-width: 1px"|32.5 AU<sup>-1</sup><br />
|-<br />
!style="border-style: solid; border-width: 1px"| S<sub>1,0</sub> <br />
|style="border-style: solid; border-width: 1px"|1.688 AU <br />
|style="border-style: solid; border-width: 1px"|1.166 AU<br />
|style="border-style: solid; border-width: 1px"|4.679 AU<br />
|style="border-style: solid; border-width: 1px"|3.003 AU<br />
|style="border-style: solid; border-width: 1px"|2.838 AU<br />
|-<br />
!style="border-style: solid; border-width: 1px"| q<sub>S,1</sub> <br />
|style="border-style: solid; border-width: 1px"|0.478 AU 10<sup>-8</sup> cell<sup>-1</sup> <br />
|style="border-style: solid; border-width: 1px"|0.319 AU 10<sup>-8</sup> cell<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|0.883 AU 10<sup>-8</sup> cell<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|0.240 AU 10<sup>-8</sup> cell<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|0.544 AU 10<sup>-8</sup> cell<sup>-1</sup><br />
|-<br />
!style="border-style: solid; border-width: 1px"| S<sub>2,0</sub> <br />
|style="border-style: solid; border-width: 1px"|16.091 AU <br />
|style="border-style: solid; border-width: 1px"|6.574 AU<br />
|style="border-style: solid; border-width: 1px"|3.873 AU<br />
|style="border-style: solid; border-width: 1px"|- <br />
|style="border-style: solid; border-width: 1px"|2.402 AU<br />
|-<br />
!style="border-style: solid; border-width: 1px"| q<sub>S,2</sub> <br />
|style="border-style: solid; border-width: 1px"|0.295 AU 10<sup>-8</sup> cell<sup>-1</sup> <br />
|style="border-style: solid; border-width: 1px"|0.191 AU 10<sup>-8</sup> cell<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|0.082 AU 10<sup>-8</sup> cell<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|-<br />
|style="border-style: solid; border-width: 1px"|0.056 AU 10<sup>-8</sup> cell<sup>-1</sup><br />
|-<br />
!style="border-style: solid; border-width: 1px"| BPA<sub>0</sub> <br />
|style="border-style: solid; border-width: 1px"|0.53 mM <br />
|style="border-style: solid; border-width: 1px"|0.53 mM<br />
|style="border-style: solid; border-width: 1px"|0.41 mM<br />
|style="border-style: solid; border-width: 1px"|0.45 mM<br />
|style="border-style: solid; border-width: 1px"|0.53 mM<br />
|-<br />
!style="border-style: solid; border-width: 1px"| q<sub>D</sub> <br />
|style="border-style: solid; border-width: 1px"|8.76 10<sup>-11</sup> mM cell<sup>-1</sup> <br />
|style="border-style: solid; border-width: 1px"|1.29 10<sup>-10</sup> mM cell<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|5.67 10<sup>-11</sup> mM cell<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|-<br />
|style="border-style: solid; border-width: 1px"|1.13 10<sup>-10</sup> mM cell<sup>-1</sup><br />
|-<br />
!style="border-style: solid; border-width: 1px"| q<sub>D,max</sub> <br />
|style="border-style: solid; border-width: 1px"|- <br />
|style="border-style: solid; border-width: 1px"|-<br />
|style="border-style: solid; border-width: 1px"|-<br />
|style="border-style: solid; border-width: 1px"|1.32 10<sup>-10</sup> mM cell<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|-<br />
|-<br />
!style="border-style: solid; border-width: 1px"| K<sub>D</sub> <br />
|style="border-style: solid; border-width: 1px"|- <br />
|style="border-style: solid; border-width: 1px"|-<br />
|style="border-style: solid; border-width: 1px"|-<br />
|style="border-style: solid; border-width: 1px"|0.121 mM<sup>-1</sup><br />
|style="border-style: solid; border-width: 1px"|-<br />
|-<br />
|}<br />
<br />
</center><br />
<br />
The specific BPA degradation rate per cell q<sub>D</sub> is about 50 % higher when using the fusion protein compared to the polycistronic ''bisdAB'' gene. On average, this results in a 9 hours faster, complete BPA degradation by ''E. coli'' carrying <partinfo>K525517</partinfo> compared to <partinfo>K525512</partinfo> as observed during [[Team:Bielefeld-Germany/Results#Bisphenol_A_degradation_with_E._coli | our cultivations]]. The fusion protein between BisdA and BisdB improves the BPA degradation by ''E. coli''. Introducing a polycistronic ferredoxin-NADP<sup>+</sup> reductase gene into these systems does not lead to a higher specific BPA degradation rate. The rates are a bit lower, though. The effect that the BisdA | BisdB fusion protein is more efficient than BisdB when expressed polycistronically with BisdA can still be observed in this setup. The cultivations with the expression of the fusion protein FNR | BisdA | BisdB differ from the expression of the other BPA degrading BioBricks. The BPA degradation rate is concentration dependent which is typical for enzymatic reactions but was not observed in the other cultivations. In addition, the growth was faster and not diauxic. The maximal specific BPA degradation rate of this BioBrick is higher than the observed specific BPA degradation rates in the other cultivations. But due to the dependence of the BPA degradation from the BPA concentration the BPA degradation with this BioBrick is not as efficient and not complete. Anyway, the fact that this BioBrick is working, is impressive.<br />
<br />
==S-layer==<br />
<br />
===Purification===<br />
The purification strategy could be accelerated by using a His-6-affinity tag and is more simple and pure now. <br />
<br />
Scheme of purification strategy for SgsE (fusion) proteins with His-tag:<br />
<br />
[[Image:IGEM-Bielefeld2011-322_Aufreinigung_symbol.png|800px|center]]<br />
<br />
By fusing the SgsE | mCitrine with a C-terminal [http://partsregistry.org/Part:BBa_K157011 His-6-tag] the S-layer protein could be simply purified by using a denaturating His-tag affinity chromatography. This purification strategy has the advantage that no time-consuming and complex inclusion body purification and filtration is necessary to decrease the amount of native ''E. coli'' proteins. Additional to the simplification, a higher purity of the S-layer protein could be reached.<br />
<br />
This purification was made using the SgsE fusion protein containing an N-terminal mCitrine for identification and a C-terminal His-6-tag. The SDS-PAGE gel (Figure 11) and the fluorescence (Figure 12) in the collected fractions showed that the majority of fusion protein was eluated with a imidazole concentration of ca. 50 mM. There was also fluorescence measurable in the flowthrough and wash fractions. This indicates that the used 1 mL ''HisTrap FF crude'' (GE Healthcare) was overloaded or the protein was bound only weakly to the affinity matrix. The resulting purity in the elution fraction, the saving of time and the easiness makes this procedure to the preferred purification method. <br />
<br />
[[Image:Bielefeld_Germany_322_Histrap.png|700px|thumb|centre|'''Figure 11: SDS-PAGE gel of a denaturating immobilized metal affinity chromatography (IMAC) of a SgsE fusion protein containing an N-terminal mCitrine and a C-terminal His-6-tag. A 1 mL ''HisTrap FF crude'' by GE Healthcare (Ni-NTA) was used for purification. The binding buffer contained 10 mM imidazole and the elution buffer (buffer B) 500 mM imidazole. The bound proteins were eluated by a stepwise increasing imidazole gradient (10 % buffer B per step). The SgsE fusion protein (111,2 kDa) was eluated in the 10 % buffer B fraction in a high purity.''']]<br />
<br />
<br />
[[Image:Bielefeld_Germany_322_fluoreszence.png|700px|thumb|centre|'''Figure 12: Fluorescence in the fractions of a denaturating immobilized metal affinity chromatography (IMAC) of a SgsE fusion protein containing an N-terminal mCitrine and a C-terminal His-6-tag. A 1 mL HisTrap FF crude by GE Healthcare (Ni-NTA) was used for purification. The binding buffer contained 10 mM imidazole and the elution buffer (buffer B) 500 mM imidazole. The bound proteins were eluated by a stepwise rising imidazole gradient (10 % buffer B per step). The SgsE fusion protein (111,2 kDa) was eluated in the 10 % buffer B fraction in a high purity.''']]<br />
<br />
Because the method is so quick and easy now we developed a [[Team:Bielefeld-Germany/Results/S-Layer/Guide | guide to do it yourself nanobiotechnology]] in which the method is presented in a vivid way.<br />
<br />
<br />
Sometimes, the inclusion body purification does not give very clean fractions. Therefore, we developed another purification strategy for <partinfo>K525405</partinfo> monomers after inclusion body purification. This strategy consists of a capture with an IEX followed by a purification with HIC. Because of issues with the PES membranes we used to rebuffer our samples (see below - PES binds most of the S-layers) we lost a lot of our proteins in this step between the two chromatographies. So there was not enough protein left to colour it with Coomassie in an SDS-PAGE and time ran out to perform a silver staining. The fluorescence of the collected fractions of the chromatographies is shown in the figures below. <br />
<br />
[[Image:Bielefeld_Germany-405IEX.png|700px|thumb|centre|'''Figure 13: Fluorescence in the fractions of an anion exchange chromatography (IEX) with monomeric SbpA <html>|</html> mCitrine solution. A 1 mL HighTrap DEAE sepharose column by GE Healthcare was used for purification. The binding buffer (buffer A) contained 25 mM sodium chloride and the elution buffer (buffer B) 1 M sodium chloride (both 25 mM acetate buffer, pH 6.0). Abbreviations: FT = flow through, W = wash, E = elution. ''']]<br />
<br />
[[Image:Bielefeld_Germany-405HIC.png|700px|thumb|centre|'''Figure 14: Fluorescence in the fractions of a hydrophobic interaction chromatography (HIC) with monomeric SbpA <html>|</html> mCitrine solution eluted from an IEX (see above). A 1 mL HighTrap butyl sepharose column by GE Healthcare was used for purification. The binding buffer (buffer A) contained 1200 mM ammonium sulfate and the elution buffer (buffer B) no ammonium sulfate (both 50 mM ammonium phosphate buffer, pH 7.0). Abbreviations: FT = flow through, E = elution. ''']]<br />
<br />
===Enzyme reaction fused to S-layer protein===<br />
To test an exemplary enzymatic reaction fused to an S-layer protein SgsE | luciferase was assembled and expressed. The fusion protein shows high luciferase activity inside the cells but unfortunately the purification and stability at room temperature has some issues which have to be solved in future applications. <br />
<br />
<br />
====Expression in ''E. coli''====<br />
For characterization experiments the SgsE gene under the control of a T7 / ''lac'' promoter (<partinfo>K525303</partinfo>) was fused to firefly luciferase (<partinfo>K525999</partinfo>) using Freiburg BioBrick assembly.<br />
<br />
The SgsE | luciferase fusion protein was overexpressed in ''E. coli'' KRX after induction of T7 polymerase with 0.2 % L-rhamnose and induction of lac operator with 1 mM IPTG. <br />
<br />
The following cultivation was carried out in a [http://www.gmi-inc.com/BioEngineering-KLF-Small-Laboratory-Fermenter.html#product_desc Bioengineering KLF] bioreactor with Bioengineering DCU and software. A sequencer which automatically pumped an inducer solution after 4 h cultivation time to start protein expression was implemented. Other parameters were: <br />
<br />
* Medium: [https://2011.igem.org/Team:Bielefeld-Germany/Protocols/Materials#HSG_medium HSG medium] with 20 mg L<sup>-1</sup> chloramphenicol<br />
* Culture volume: 2.5 L<br />
* Starting OD<sub>600</sub>: 0.4<br />
* DO: 60 % air saturation (controlled with stirrer cascade starting with 200 rpm)<br />
* pH: 7.0 (controlled with 20 % phosphoric acid and 2 M NaOH)<br />
* Antifoam: BASF pluronic PE-8100<br />
* Induction solution: 0.2 % L-rhamnose and 1 mM IPTG<br />
<br />
<br />
The following figure shows the expression of the SgsE | luciferase S-layer fusion protein (<partinfo>K525311</partinfo>) in ''E. coli'' KRX in HSG medium with autoinduction sequencer as described above. Optical density, activity of the fused luciferase, dissolved oxygen and agitator speed are plotted against the cultivation time. <br />
<br />
[[Image:IGEM-Bielefeld2011-Cultivation311.jpg|750px|thumb|center|'''Figure 15: Bioreactor cultivation of ''E. coli'' KRX expressing the fusion protein SgsE :: luciferase <partinfo>K525311</partinfo> under the control of a T7 / ''lac'' promoter. Induction with 1 mM IPTG and 0.2 % L-rhamnose after 4 h controlled by sequencer. Cultivation in [http://www.gmi-inc.com/BioEngineering-KLF-Small-Laboratory-Fermenter.html#product_desc Bioengineering KLF] bioreactor with 2.5 L [https://2011.igem.org/Team:Bielefeld-Germany/Protocols/Materials#HSG_medium HSG medium] with 20 mg L<sup>-1</sup> chloramphenicol starting with OD<sub>600</sub> = 0.4. Dissolved oxygen was regulated to 60 % air saturation (controlled with stirrer cascade starting with 200 rpm) and the pH to 7.0 (controlled with 20 % phosphoric acid and 2 M NaOH). The dissolved oxygen sensor had to be recalibrated after about 140 min. ''']]<br />
<br />
====Purification====<br />
After the analysis of cultivations with expression of SgsE | luciferase fusion proteins, different cell fractions were analyzed. It could be seen that the proteins form inclusion bodies in ''E. coli'' but that there are some soluble proteins left. This has the advantage that the proteins carrying an enzyme as fusion protein do not have to be treated with denaturating agents like urea which destroys the enzyme (data not shown).<br />
<br />
To capture the protein from the cell lysate an ion exchange chromatography (IEX) was carried out (binding with pH 7.0, 25 mM NaCl, quaternary amine beads, elution with 100 mM NaCl). A lot of protein was found in the flow-through. When concentrating and rebuffering the proteins with PES (polyethylene sulfone) membranes a lot of protein was lost. The S-layer proteins stuck to the membrane. Some could be removed again from the membrane after cutting out the filter and incubate it in ddH<sub>2</sub>O over-night. This problem has to be kept in mind when using this S-layer.<br />
<br />
The results of the purification approach are shown in Figure 16:<br />
<br />
[[Image:IGEM-Bielefeld2011-311purification.png|500px|thumb|center|'''Figure 16: Luminescence corrected with the volume of the collected fraction plotted against the fraction during purification of <partinfo>K525311</partinfo>. Abbreviations: s.n. = supernatant, F = filtrate, R = retentate, FT = flow through, E10 = elution with 100 mM NaCl.''']]<br />
<br />
The purification strategy has to be improved. The inclusion bodies cannot be purified because urea damages the luciferase irreversible (data not shown). The loss due to adsorption of the SgsE | luciferase fusion protein to PES membranes could be avoided by using different membranes. The binding conditions of the IEX have to be improved as well. Anyway, the idea behind this purification strategy could be a starting point for a better strategy. Possibilities for improvement are:<br />
* Different membranes for ultra- / diafiltration<br />
* Other binding conditions for the IEX capturing step (higher pH)<br />
* Hydrophobic interaction chromatography as purification step after IEX (works in general, data not shown)<br />
* Size exclusion chromatography (SEC) for polishing<br />
<br />
====Stability====<br />
To test whether the S-layer protein enhances the half-life of the firefly luciferase, immobilization experiments were carried out. After the IEX described above, the elution fraction was immobilized on silicon dioxide beads or just diluted with HBSS buffer which is used for the immobilization / recrystallization of SgsE S-layer proteins. The immobilization is carried out at room temperature for 4 h. It could be seen that the luciferase activity nearly expired during this time (in the positive and the negative control). So the S-layer SgsE could not stabilize the luciferase at room temperature. The results of this experiment is shown in Figure 17:<br />
<br />
[[Image:IGEM-Bielefeld2011-311immobilization.png|500px|thumb|center|'''Figure 17: Results of immobilization experiments with <partinfo>K525311</partinfo>. The stability of the luciferase activity at room temperature is decreasing quickly.''']]<br />
<br />
==NAD<sup>+</sup> detection==<br />
<br />
===Identification of LigA===<br />
The gel bands of SDS-PAGE for His-tag purified LigA were analysed by MALDI-TOF MS and the comparison with the Swiss-Prot database clearly identified the purified protein as LigA (Figure 18, Table 2). As it is reported in literature the majority of LigA usually occurs in its adenylated form after extraction from ''E. coli''. An indication for this is the double band on the right edge of Figure 18. The proteins in both gel bands were identified as LigA from ''E. coli'' suggesting that gel band&nbsp;3 is the adenylated form and gel band&nbsp;4 is the deadenylated form. <br />
<br />
[[Image:Bielefeld-Germany-2011 LigA-purification_MALDI.png|600px|thumb|centre| '''Figure 18: SDS-PAGE analysis of LigA after His-tag purification as a preparation for MALDI-TOF MS analysis. The cleared lysate from <partinfo>BBa_K525710</partinfo> overexpressing ''E. coli'' KRX cells was loaded on a HisTrap™ FF crude column (1&nbsp;mL), the protein was eluted with 100 mM imidazole and collected in 1&nbsp;mL fractions. The framed and numbered fields indicate the gel bands which were analysed by MALDI-TOF MS. M: [http://www.fermentas.de/admin/images/media/labaid_pageruler_L7P.pdf prestained protein marker].''']]<br />
<br style="clear: both" /><br />
<br />
'''Table 2: Identification of LigA by MALDI-TOF MS. The values correspond to the framed and numbered gel bands in Figure 18. The threshold for significance of the Mascot Score for MS is 63 and the one for MS/MS is 26. The MS-Coverage represents the sequence coverage of the investigated protein with the corresponding entry in the ''E. coli'' Swiss-Prot data base. The Sequence-Coverage shows the percentage of similarity to the translated BioBrick <partinfo>BBa_K525710</partinfo> (translation was perfomed with the [http://web.expasy.org/translate/ ExPASy Translate] tool).'''<br />
<center><br />
{|cellspacing=0, spacepadding=0, border=1}<br />
!Number<br />
!Method<br />
!Swiss-Prot<br> number<br />
!Protein<br />
!Protein <br> Mascot <br> Score<br />
!Protein MW<br />
!pI-Value<br />
!MS-<br>Coverage [%]<br />
!Sequence-<br>Coverage [%]<br />
|-<br />
|1 <br />
|MS<br />
|B1XA82<br />
|DNA ligase <br> OS=Escherichia coli <br> GN=ligA<br />
|96<br />
|73560<br />
|5.3<br />
|17<br />
|25.8<br />
|-<br />
|2<br />
|MS<br />
|B1XA82<br />
|DNA ligase <br> OS=Escherichia coli <br> GN=ligA<br />
|109<br />
|73560<br />
|5.3<br />
|21<br />
|28.7<br />
|-<br />
|3<br />
|MS<br />
|A7ZPL2<br />
|DNA ligase <br> OS=Escherichia coli <br> GN=ligA<br />
|41<br />
|73618<br />
|5.2<br />
|7<br />
|10.2<br />
|-<br />
|4<br />
|MS<br />
|B1XA82<br />
|DNA ligase <br> OS=Escherichia coli <br> GN=ligA<br />
|134<br />
|73560<br />
|5.3<br />
|29<br />
|36.6<br />
|-<br />
|1<br />
|MS/MS<br />
|Q0TF55<br />
|DNA ligase <br> OS=Escherichia coli <br> GN=ligA<br />
|54<br />
|73602<br />
|5.2<br />
|6<br />
|/<br />
|-<br />
|2<br />
|MS/MS<br />
|Q0TF55<br />
|DNA ligase <br> OS=Escherichia coli <br> GN=ligA<br />
|63<br />
|73602<br />
|5.2<br />
|6<br />
|/<br />
|-<br />
|3<br />
|MS/MS<br />
|Q0TF55<br />
|DNA ligase <br> OS=Escherichia coli <br> GN=ligA<br />
|33<br />
|73602<br />
|5.2<br />
|2<br />
|/<br />
|-<br />
|4<br />
|MS/MS<br />
|Q0TF55<br />
|DNA ligase <br> OS=Escherichia coli <br> GN=ligA<br />
|36<br />
|73602<br />
|5.2<br />
|2<br />
|/<br />
|-<br />
|}<br />
</center><br />
<br />
<br />
===Limit of detection===<br />
To determine the limit of detection of the NAD<sup>+</sup> bioassay the NAD<sup>+</sup> concentrations were decreased until the fluorescence enhancement was no longer distinguishable from added H<sub>2</sub>O. This was the case as soon as the NAD<sup>+</sup> concentration came below 2 nM. Deductively, the limit of detection of the molecular beacon based and LigA associated NAD<sup>+</sup> bioassay was set at 2 nM NAD<sup>+</sup>. Furthermore, this confirmed the linear dependence between the initial velocity and the NAD<sup>+</sup> concentration (Figure 19).<br />
<br />
[[Image:Bielefeld-Germany-2011 NAD+ calibrationcurve.png|650px|thumb|centre| '''Figure 19: Initial velocity calibration curve for NAD<sup>+</sup>. The initial velocity, calculated from the average fluorescence enhancement rate in 200&nbsp;s after NAD<sup>+</sup> addition, is plotted versus the employed NAD<sup>+</sup> concentration. Data is fitted with linear regression for NAD<sup>+</sup> concentrations ranging from 0 to 200&nbsp;nM (R²&nbsp;=&nbsp;0.991, n&nbsp;=&nbsp;4).''']]<br />
<br />
<br />
===Selectivity test===<br />
The specifity of LigA for its substrate NAD<sup>+</sup> is important in order to couple the NAD<sup>+</sup> detection with investigated processes including NADH-dependent enzyme reactions. The verification of the selectivity is an important matter to exclude any unspecific reactions which might result in a NAD<sup>+</sup>-independent fluorescence enhancement. Hence, a selectivity test for LigA was performed with the analytes NADH, NADP<sup>+</sup>, NADPH, 3-ADAP (NAD<sup>+</sup> with an exchanged functional group at the nicotinamide ring system), ATP and ADP, and the relative fluorescence enhancement rates in a NAD<sup>+</sup> bioassay were compared with the one for NAD<sup>+</sup> (Figure&nbsp;20).<br />
<br />
[[Image:Bielefeld-Germany-2011_Selectivity-of-LigA.png|650px|thumb|centre| '''Figure 20: LigA shows high selectivity for NAD<sup>+</sup>. The final concentration of all analytes was 100&nbsp;nM. The responses were evaluated on the basis of the average fluorescence enhancement rate in a range of 200&nbsp;s after addition of each analyte into the NAD<sup>+</sup> bioassay. The dotted line marks the threshold indicating the intensity of background signal. All data are normalized to the NAD<sup>+</sup> value (n&nbsp;=&nbsp;2).''']]<br />
<br />
<br />
The negative control (H<sub>2</sub>O) shows that there was a small background signal about 5&nbsp;% of the signal that was produced by NAD<sup>+</sup> when using 100&nbsp;nM of analytes. This marks the threshold for fluorescence enhancement which is caused by the employed analyte. NADH, NADPH and ATP were similar to the negative control and can thereby seen as analytes that do not enable DNA ligation by LigA. Only the values for the three analytes NADP<sup>+</sup>, 3-APAD and ADP were above the threshold, but they were constantly below 10&nbsp;% of the NAD<sup>+</sup> signal. This leads to the suggestion that LigA ist highly selective for NAD<sup>+</sup> even in presence of structurally similar analytes. This makes the NAD<sup>+</sup> bioassay and the associated enzyme LigA suitable for investigating NADH-dependent enzyme reactions or measuring NAD<sup>+</sup> in biological analyte mixtures such as cell lysates.<br />
<br />
<br />
===Coupled enzyme reaction===<br />
The coupling of the LigA-including NAD<sup>+</sup> detection system was performed with a NADH-dependent enzymatic reaction which was in concrete the conversion of pyruvate to L-lactate by lactic acid dehydrogenase (LDH) from ''E. coli''. For this the LDH reaction was done with an excess of NADH and various pyruvate concentrations. Afterwards, the reaction mix was transferred to a NAD<sup>+</sup> bioassay and the fluorescence intensity was monitored. In Figure 21 the normalized initial fluorescence enhancement rates for the employed pyruvate concentrations are indicated. The calculated initial velocity was then plotted against the pyruvate concentration (Figure&nbsp;22).<br />
<br />
[[Image:Bielefeld-Germany-2011_Fluorescence-enhancement-after addition-of-LDH-reaction-mix.png|650px|thumb|centre| '''Figure 21: Fluorescence enhancement rate after addition of LDH reaction mix with various pyruvate concentrations into a LigA-dependent NAD<sup>+</sup> bioassay. 50&nbsp;ng/µL LDH was preincubated with 100&nbsp;µM NADH and different pyruvate concentrations for 2&nbsp;min at 37&nbsp;°C. 1&nbsp;µL of the LDH reaction mix was then added to a NAD<sup>+</sup> bioassay, composed of a nicked DNA substrate (250&nbsp;nM molecular beacon hybridized to a split target) and 6.5&nbsp;ng/µL LigA, and the fluorescence intensity was monitored. For illustration the fluorescence intensity is normalized for each measurement series to the first measured fluorescence value after LDH reaction mix addition (n&nbsp;=&nbsp;4).''']]<br />
<br />
[[Image:Bielefeld-Germany-2011_Pyruvate-calibration-curve.png|650px|thumb|centre| '''Figure 22: Initial velocity calibration curve for pyruvate. The initial velocity, calculated from the average fluorescence enhancement rate in 100&nbsp;s after LDH reaction mix addition into a LigA-dependent NAD<sup>+</sup> bioassay, is plotted versus the employed pyruvate concentration. Data is fitted with linear regression for pyruvate concentrations ranging from 0 to 10&nbsp;µM (R²&nbsp;=&nbsp;0.951, n&nbsp;=&nbsp;4).''']]<br />
<br />
<br />
The addition of the LDH reaction mix resulted in a characteristic fluorescence enhancement rate depending on the employed pyruvate concentration. The existing correlation between both parameters seemed to be a linear. That the signal for 0&nbsp;µM pyruvate was remarkably high could be the result of an pyruvate-independent transfer of electrons from NADH to the active site histidine of LDH under formation of NAD<sup>+</sup>. The limit of detection for pyruvate seemed to be near 1&nbsp;µM pyruvate. That this value was not in nano molarity scale is firstly caused by 100-fold dilution of the LDH reaction mix after addition to the NAD<sup>+</sup> bioassay and secondly by the fact that LDH does not necessarily converte 100&nbsp;% of the pyruvate to L-lactate. Nevertheless, for the LigA based NAD<sup>+</sup> detection system it has been proven that it can be coupled to NADH-dependent enzymatic reactions. This makes the NAD<sup>+</sup> bioassay suitable for a wide range of biological studies dealing with the ubiquitous cofactors NADH/NAD<sup>+</sup>.<br />
<br />
<br />
===Molecular cloning===<br />
Because DNA ligase from ''E. coli'' is commercially aquirable for cloning purposes LigA (<partinfo>BBa_K525710</partinfo>) was tested whether it is also suitable for molecular cloning procedures. Hence, cloning of ''rfp'' into a pSB1C3 vector was performed with LigA. Ligation was done with NAD<sup>+</sup> bioassay buffer containing 10&nbsp;mM NAD<sup>+</sup> at 37&nbsp;°C or 22&nbsp;°C and the vectors were transformed into ''E. coli'' KRX by electroporation. After growth over night at 37&nbsp;°C on petri dishes the results were documented (Figure&nbsp;23).<br />
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[[Image:Bielefeld-Germany-2011_LigAmolecularcloning-control.JPG|700px|thumb|centre| '''Figure 23: ''rfp'' cloning without any ligase (A) or with ligation by DNA ligase from ''E. coli'' (LigA) at 37&nbsp;°C (B) and 22&nbsp;°C (C). The picture shows the grown colonies after transformation of a control sample (''rfp'' and cut pSB1C3) or ligation sample (''rfp'' and cut pSB1C3 with LigA) into ''E. coli'' KRX.''']]<br />
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By analyzing the number of colony forming units and color of the colonies the ligation efficiency of LigA should be assessable. As shown for the sample without any ligase only few clones were able to grow on chloramphenicol supplemented LB medium. A much bigger number of clones was observed when ligation by LigA was performed at 22&nbsp;°C. Furthermore, there were a lot of red colonies indicating positive clones. Deductively, LigA might be useful for (large-scale) molecular cloning procedures which could be beneficial because of its easy production in ''E. coli'' and simple purification.<br />
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== Bielefeld University ==<br />
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Founded in the late 60`s [http://www.uni-bielefeld.de/%28en%29/ Bielefeld University] is one of the youngest members in the system of higher education in Germany. It is the largest of the six universities in Bielefeld with its 17.500 students and 2,600 staff (including approx. 1,480 academic staff). The university is divided into 13 different faculties, covering a broad spectrum of disciplines in the humanities, natural sciences, social sciences, and technology. Well known for its outstanding disciplinary social science faculty, the university also turned into a lighthouse of biotechnology during the last few years.<br />
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Since the founding of the so called [http://www.cebitec.uni-bielefeld.de/ CeBiTec (Center for Biotechnology]) in 1998 by the senate of Bielefeld University a huge range of techniques and equipment to cover all „omics“ research fields have been established. In the summer of 2007 the CeBiTec resided into a new building on the campus of Bielefeld University. The building offers laboratories for the Institute for Genome Research and Systems Biology, in particular for the Technology Platform Genomics. With focus on international collaborations, the platform hosts several techniques including sequencing (454 as well as illumina), transcriptomics and annotations over proteomics (GC-GC-MS, MALDI-TOF-MS, qTOF-MS) up to the metabolomics. Furthermore, the Chair of Genome Research is based in the building. Also the Institute for Biophysics and Nanosciences and the Institute for Biochemistry and Bioengineering run specific laboratories. This way, the CeBiTec creates an unique environment of excellent competence.<br />
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''' <html><div align="center"><span style="font-size:24px;"> We successfully participated at the Europe Regional Jamboree </span style></div></html>'''<br />
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[[File:iGEM-Bielefeld_gold.png|410px|frameless|right]]<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] <b>We were able to present our project to many interested people <br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We received inspiring and motivating feedback<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We won a gold medal<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We qualified for the iGEM World Championship at the MIT<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] And of course we had a lot of fun and an awesome good time</b><br />
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''' <html><div align="center"><span style="font-size:24px;">Project Achievements </span style></div></html>'''<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] <b>We successfully laid the foundation for cell-free biosensors <br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We were the first team to immobilize functionalized S-layers and thereby creating defined nano structures ([[Team:Bielefeld-Germany/Results/S-Layer|Results]])<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We provided a system to quantify NAD<sup>+</sup> highly sensitive and selective by the use of a ligase and molecular beacons ([[Team:Bielefeld-Germany/Results/NAD|Results]])<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We were able to degrade Bisphenol A quickly and selectively with ''E. coli'' ([[Team:Bielefeld-Germany/Results/BPA|Results]])<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We could show that all of our three subprojects function properly<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We have created several new and working BioBricks (e.g. <partinfo>BBa_K525121</partinfo>, <partinfo>K525222</partinfo>, <partinfo>BBa_K525305</partinfo>, <partinfo>BBa_K525405</partinfo>, <partinfo>BBa_K525512</partinfo>, <partinfo>BBa_K525515</partinfo>, <partinfo>BBa_K525710</partinfo>)<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We showed that our BioBricks work as expected<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We could successfully characterize these BioBricks and added the information to the Partsregistry<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We improved the existing Bispenol A degrading BioBricks (<partinfo>BBa_K123000</partinfo>, <partinfo>BBa_K123001</partinfo>)<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We established a versatile NAD<sup>+</sup> bioassay for future iGEM teams<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We provided a nanobiotechnological S-layer protein toolkit for the iGEM community<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We modeled our cell-free biosensor system and could show a correlation between BPA degradation and signal output ([[Team:Bielefeld-Germany/Modell|Model]])<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We organized and participated in various events to improve the iGEM outreach ([[Team:Bielefeld-Germany/Public_relations_Overview|Overview]])<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We implemented safety considerations in our project by working on a cell-free system<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We got much attention in Germany shown by press reports and invitations to various events<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We were able to manage the project on our own from the sketchbook to the final result<br />
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[[File:Bielefeld-Germany2011-check.jpg|30px|frameless|left]] We managed to completely finance our project through sponsoring ([[Team:Bielefeld-Germany/Partner|Sponsors]]) </b></div>Panahttp://2011.igem.org/File:Bielefeld-header-achiefments.pngFile:Bielefeld-header-achiefments.png2011-10-29T02:34:45Z<p>Pana: </p>
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==S-layer==<br />
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===Applications===<br />
The ability of self-assembly and forming defined nanostructures enables the use of S-layer proteins as building blocks in a biomolecular construction kit for the generation of a universal nanobiotechnological matrix. By using the matrix as a nano pinboard for further components or just the nanostructure itself, a great variety of applications in modular nanobiotechnology, biomimetics, bioanalytics and medical diagnostic are imaginable.<br />
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Concentrating on the repetitive features of S-layer proteins to form a well defined nano lattice, the use as filtration units become reasonable. So-called isoporous S-layer ultrafiltration membrane (SUM) combine the advantages of a constant lattice with a well-defined pore size and the presence of functional groups in defined positions and orientations which are a common problem of current ultrafiltration membranes.<br />
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A current topic in S-layer research is the design of more sensitive and improved optical and electrochemical sensors. One example is the luminescence lifetime based oxygen sensor. The key benefits of using S-layer proteins for this type of sensor are the imparted anti-fouling properties and the good biocompatibility. This is especially valuable for sensing applications in complex biological fluids or implantable sensors.<br />
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The self-assembly and immobilization properties of S-layer fusion proteins also open new innovative perspectives in clinical applications like immune therapy, blood purification and drug targeting. The genetic fusion of an allergen to SbsC S-layer proteins from Geobacillus stearothermophilus combine reduced allergenicity with immunomodulatory capacity. These qualities make them to an ideal allergen carrier/adjuvants in specific immunotherapy, which is the only causative treatment for type I allergy.<br />
In conclusion, S-layer proteins are one of the most innovative and promising discoveries for combining biology with engineering in the new field of nanobiotechnology.<br />
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[https://2011.igem.org/Team:Bielefeld-Germany/Project/Description#S-layer Read a more detailed S-layer description]<br />
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===Impact on the Parts Registry===<br />
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With our approach in the field of functional S-layer proteins we made it easy for future iGEM teams to create compact, well defined nanobiotechnological surfaces with chosen characteristics and to realize their own projects in a cell-free and biosafe way.<br />
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We provided two different fully BioBrick-compatible S-layer genes to the partsregistry (<partinfo>K525301</partinfo> and <partinfo>K525401</partinfo>) which cover the lattice geometries oblique and square, so future iGEM-Teams are able to choose the one they like to use for their application. <br />
Because of the [http://partsregistry.org/Assembly_standard_25 Freiburg assembly standard] it is easy to create fusion proteins with the S-layer self-assembly domain on the one side and a functional domain on the other side.<br />
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Take a look at our [https://2011.igem.org/Team:Bielefeld-Germany/Results/S-Layer/Guide practical guide to do it yourself nanobiotechnology] to plan and realize your project with our S-layer proteins.<br />
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==NAD<sup>+</sup> detection==<br />
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===Applications===<br />
The reviewed molecular beacon based NAD<sup>+</sup> bioassay can be applied to biochemical and biomedical studies. Accordingly, it can be utilized to detect NAD<sup>+</sup>/NADH-dependent enzymatic processes. The low limit of detection, its reliability and manageability provides a practical alternative to present colorimetric, fluorometric, chemiluminescent, electrochemical or mass spectrometric methods detecting NAD<sup>+</sup> or NADH. In context of clinical applications and therapeutics the NAD<sup>+</sup> bioassay can be useful to monitor cellular NAD<sup>+</sup> levels during treatments of tissues or cell cultures for identification of drug targets, for instance. It may also be applied in the field of diagnostics. Finally, the principle of the proposed NAD<sup>+</sup> bioassay can be used for cell-free optical biosensors by taking molecular beacons either immobilized on the sensor surface or being present in the reaction medium.<br />
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[https://2011.igem.org/Team:Bielefeld-Germany/Project/Description#NAD.2B_detection Read a more detailed NAD<sup>+</sup> detection description]<br />
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With our provided NAD<sup>+</sup> dependent Ligase (<partinfo>K525710</partinfo>) in the combination with a [https://2011.igem.org/Team:Bielefeld-Germany/Project/Description#NAD.2B_detection molecular beacon], we made it possible for future iGEM teams to detect and meassure any NAD<sup>+</sup> producing reaction. As shown in the [https://2011.igem.org/Team:Bielefeld-Germany/Results/NAD results] the detection is highly sensitive and selective.<br />
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By fusing your NAD<sup>+</sup> producing enzymes to our provided S-layer proteins (<partinfo>K525301</partinfo> and <partinfo>K525401</partinfo>), it is possible to combine these fusion protein with our provided S-layer fused Ligase (<partinfo>K525005</partinfo> and <partinfo>K525006</partinfo>) to create a nanobiotechnological surface and to establish a biosensor with close proximity of the detecting and the indicating part.</div>Panahttp://2011.igem.org/File:Bielefeld-header-future.pngFile:Bielefeld-header-future.png2011-10-29T02:33:54Z<p>Pana: </p>
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==Project description==<br />
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The development of sensitive and selective <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/Biosensor" target="_blank" title="A biosensor is an analytical device for the detection of an analyte that combines a biological component with a physicochemical detector component.">biosensors</a></span></html> is an important topic in <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/Synthetic_biology" target="_blank" title="Synthetic biology is the newest development in modern biology. It connects principles of engineering and informatics with genetic engineering, molecular biology and organic chemistry to build biological systems with novel functions. ">synthetic biology</a></span></html>. Biosensors can be applied in a wide range - from the detection of environmental toxics up to clinical diagnostics. Because cells have to sense their surroundings, there are a lot of natural systems that are similar to a biosensor. Prejudicial cellular biosensors often show negative side effects that complicate any practical application. Common problems are the <html><span id="igem-tooltip"><a href="http://europa.eu/legislation_summaries/agriculture/food/l28130_en.htm" target="_blank" title="In Europe, it is not allowed to release genetically modified organisms (GMOs) into the environment or place them on the market. You are only allowed to work with GMOs inside of laboratories that fit special requirements to avoid their release (EU directive 2001/18/EC and 90/220/EEC).">limited use</a></span></html> outside a gene laboratory due to the use of genetically engineered cells, the low durability because of the usage of living cells and the appearance of undesired signals induced by endogenous metabolic pathways.<br />
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To solve these problems, the [[Team:Bielefeld-Germany/Team | iGEM-Team Bielefeld 2011]] aims at developing a cell-free <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/Bisphenol_a" target="_blank" title="Bisphenol A (abbreviated BPA) is a potentially harmful substance used in the production of polycarbonates and epoxy resins. These plastics are used in many food containing bins so we are exposed to BPA every day. ">bisphenol A</a></span></html> (BPA) biosensor based on a coupled enzyme reaction fused to S-layer proteins for everyday use. Bisphenol A is a supposedly harmful substance which is used in the production of polycarbonate. To detect BPA it is degraded by a <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/Fusion_protein" target="_blank" title="Fusion proteins are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins. Recombinant fusion proteins are created artificially by recombinant DNA technology. ">fusion protein</a></span></html> under formation of <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/NAD%2B" target="_blank" title="Nicotinamide adenine dinucleotide, abbreviated NAD<sup>+</sup>, is a coenzyme found in all living cells. In metabolism, NAD<sup>+</sup> is involved in redox reactions, carrying electrons from one reaction to another. The enzymes that make and use NAD<sup>+</sup> and NADH are important in both current pharmacology and the research into future treatments for disease.">NAD<sup>+</sup></a></span></html> which is detected by an NAD<sup>+</sup>-dependent enzymatic reaction with a <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/Molecular_beacon" target="_blank" title="Molecular beacons are oligonucleotide hybridization probes that can report the presence of specific nucleic acids in homogenous solutions. The terms more often used is molecular beacon probes. Molecular beacons are hairpin shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid sequence.">molecular beacon</a></span></html>. Both enzymes are fused to S-layer proteins which build up well-defined nanosurfaces and are attached to the surface of beads. By providing these nanobiotechnological building blocks the system is expandable to other applications.<br />
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An overview of our project is shown in figure 1. The background and the state of the art of each subproject is described [[Team:Bielefeld-Germany/Project/Description#S-layer | below this figure]]. To have a quick insight of what is happining in our lab take a look in our [[Team:Bielefeld-Germany/Labjournal | labjournal]].<br />
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[[Image:IGEM_Bielefeld_Project.jpg|930px|thumb|centre|'''Figure 1: Overview of the projectidea of iGEM team Bielefeld 2011.''' Bisphenol A (BPA) is reduced by the electrons from NADH transferred by the ferredoxin-NADP<sup>+</sup> oxidoreductase (FNR), ferredoxin (Fd) and cytochrome P450 (CYP) which are fused to a S-Layer protein. The molecular beacon (hairpin structure) binds two short DNA oligos. The NAD<sup>+</sup>-dependent ligase (LigA), which is also fused to a S-Layer protein, ligates the two oligos so that the hairpin structure opens up and the fluorophore is able to emit light after extinction.]]<br />
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==S-layer==<br />
S-layers (crystalline bacterial surface layer) are <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/Crystal_structure" target="_blank" title="The crystal structure of a material can be described in terms of its unit cell. The unit cell is a small box containing one or more atoms, a spatial arrangement of atoms. The unit cells stacked in three-dimensional space describe the bulk arrangement of atoms of the crystal. The unit cell is given by its lattice parameters which are the length of the cell edges and the angles between them.">crystal-like</a></span></html> layers consisting of multiple protein <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/Monomer" target="_blank" title="A monomer is an atom or a small molecule that may bind chemically to other monomers to form a polymer. An example is the monomer bisphenol A that forms the plastic polycarbonate, a polymer. The term monomeric protein may also be used to describe one of the proteins making up a multiprotein complex.">monomers</a></span></html> and can be found in various (archae-)bacteria. They constitute the outermost part of the cell wall. Especially their ability for <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/Self-assembly" target="_blank" title="Self-assembly is a term used to describe processes in which a disordered system of pre-existing components forms an organized structure or pattern as a consequence of specific, local interactions among the components themselves, without external direction.">self-assembly</a></span></html> into distinct geometries is of scientific interest. At phase boundaries, in solutions and on a variety of surfaces they form different lattice structures. The geometry and arrangement is determined by the C-terminal self assembly-domain, which is specific for each S-layer protein. The most common lattice geometries are oblique, square and hexagonal. By modifying the characteristics of the S-layer through combination with functional groups and <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/Protein_domain" target="_blank" title="A protein domain is a part of a protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins in nature consist of several structural domains. ">protein domains</a></span></html> as well as their defined position and orientation to each other (determined by the S-layer geometry) it is possible to realize various practical applications ([http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2006.00573.x/full Sleytr ''et al.'', 2007]). The usability of such well defined nano-lattice structures is far-reaching from ultrafiltration membranes to the development of immobilized biosensors.<br />
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Especially for the production of cell-free biosensors, functional fusion proteins are of great importance. [http://pubs.acs.org/doi/abs/10.1021/bm901071b Kainz ''et al.'' (2010)] fused fluorescent proteins with an S-layer glycoprotein from [http://en.wikipedia.org/wiki/Geobacillus_stearothermophilus ''Geobacillus stearothermophilus'']. They demonstrated that the properties of the fusion protein were similar to the native fluorescent protein. The intensity of the fluorescence, the lifetime and the adsorption spectra showed comparable behavior at different pH-values. Enzymes fused to immobilized S-layers showed a significantly longer durability and were more stable against physical and chemical treatment.([http://onlinelibrary.wiley.com/doi/10.1002/smll.200700200/pdf Schäffer ''et al.'', 2007], [http://www.sciencedirect.com/science/article/pii/S0168165607016070 Tschiggerl ''et al.'', 2008]).<br />
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The iGEM-Team Bielefeld aims at the assembly, production and immobilization of S-layer fusion proteins for the detection of BPA by a coupled enzymatic reaction. S-layers from five different organisms are employed. The provision of various S-layers with different geometries offers the possibility for the scientific community to create functional <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/Nanobiotechnology#Nanobiotechnology" target="_blank" title="Bionanotechnology and nanobiotechnology are terms that refer to the intersection of nanotechnology and biology. Many new medical technologies involving nanoparticles as delivery systems or as sensors would be examples of nanobiotechnology since they involve using nanotechnology to advance the goals of biology. ">nanobiotechnological</a></span></html> surfaces with simple and standardized methods, quasi <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/DIY_biology" target="_blank" title="Do it yourself biology (DIY biology, DIY bio) is a growing movement in which individuals, or sometimes small informal organizations, change the genetics of life forms, with small resources, and often little or no formal training, oversight by professionals, or regulation by governments. This may be done as a hobby, sometimes called bio-hacking, or for profit, to start a business. ">do it yourself nanobiotechnology</a></span></html>. First, different fusion proteins with fluorescent proteins and a luciferase are created. The functionality and efficiency of the immobilization to various materials such as silicon dioxide or cellulose is then characterized by measuring the fluorescence and luminescence, respectively.<br />
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==Bisphenol A degradation==<br />
In 2005, [http://www.springerlink.com/content/q7864l02734wg32m/ Sasaki ''et al.''] isolated a soil bacterium from the ''Sphingomonas'' genus which is able to degrade the environmental poison <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/Bisphenol_a" target="_blank" title="Bisphenol A (abbreviated BPA) is a potentially harmful substance used in the production of polycarbonates and epoxy resins. These plastics are used in many food containing bins so we are exposed to BPA every day. ">bisphenol A</a></span></html> (BPA) with a unique rate and efficiency compared to other BPA degrading organisms. This strain was called ''Sphingomonas bisphenolicum'' AO1 and is able to completely decompose 120 mg BPA L<sup>-1</sup> in about 6 hours. Three genes which are responsible for the first step of this effective BPA degradation by ''S. bisphenolicum'' AO1 were identified: a <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/Cytochrome_P450" target="_blank" title="The cytochrome P450 superfamily (officially abbreviated as CYP) is a large and diverse group of enzymes. The function of most CYP enzymes is to catalyze the oxidation of organic substances. The substrates of CYP enzymes include metabolic intermediates such as lipids and steroidal hormones, as well as xenobiotic substances such as drugs and other toxic chemicals. ">cytochrome P450</a></span></html> (''bisdB''), a <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/Ferredoxin" target="_blank" title="Ferredoxins (often abbreviated Fd) are small proteins containing iron and sulfur atoms organized as iron-sulfur clusters. These biological capacitors can accept or discharge electrons, the effect being change in the oxidation states (+2 or +3) of the iron atoms. This way, ferredoxin acts as electron transfer agents in biological redox reactions. ">ferredoxin</a></span></html> (''bisdA'') and a <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/Ferredoxin—NAD(P)(%2B)_reductase" target="_blank" title="Ferredoxin NADP<sup>+</sup> reductase (abbreviated FNR) is the last enzyme in the transfer of electrons during photosynthesis from photosystem I to NADPH. In nonphotosynthetic organisms, the FNR primarily works in reverse to provide reduced ferredoxin for various metabolic pathways. These pathways include nitrogen fixation, steroid metabolism, oxidative stress response, and iron–sulfur protein biogenesis. ">ferredoxin-NADP<sup>+</sup> oxidoreductase</a></span></html> (''FNR'') ([http://aem.asm.org/cgi/content/abstract/71/12/8024 Sasaki ''et al.'', 2005b]). The ''bisdAB'' genes from ''S. bisphenolicum'' AO1 were isolated, transformed into and expressed in ''E. coli'' and enabled this bacterium to degrade BPA, too ([http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2008.03843.x/full Sasaki ''et al.'', 2008]). In addition, the BisdAB proteins from ''S. bisphenolicum'' AO1 were able to degrade BPA in a cell free system in which spinach reductase was added ([http://aem.asm.org/cgi/content/abstract/71/12/8024 Sasaki ''et al.'', 2005b]). So we assume that the BisdAB proteins also work in a cell free system together with the ferredoxin-NAD(P)<sup>+</sup> oxidoreductase from ''E. coli''. The suggested reaction mechanism of the first BPA degradation step is shown in the project overview image above or in the animated picture of figure 2.<br />
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[[Image:Bielefeld-Germany-2011-BPA-Degradation-Animation.gif|center|700px|thumb|'''Figure 2: Suggested reaction mechanism of bisphenol A degradation and BioBricks needed for this reaction ''in vitro''. ''']]<br />
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In 2008, the iGEM team from the [https://2008.igem.org/Team:The_University_of_Alberta/Parts University of Alberta] submitted the codon usage optimized ''bisdAB'' genes from ''S. bisphenolicum'' AO1 to the registry of standard biological parts in the so called [http://partsregistry.org/Assembly_standard_25 Freiburg BioBrick assembly standard] (<partinfo>K123000</partinfo> and <partinfo>K123001</partinfo>). Via this assembly standard it is very easy to build fusion proteins. These already existing protein domains will be fused together with the NAD(P)<sup>+</sup> oxidoreductase gene from ''E. coli'' to the fusion protein FNR:Fd<sub>bisd</sub>:P450<sub>bisd</sub> which subsequently will be fused to an S-layer gene. We have already shown that the Fd<sub>bisd</sub>:P450<sub>bisd</sub> fusion protein is degrading BPA more effective in ''E. coli'' than the polycistronic ''bisdAB'' gene ([[Team:Bielefeld-Germany/Labjournal#Week_15:_8th_august_-_14th_august |data here]]).<br />
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==NAD<sup>+</sup> detection==<br />
<br />
Our selected NAD<sup>+</sup> detection method displays a <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/Molecular_beacon" target="_blank" title="Molecular beacons are oligonucleotide hybridization probes that can report the presence of specific nucleic acids in homogenous solutions. The terms more often used is molecular beacon probes. Molecular beacons are hairpin shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid sequence.">molecular beacon</a></span></html> based approach. These have been initially described in 1996 as nucleic acid probes that fluoresce upon hybridization ([http://www.nature.com/nbt/journal/v14/n3/abs/nbt0396-303.html Tyagi ''et al.'', 1996]). For this effect the ends of a single-stranded DNA molecule are labeled with a fluorophore as well as with an appropriate quencher. Both are in close proximity to each other due to a formed stem-loop, so that the detection of any fluorescence signal is prevented.<br />
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The molecular beacons closed state can be applied to a bioassay detecting <html><span id="igem-tooltip"><a href="http://en.wikipedia.org/wiki/NAD%2B" target="_blank" title="Nicotinamide adenine dinucleotide, abbreviated NAD<sup>+</sup>, is a coenzyme found in all living cells. In metabolism, NAD<sup>+</sup> is involved in redox reactions, carrying electrons from one reaction to another. The enzymes that make and use NAD<sup>+</sup> and NADH are important in both current pharmacology and the research into future treatments for disease.">NAD<sup>+</sup></a></span></html> even in very low concentrations ([http://pubs.acs.org/doi/abs/10.1021/ac102742k Tang ''et al.'', 2011]). Using two complementary targets hybridizing side-by-side with the hairpin enables NAD<sup>+</sup>-dependent DNA ligation by ''E. coli'' DNA ligase. Only after closing the gap between both hybridized targets the stem melts and the secondary structure gets broken down to a linearized probe-target hybrid. The immediate consequence is a disruption of the close proximity of the fluorophore and the quencher, so that an excitation with light is converted into a visible fluorescence signal. Hence, NAD<sup>+</sup> concentration determines DNA ligase activity, which is responsible for the formation of the molecular beacons open state and therefore directly correlates with the emerging fluorescence signal. Additionally, the highly selective [http://pubs.acs.org/doi/abs/10.1021/ac102742k NAD<sup>+</sup> bioassay] has a low limit of detection compared to other methods.<br />
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Because of the signal’s stability and the suitability for daily use the NAD<sup>+</sup> bioassay can be coupled to NADH-dependent BPA degradation in the context of biosensing.<br />
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==References==<br />
<br />
Kainz B, Steiner K, Möller M, Pum D, Schäffer C, Sleytr UB, Toca-Herrera JL (2010) Absorption, Steady-State Fluorescence, Fluorescence Lifetime, and 2D Self-Assembly Properties of Engineered Fluorescent S-Layer Fusion Proteins of ''Geobacillus stearothermophilus'' NRS 2004/3a, [http://pubs.acs.org/doi/abs/10.1021/bm901071b ''Biomacromolecules'' 11(1):207-214].<br />
<br />
Sasaki M, Maki J, Oshiman K, Matsumura Y, Tsuchido T (2005a) Biodegradation of bisphenol A by cells and cell lysate from ''Sphingomonas'' sp. strain AO1, [http://www.springerlink.com/content/q7864l02734wg32m/ ''Biodegradation'' 16(5):449-459].<br />
<br />
Sasaki M, Akahira A, Oshiman K, Tsuchido T, Matsumura Y (2005b) Purification of Cytochrome P450 and Ferredoxin, Involved in Bisphenol A Degradation, from ''Sphingomonas'' sp. Strain AO1, [http://aem.asm.org/cgi/content/abstract/71/12/8024 ''Appl Environ Microbiol'' 71(12):8024-8030].<br />
<br />
Sasaki M, Tsuchido T, Matsumura Y (2008) Molecular cloning and characterization of cytochrome P450 and ferredoxin genes involved in bisphenol A degradation in ''Sphingomonas bisphenolicum'' strain AO1, [http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2672.2008.03843.x/full ''J Appl Microbiol'' 105(4):1158-1169].<br />
<br />
Schäffer C, Novotny R, Küpcü R, Zayni S, Scheberl A, Friedmann J, Sleytr UB, Messner P (2007) Novel Biocatalysts Based on S-Layer Self-Assembly of ''Geobacillus Stearothermophilus'' NRS 2004/3a: A Nanobiotechnological Approach, [http://onlinelibrary.wiley.com/doi/10.1002/smll.200700200/pdf ''Small'' 3(9):1549-1559].<br />
<br />
Sleytr UB, Huber C, Ilk N, Pum D, Schuster B, Egelseer EM (2007) S-layers as a tool kit for nanobiotechnological applications, [http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2006.00573.x/full ''FEMS Microbiol Lett'' 267(2):131-144].<br />
<br />
Tang Z, Liu P, Ma C, Yang X, Wang K, Tan W, Lv X (2011) Molecular Beacon Based Bioassay for Highly Sensitive and Selective<br />
Detection of Nicotinamide Adenine Dinucleotide and the Activity of Alanine Aminotransferase, [http://pubs.acs.org/doi/abs/10.1021/ac102742k ''Anal Chem'' 83(7):2505-2510].<br />
<br />
Tschiggerl H, Breitwieser A, de Roo G, Verwoerd T, Schäffer C, Sleytr U B (2008) Exploitation of the S-layer self-assembly system for site directed immobilization of enzymes demonstrated for an extremophilic laminarinase from ''Pyrococcus furiosus'', [http://www.sciencedirect.com/science/article/pii/S0168165607016070 ''Journal of Biotechnology'' 133:403-411]<br />
<br />
Tyagi S, Kramer FR (1996) Molecular beacons: probes hat fluoresce upon hybridization, [http://www.nature.com/nbt/journal/v14/n3/abs/nbt0396-303.html ''Nature Biotechnology'' 14:303-308].<br />
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</html></div>Panahttp://2011.igem.org/File:IGEM-Bielefeld-2011_Beacon_play_again.swfFile:IGEM-Bielefeld-2011 Beacon play again.swf2011-10-28T09:51:09Z<p>Pana: </p>
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<div></div>Panahttp://2011.igem.org/Team:Bielefeld-Germany/Bielefeld-UniversityTeam:Bielefeld-Germany/Bielefeld-University2011-10-28T07:11:16Z<p>Pana: </p>
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== Bielefeld University ==<br />
[[File:Bielefeld-Germany2011-logo-Bielefeld-Univeristy.jpg|400px|frameless|left|]]<br />
Founded in the late 60`s Bielefeld University is one of the youngest members in the system of higher education in Germany. It is the largest of the six universities in Bielefeld with its 17.500 students. The university is divided into 13 different faculties. Well known for its outstanding disciplinary social science faculty, the university also turned into a lighthouse of biotechnology during the last few years.<br />
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[[File:Bielefeld-Germany-CeBiTec.jpg|400px|frameless|left|]]<br />
Since the founding of the so called CeBiTec (Center for Biotechnology) in 1998 by the senate of Bielefeld the university established a huge range of techniques and equipment to cover all „omics“ research fields. Since the summer of 2007 the CeBiTec resides in a new building on the campus of Bielefeld University. The building offers laboratories for the Institute for Genome Research and Systems Biology, in particular for the Technology Platform Genomics. With focus on international collaborations , the platform hosts several techniques including sequencing (454 as well as illumina), transcriptomics, and annotations over proteomics (GC-GC-MS, MALDI-TOF-MS, qTOF-MS) up to the metabolomics took place here in the CeBiTec. Furthermore, the Chair of Genome Research resides in the building. Also the Institute for Biophysics and Nanosciences and the Institute for Biochemistry and Bioengineering run specific laboratories. This way, the CeBiTec creates an unique environment of excellent competence.<br />
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[[File:Bielefeld-Germany2011-CeBiTec.jpg|400px|thumb|left|The Center for Biotechnology - CeBiTec]]</div>Panahttp://2011.igem.org/Team:Bielefeld-Germany/Public_relations_OverviewTeam:Bielefeld-Germany/Public relations Overview2011-09-22T03:54:17Z<p>Pana: </p>
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<a href="https://2011.igem.org/Team:Bielefeld-Germany/Public_relations_Summary"><img src="https://static.igem.org/mediawiki/2011/6/6f/Bielefeld-Germany-2011-Science-Cafe-Titel.jpg" /></a> <br />
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<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Public_relations_Summary">Summary</a></h3><br />
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In Germany there is an ongoing debate about synthetic biology and iGEM. This discussion is often affected by concerns, skepticisms, fear and the criticism about “playing god”..<br />
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Many big German newspapers published about these topics (Der Spiegel, Der Tagesspiegel, Die Zeit), there are even political movements which want to prevent the application of synthetic biology. These movements are for example initiating petitions with the collection of signatures against the design of synthetic organisms (Testbiotech). Even the German government had to face questions about synthetic biology and iGEM by members of the German parliament. The questions were answered by the State Secretary Dr. Helge Braun of the Federal Ministry of Education and Research.<a href="https://2011.igem.org/Team:Bielefeld-Germany/Public_relations_Summary">read more</a><br />
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<a href="https://2011.igem.org/Team:Bielefeld-Germany/cebitec-symposium"><img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/symposium-big.png" /></a> <br />
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European iGEM teams meet in Bielefeld, Germany. A review on the 6th CeBiTec Symposium Genome-Based Microbiology: From -omics Research to Systems and Synthetic Biology<br />
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The annual CeBiTec Symposium, first held in 2006, deals with prospective topics in the broad spectrum of biotechnological research. Each year international scientists come together in Bielefeld setting the focus on current innovations, approaches and methodologies. The issues so far included Solar Bio-Fuels(2008), bioIMAGING (2009) and New Frontiers in Microbial Genome Research (2010). The 6th CeBiTec Symposium was held under the topic Genome-Based Microbiology: From -omics Research to Systems and Synthetic Biology took place between the 18th and 20th of July, 2011. <a href="https://2011.igem.org/Team:Bielefeld-Germany/cebitec-symposium">read more</a><br />
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<a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_lab_course"><img src="https://static.igem.org/mediawiki/2011/6/6a/Bielefeld-Germany2011-labcourse14.jpg" /></a> <br />
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<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_lab_course">GENIALE Open Lab Course</a></h3><br />
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We organized a lab course and lab tour for over 150 children, adolescents and their parents.<br />
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One of our goals is an objective science communication in the fields of biotechnology and especially synthetic biology. To improve and expand our outreach we had the opportunity to take part in the GENIALE. This is one of the biggest European science fairs with over 50,000 participants, funded by the European Union and the state of North Rhine Westphalia. The iGEM team Bielefeld took the chance to participate with an open lab course for all ages and an in depth science café in the evening. <a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_lab_course">read more</a><br />
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We hosted a science café about synthetic biology, iGEM and our projects.<br />
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The science café is an event organized by Bielefeld Marketing in a restaurant in downtown Bielefeld. In a comfortable setting we were able to give a presentation, followed by a moderated panel discussion with some team members and questions from the interested audience. Our goal was to show that we want to get in contact with the public, in particular present our project and get some feedback. <a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_science_cafe">read more</a><br />
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<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/student-convention">Student Convention</a></h3><br />
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The iGEM Team Bielefeld was honored with an invitation to the 2nd BIO.NRW (PhD) Student Convention at the BayArena in Leverkusen.<br />
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The congress gives lifesience (PhD) students the opportunity to meet representatives of industry and academia, get information for career entry as well as improve their soft skills in workshops. Apart from the opportunity to participate in the convention, the role of the iGEM Team Bielefeld was to give a project presentation about iGEM as an example for remarkable scientific dedication during academic studies. The VIP-lounge in the stadium of the national league soccer team Bayer Leverkusen served as the venue for the two day congress. <a href="https://2011.igem.org/Team:Bielefeld-Germany/student-convention">read more</a><br />
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<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/high-school">The iGEM Team Bielefeld at a local high school</a></h3><br />
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Though synthetic biology (SB) is going to be the next big thing in biology, its researches are always raising public concerns respectivly are always in the focus of a public opinion.<br />
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Therefore we got in contact with a local high school, in order to get an impression of the public awareness concerning synthetic biology. Hence we discussed with pupils about pros and cons of Synthetic Biology, iGEM and our projects as examples for applications. About 80 seniors from different biology and chemistry A-level courses came to the school's assembly hall and listened to our presentation, followed by questions. Afterwards the students were split into smaller groups of ten to twenty people and got accompanied by members of the Bielefeld iGEM team to discuss about iGEM and SB. <a href="https://2011.igem.org/Team:Bielefeld-Germany/high-school">read more</a><br />
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The German Federal Ministry of Education and Research manages the Strategic Process Biotechnology 2020+. <br />
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Together with research organizations and universities the vision is to develop the next generation of biotechnological processes as well as initiate their realization. Apart from funding of research projects, expert talks on the topics medicine, energy, environment and industry are arranged. Every year a highlight congress is organized, where representatives from academia, industry and politics meet to discuss the future of biotechnology. This year, parts of the iGEM Team Bielefeld were invited to actively participate and present posters about the 2010 and 2011 projects. <a href="https://2011.igem.org/Team:Bielefeld-Germany/strategic-process">read more</a><br />
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<a href="https://2011.igem.org/Team:Bielefeld-Germany/Black-box" ><img src="https://static.igem.org/mediawiki/2011/e/e6/Bielefeld-Germany2011-circuitboard-240px.png"></a><br />
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A prototype to show how the fluorescence would look like when using our cell-free bisphenol A biosensor system.<br />
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We build this "Beacon Box" to show the audience at the Science Cafe presentation the fluorescence light which would be emitted by our cell-free bishphenol A biosensor system. It is a simple prototype which can easyly be build by everyone for home-use. To see the specifications and construction manual for our "Beacon Box" <a href="https://2011.igem.org/Team:Bielefeld-Germany/Black-box">read more</a><br />
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<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Press">Press</a></h3><br />
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A comprehensive list of all press articles about our iGEM team.<br />
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This page gives a list of all news paper articles, internet news spots, radio feature, etc. about our iGEM team project. <a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Press">read more</a><br />
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<li><br />
<a href="#5"><br />
<img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/convention-small.png"/><br />
<strong>Student Convention</strong><br />
Leverkusen, Germany<br />
</a><br />
<br />
</li><br />
<li><br />
<a href="#6"><br />
<img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/school-small.png" /><br />
<strong>High School</strong><br />
Herford, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#7"><br />
<img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/2020+-small.png" /><br />
<strong>Strategic Process</strong><br />
Berlin, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#8"><br />
<img src="https://static.igem.org/mediawiki/2011/b/be/Bielefeld-Germany-2011-Beacon-Box-75.JPG" /><br />
<strong>Beacon Box</strong><br />
Bielefeld, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#9"><br />
<img src="https://static.igem.org/mediawiki/2011/0/03/Bielefeld-Germany-2011-Timo-Interview.geaendert.jpg" /><br />
<strong>Press</strong><br />
Bielefeld, Germany<br />
</a><br />
</li><br />
</ul><br />
<br />
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</html></div>Panahttp://2011.igem.org/Team:Bielefeld-Germany/Public_relations_OverviewTeam:Bielefeld-Germany/Public relations Overview2011-09-22T03:53:38Z<p>Pana: </p>
<hr />
<div>{{Bielefeld_2011_Header}}<br />
<html><img src="https://static.igem.org/mediawiki/2011/c/cb/Bielefeld-header-hp-overview.png"/><p></p></html><br />
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<a href="https://2011.igem.org/Team:Bielefeld-Germany/Public_relations_Summary"><img src="https://static.igem.org/mediawiki/2011/6/6f/Bielefeld-Germany-2011-Science-Cafe-Titel.jpg" /></a> <br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Public_relations_Summary">Summary</a></h3><br />
<br />
<p class="more"><br />
In Germany there is an ongoing debate about synthetic biology and iGEM. This discussion is often affected by concerns, skepticisms, fear and the criticism about “playing god”..<br />
</p><br />
<br />
<p><br />
Many big German newspapers published about these topics (Der Spiegel, Der Tagesspiegel, Die Zeit), there are even political movements which want to prevent the application of synthetic biology. These movements are for example initiating petitions with the collection of signatures against the design of synthetic organisms (Testbiotech). Even the German government had to face questions about synthetic biology and iGEM by members of the German parliament. <a href="https://2011.igem.org/Team:Bielefeld-Germany/Public_relations_Summary">read more</a><br />
</p><br />
</div><br />
<br />
<div><br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/cebitec-symposium"><img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/symposium-big.png" /></a> <br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/cebitec-symposium">Cebitec Symposium</a></h3><br />
<br />
<p class="more"><br />
European iGEM teams meet in Bielefeld, Germany. A review on the 6th CeBiTec Symposium Genome-Based Microbiology: From -omics Research to Systems and Synthetic Biology<br />
</p><br />
<br />
<p><br />
The annual CeBiTec Symposium, first held in 2006, deals with prospective topics in the broad spectrum of biotechnological research. Each year international scientists come together in Bielefeld setting the focus on current innovations, approaches and methodologies. The issues so far included Solar Bio-Fuels(2008), bioIMAGING (2009) and New Frontiers in Microbial Genome Research (2010). The 6th CeBiTec Symposium was held under the topic Genome-Based Microbiology: From -omics Research to Systems and Synthetic Biology took place between the 18th and 20th of July, 2011. <a href="https://2011.igem.org/Team:Bielefeld-Germany/cebitec-symposium">read more</a><br />
</p><br />
</div><br />
<br />
<div><br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_lab_course"><img src="https://static.igem.org/mediawiki/2011/6/6a/Bielefeld-Germany2011-labcourse14.jpg" /></a> <br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_lab_course">GENIALE Open Lab Course</a></h3><br />
<br />
<p class="more"><br />
We organized a lab course and lab tour for over 150 children, adolescents and their parents.<br />
</p><br />
<br />
<p><br />
One of our goals is an objective science communication in the fields of biotechnology and especially synthetic biology. To improve and expand our outreach we had the opportunity to take part in the GENIALE. This is one of the biggest European science fairs with over 50,000 participants, funded by the European Union and the state of North Rhine Westphalia. The iGEM team Bielefeld took the chance to participate with an open lab course for all ages and an in depth science café in the evening. <a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_lab_course">read more</a><br />
</p><br />
</div><br />
<br />
<div><br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_science_cafe"><img src="https://static.igem.org/mediawiki/2011/8/8a/Bielefeld-Germany2011-science-cafe11.jpg" /></a> <br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_science_cafe">Science Cafe</a></h3><br />
<br />
<p class="more"><br />
We hosted a science café about synthetic biology, iGEM and our projects.<br />
</p><br />
<br />
<p><br />
The science café is an event organized by Bielefeld Marketing in a restaurant in downtown Bielefeld. In a comfortable setting we were able to give a presentation, followed by a moderated panel discussion with some team members and questions from the interested audience. Our goal was to show that we want to get in contact with the public, in particular present our project and get some feedback. <a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_science_cafe">read more</a><br />
</p><br />
</div><br />
<br />
<br />
<div><br />
<br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/student-convention"><img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/convention-big.png" /></a><br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/student-convention">Student Convention</a></h3><br />
<br />
<p class="more"><br />
The iGEM Team Bielefeld was honored with an invitation to the 2nd BIO.NRW (PhD) Student Convention at the BayArena in Leverkusen.<br />
</p><br />
<br />
<p><br />
The congress gives lifesience (PhD) students the opportunity to meet representatives of industry and academia, get information for career entry as well as improve their soft skills in workshops. Apart from the opportunity to participate in the convention, the role of the iGEM Team Bielefeld was to give a project presentation about iGEM as an example for remarkable scientific dedication during academic studies. The VIP-lounge in the stadium of the national league soccer team Bayer Leverkusen served as the venue for the two day congress. <a href="https://2011.igem.org/Team:Bielefeld-Germany/student-convention">read more</a><br />
</p><br />
<br />
</div><br />
<br />
<div><br />
<br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/high-school"><img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/school-big.png" /></a><br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/high-school">The iGEM Team Bielefeld at a local high school</a></h3><br />
<br />
<p class="more"><br />
Though synthetic biology (SB) is going to be the next big thing in biology, its researches are always raising public concerns respectivly are always in the focus of a public opinion.<br />
</p><br />
<br />
<p><br />
Therefore we got in contact with a local high school, in order to get an impression of the public awareness concerning synthetic biology. Hence we discussed with pupils about pros and cons of Synthetic Biology, iGEM and our projects as examples for applications. About 80 seniors from different biology and chemistry A-level courses came to the school's assembly hall and listened to our presentation, followed by questions. Afterwards the students were split into smaller groups of ten to twenty people and got accompanied by members of the Bielefeld iGEM team to discuss about iGEM and SB. <a href="https://2011.igem.org/Team:Bielefeld-Germany/high-school">read more</a><br />
</p><br />
<br />
</div><br />
<br />
<div> <br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/strategic-process"><img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/2020+-big.png" /></a><br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/strategic-process">Strategic Process</a></h3><br />
<br />
<p class="more"><br />
The German Federal Ministry of Education and Research manages the Strategic Process Biotechnology 2020+. <br />
</p><br />
<br />
<p><br />
Together with research organizations and universities the vision is to develop the next generation of biotechnological processes as well as initiate their realization. Apart from funding of research projects, expert talks on the topics medicine, energy, environment and industry are arranged. Every year a highlight congress is organized, where representatives from academia, industry and politics meet to discuss the future of biotechnology. This year, parts of the iGEM Team Bielefeld were invited to actively participate and present posters about the 2010 and 2011 projects. <a href="https://2011.igem.org/Team:Bielefeld-Germany/strategic-process">read more</a><br />
</p> <br />
<br />
</div><br />
<br />
<div> <br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/Black-box" ><img src="https://static.igem.org/mediawiki/2011/e/e6/Bielefeld-Germany2011-circuitboard-240px.png"></a><br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Black-box">Beacon Box</a></h3><br />
<br />
<p class="more"><br />
A prototype to show how the fluorescence would look like when using our cell-free bisphenol A biosensor system.<br />
</p><br />
<br />
<p><br />
We build this "Beacon Box" to show the audience at the Science Cafe presentation the fluorescence light which would be emitted by our cell-free bishphenol A biosensor system. It is a simple prototype which can easyly be build by everyone for home-use. To see the specifications and construction manual for our "Beacon Box" <a href="https://2011.igem.org/Team:Bielefeld-Germany/Black-box">read more</a><br />
</p> <br />
<br />
</div><br />
<div> <br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Press"><img src="https://static.igem.org/mediawiki/2011/7/75/Bielefeld-Germany-2011-Timo-2020gross.jpg" /></a><br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Press">Press</a></h3><br />
<br />
<p class="more"><br />
A comprehensive list of all press articles about our iGEM team.<br />
</p><br />
<br />
<p><br />
This page gives a list of all news paper articles, internet news spots, radio feature, etc. about our iGEM team project. <a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Press">read more</a><br />
</p> <br />
<br />
</div><br />
<br />
</div><br />
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<br />
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<div id="nav" style="text-align:center;"><br />
<ul><br />
<br />
<li><br />
<a href="#1"><br />
<img src="https://static.igem.org/mediawiki/2011/7/72/Bielefeld-Germany-2011-Pana-kittel-klein.jpg"/><br />
<strong>Summary</strong><br />
<br />
Bielefeld, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#2"><br />
<img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/symposium-small.png"/><br />
<strong>Cebitec Symposium</strong><br />
<br />
Bielefeld, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#3"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c9/Bielefeld-Germany2011-labcourse15.jpg"/><br />
<strong>Open Lab Course</strong><br />
<br />
Bielefeld, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#4"><br />
<img src="https://static.igem.org/mediawiki/2011/d/d0/Bielefeld-Germany2011-science-cafe10.jpg"/><br />
<strong>Science Cafe</strong><br />
<br />
Bielefeld, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#5"><br />
<img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/convention-small.png"/><br />
<strong>Student Convention</strong><br />
Leverkusen, Germany<br />
</a><br />
<br />
</li><br />
<li><br />
<a href="#6"><br />
<img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/school-small.png" /><br />
<strong>High School</strong><br />
Herford, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#7"><br />
<img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/2020+-small.png" /><br />
<strong>Strategic Process</strong><br />
Berlin, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#8"><br />
<img src="https://static.igem.org/mediawiki/2011/b/be/Bielefeld-Germany-2011-Beacon-Box-75.JPG" /><br />
<strong>Beacon Box</strong><br />
Bielefeld, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#9"><br />
<img src="https://static.igem.org/mediawiki/2011/0/03/Bielefeld-Germany-2011-Timo-Interview.geaendert.jpg" /><br />
<strong>Press</strong><br />
Bielefeld, Germany<br />
</a><br />
</li><br />
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</html></div>Panahttp://2011.igem.org/Team:Bielefeld-Germany/Public_relations_OverviewTeam:Bielefeld-Germany/Public relations Overview2011-09-22T03:50:57Z<p>Pana: </p>
<hr />
<div>{{Bielefeld_2011_Header}}<br />
<html><img src="https://static.igem.org/mediawiki/2011/c/cb/Bielefeld-header-hp-overview.png"/><p></p></html><br />
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<a href="https://2011.igem.org/Team:Bielefeld-Germany/Public_relations_Summary"><img src="https://static.igem.org/mediawiki/2011/6/6f/Bielefeld-Germany-2011-Science-Cafe-Titel.jpg" /></a> <br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Public_relations_Summary">Summary</a></h3><br />
<br />
<p class="more"><br />
This is an overview about our motivation and a general summary about our Human Practice Advance.<br />
</p><br />
<br />
<p><br />
Our goals are to awake the public awareness, start public discussions and participate in the outreach about iGEM. Further we want to promote the open source principle behind iGEM, arouse interest and hopefully prevented fear when facing the principles of synthetic biology. <a href="https://2011.igem.org/Team:Bielefeld-Germany/Public_relations_Summary">read more</a><br />
</p><br />
</div><br />
<br />
<div><br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/cebitec-symposium"><img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/symposium-big.png" /></a> <br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/cebitec-symposium">Cebitec Symposium</a></h3><br />
<br />
<p class="more"><br />
European iGEM teams meet in Bielefeld, Germany. A review on the 6th CeBiTec Symposium Genome-Based Microbiology: From -omics Research to Systems and Synthetic Biology<br />
</p><br />
<br />
<p><br />
The annual CeBiTec Symposium, first held in 2006, deals with prospective topics in the broad spectrum of biotechnological research. Each year international scientists come together in Bielefeld setting the focus on current innovations, approaches and methodologies. The issues so far included Solar Bio-Fuels(2008), bioIMAGING (2009) and New Frontiers in Microbial Genome Research (2010). The 6th CeBiTec Symposium was held under the topic Genome-Based Microbiology: From -omics Research to Systems and Synthetic Biology took place between the 18th and 20th of July, 2011. <a href="https://2011.igem.org/Team:Bielefeld-Germany/cebitec-symposium">read more</a><br />
</p><br />
</div><br />
<br />
<div><br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_lab_course"><img src="https://static.igem.org/mediawiki/2011/6/6a/Bielefeld-Germany2011-labcourse14.jpg" /></a> <br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_lab_course">GENIALE Open Lab Course</a></h3><br />
<br />
<p class="more"><br />
We organized a lab course and lab tour for over 150 children, adolescents and their parents.<br />
</p><br />
<br />
<p><br />
One of our goals is an objective science communication in the fields of biotechnology and especially synthetic biology. To improve and expand our outreach we had the opportunity to take part in the GENIALE. This is one of the biggest European science fairs with over 50,000 participants, funded by the European Union and the state of North Rhine Westphalia. The iGEM team Bielefeld took the chance to participate with an open lab course for all ages and an in depth science café in the evening. <a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_lab_course">read more</a><br />
</p><br />
</div><br />
<br />
<div><br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_science_cafe"><img src="https://static.igem.org/mediawiki/2011/8/8a/Bielefeld-Germany2011-science-cafe11.jpg" /></a> <br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_science_cafe">Science Cafe</a></h3><br />
<br />
<p class="more"><br />
We hosted a science café about synthetic biology, iGEM and our projects.<br />
</p><br />
<br />
<p><br />
The science café is an event organized by Bielefeld Marketing in a restaurant in downtown Bielefeld. In a comfortable setting we were able to give a presentation, followed by a moderated panel discussion with some team members and questions from the interested audience. Our goal was to show that we want to get in contact with the public, in particular present our project and get some feedback. <a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_science_cafe">read more</a><br />
</p><br />
</div><br />
<br />
<br />
<div><br />
<br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/student-convention"><img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/convention-big.png" /></a><br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/student-convention">Student Convention</a></h3><br />
<br />
<p class="more"><br />
The iGEM Team Bielefeld was honored with an invitation to the 2nd BIO.NRW (PhD) Student Convention at the BayArena in Leverkusen.<br />
</p><br />
<br />
<p><br />
The congress gives lifesience (PhD) students the opportunity to meet representatives of industry and academia, get information for career entry as well as improve their soft skills in workshops. Apart from the opportunity to participate in the convention, the role of the iGEM Team Bielefeld was to give a project presentation about iGEM as an example for remarkable scientific dedication during academic studies. The VIP-lounge in the stadium of the national league soccer team Bayer Leverkusen served as the venue for the two day congress. <a href="https://2011.igem.org/Team:Bielefeld-Germany/student-convention">read more</a><br />
</p><br />
<br />
</div><br />
<br />
<div><br />
<br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/high-school"><img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/school-big.png" /></a><br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/high-school">The iGEM Team Bielefeld at a local high school</a></h3><br />
<br />
<p class="more"><br />
Though synthetic biology (SB) is going to be the next big thing in biology, its researches are always raising public concerns respectivly are always in the focus of a public opinion.<br />
</p><br />
<br />
<p><br />
Therefore we got in contact with a local high school, in order to get an impression of the public awareness concerning synthetic biology. Hence we discussed with pupils about pros and cons of Synthetic Biology, iGEM and our projects as examples for applications. About 80 seniors from different biology and chemistry A-level courses came to the school's assembly hall and listened to our presentation, followed by questions. Afterwards the students were split into smaller groups of ten to twenty people and got accompanied by members of the Bielefeld iGEM team to discuss about iGEM and SB. <a href="https://2011.igem.org/Team:Bielefeld-Germany/high-school">read more</a><br />
</p><br />
<br />
</div><br />
<br />
<div> <br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/strategic-process"><img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/2020+-big.png" /></a><br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/strategic-process">Strategic Process</a></h3><br />
<br />
<p class="more"><br />
The German Federal Ministry of Education and Research manages the Strategic Process Biotechnology 2020+. <br />
</p><br />
<br />
<p><br />
Together with research organizations and universities the vision is to develop the next generation of biotechnological processes as well as initiate their realization. Apart from funding of research projects, expert talks on the topics medicine, energy, environment and industry are arranged. Every year a highlight congress is organized, where representatives from academia, industry and politics meet to discuss the future of biotechnology. This year, parts of the iGEM Team Bielefeld were invited to actively participate and present posters about the 2010 and 2011 projects. <a href="https://2011.igem.org/Team:Bielefeld-Germany/strategic-process">read more</a><br />
</p> <br />
<br />
</div><br />
<br />
<div> <br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/Black-box" ><img src="https://static.igem.org/mediawiki/2011/e/e6/Bielefeld-Germany2011-circuitboard-240px.png"></a><br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Black-box">Beacon Box</a></h3><br />
<br />
<p class="more"><br />
A prototype to show how the fluorescence would look like when using our cell-free bisphenol A biosensor system.<br />
</p><br />
<br />
<p><br />
We build this "Beacon Box" to show the audience at the Science Cafe presentation the fluorescence light which would be emitted by our cell-free bishphenol A biosensor system. It is a simple prototype which can easyly be build by everyone for home-use. To see the specifications and construction manual for our "Beacon Box" <a href="https://2011.igem.org/Team:Bielefeld-Germany/Black-box">read more</a><br />
</p> <br />
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</div><br />
<div> <br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Press"><img src="https://static.igem.org/mediawiki/2011/7/75/Bielefeld-Germany-2011-Timo-2020gross.jpg" /></a><br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Press">Press</a></h3><br />
<br />
<p class="more"><br />
A comprehensive list of all press articles about our iGEM team.<br />
</p><br />
<br />
<p><br />
This page gives a list of all news paper articles, internet news spots, radio feature, etc. about our iGEM team project. <a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Press">read more</a><br />
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<img src="https://static.igem.org/mediawiki/2011/7/72/Bielefeld-Germany-2011-Pana-kittel-klein.jpg"/><br />
<strong>Summary</strong><br />
<br />
Bielefeld, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#2"><br />
<img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/symposium-small.png"/><br />
<strong>Cebitec Symposium</strong><br />
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Bielefeld, Germany<br />
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<a href="#3"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c9/Bielefeld-Germany2011-labcourse15.jpg"/><br />
<strong>Open Lab Course</strong><br />
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Bielefeld, Germany<br />
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<a href="#4"><br />
<img src="https://static.igem.org/mediawiki/2011/d/d0/Bielefeld-Germany2011-science-cafe10.jpg"/><br />
<strong>Science Cafe</strong><br />
<br />
Bielefeld, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#5"><br />
<img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/convention-small.png"/><br />
<strong>Student Convention</strong><br />
Leverkusen, Germany<br />
</a><br />
<br />
</li><br />
<li><br />
<a href="#6"><br />
<img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/school-small.png" /><br />
<strong>High School</strong><br />
Herford, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#7"><br />
<img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/2020+-small.png" /><br />
<strong>Strategic Process</strong><br />
Berlin, Germany<br />
</a><br />
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<li><br />
<a href="#8"><br />
<img src="https://static.igem.org/mediawiki/2011/b/be/Bielefeld-Germany-2011-Beacon-Box-75.JPG" /><br />
<strong>Beacon Box</strong><br />
Bielefeld, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#9"><br />
<img src="https://static.igem.org/mediawiki/2011/0/03/Bielefeld-Germany-2011-Timo-Interview.geaendert.jpg" /><br />
<strong>Press</strong><br />
Bielefeld, Germany<br />
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</html></div>Panahttp://2011.igem.org/File:Bielefeld-Germany-2011-Science-Cafe-Titel.jpgFile:Bielefeld-Germany-2011-Science-Cafe-Titel.jpg2011-09-22T03:50:26Z<p>Pana: </p>
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<div></div>Panahttp://2011.igem.org/Team:Bielefeld-Germany/Public_relations_OverviewTeam:Bielefeld-Germany/Public relations Overview2011-09-22T03:50:08Z<p>Pana: </p>
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<a href="https://2011.igem.org/Team:Bielefeld-Germany/Public_relations_Summary"><img src="https://static.igem.org/mediawiki/2011/5/52/Bielefeld-Germany-2011-Science-Cafe-Titel.geaendert.jpg" /></a> <br />
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<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Public_relations_Summary">Summary</a></h3><br />
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<p class="more"><br />
This is an overview about our motivation and a general summary about our Human Practice Advance.<br />
</p><br />
<br />
<p><br />
Our goals are to awake the public awareness, start public discussions and participate in the outreach about iGEM. Further we want to promote the open source principle behind iGEM, arouse interest and hopefully prevented fear when facing the principles of synthetic biology. <a href="https://2011.igem.org/Team:Bielefeld-Germany/Public_relations_Summary">read more</a><br />
</p><br />
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<div><br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/cebitec-symposium"><img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/symposium-big.png" /></a> <br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/cebitec-symposium">Cebitec Symposium</a></h3><br />
<br />
<p class="more"><br />
European iGEM teams meet in Bielefeld, Germany. A review on the 6th CeBiTec Symposium Genome-Based Microbiology: From -omics Research to Systems and Synthetic Biology<br />
</p><br />
<br />
<p><br />
The annual CeBiTec Symposium, first held in 2006, deals with prospective topics in the broad spectrum of biotechnological research. Each year international scientists come together in Bielefeld setting the focus on current innovations, approaches and methodologies. The issues so far included Solar Bio-Fuels(2008), bioIMAGING (2009) and New Frontiers in Microbial Genome Research (2010). The 6th CeBiTec Symposium was held under the topic Genome-Based Microbiology: From -omics Research to Systems and Synthetic Biology took place between the 18th and 20th of July, 2011. <a href="https://2011.igem.org/Team:Bielefeld-Germany/cebitec-symposium">read more</a><br />
</p><br />
</div><br />
<br />
<div><br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_lab_course"><img src="https://static.igem.org/mediawiki/2011/6/6a/Bielefeld-Germany2011-labcourse14.jpg" /></a> <br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_lab_course">GENIALE Open Lab Course</a></h3><br />
<br />
<p class="more"><br />
We organized a lab course and lab tour for over 150 children, adolescents and their parents.<br />
</p><br />
<br />
<p><br />
One of our goals is an objective science communication in the fields of biotechnology and especially synthetic biology. To improve and expand our outreach we had the opportunity to take part in the GENIALE. This is one of the biggest European science fairs with over 50,000 participants, funded by the European Union and the state of North Rhine Westphalia. The iGEM team Bielefeld took the chance to participate with an open lab course for all ages and an in depth science café in the evening. <a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_lab_course">read more</a><br />
</p><br />
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<br />
<div><br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_science_cafe"><img src="https://static.igem.org/mediawiki/2011/8/8a/Bielefeld-Germany2011-science-cafe11.jpg" /></a> <br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_science_cafe">Science Cafe</a></h3><br />
<br />
<p class="more"><br />
We hosted a science café about synthetic biology, iGEM and our projects.<br />
</p><br />
<br />
<p><br />
The science café is an event organized by Bielefeld Marketing in a restaurant in downtown Bielefeld. In a comfortable setting we were able to give a presentation, followed by a moderated panel discussion with some team members and questions from the interested audience. Our goal was to show that we want to get in contact with the public, in particular present our project and get some feedback. <a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Geniale_science_cafe">read more</a><br />
</p><br />
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<br />
<div><br />
<br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/student-convention"><img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/convention-big.png" /></a><br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/student-convention">Student Convention</a></h3><br />
<br />
<p class="more"><br />
The iGEM Team Bielefeld was honored with an invitation to the 2nd BIO.NRW (PhD) Student Convention at the BayArena in Leverkusen.<br />
</p><br />
<br />
<p><br />
The congress gives lifesience (PhD) students the opportunity to meet representatives of industry and academia, get information for career entry as well as improve their soft skills in workshops. Apart from the opportunity to participate in the convention, the role of the iGEM Team Bielefeld was to give a project presentation about iGEM as an example for remarkable scientific dedication during academic studies. The VIP-lounge in the stadium of the national league soccer team Bayer Leverkusen served as the venue for the two day congress. <a href="https://2011.igem.org/Team:Bielefeld-Germany/student-convention">read more</a><br />
</p><br />
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</div><br />
<br />
<div><br />
<br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/high-school"><img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/school-big.png" /></a><br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/high-school">The iGEM Team Bielefeld at a local high school</a></h3><br />
<br />
<p class="more"><br />
Though synthetic biology (SB) is going to be the next big thing in biology, its researches are always raising public concerns respectivly are always in the focus of a public opinion.<br />
</p><br />
<br />
<p><br />
Therefore we got in contact with a local high school, in order to get an impression of the public awareness concerning synthetic biology. Hence we discussed with pupils about pros and cons of Synthetic Biology, iGEM and our projects as examples for applications. About 80 seniors from different biology and chemistry A-level courses came to the school's assembly hall and listened to our presentation, followed by questions. Afterwards the students were split into smaller groups of ten to twenty people and got accompanied by members of the Bielefeld iGEM team to discuss about iGEM and SB. <a href="https://2011.igem.org/Team:Bielefeld-Germany/high-school">read more</a><br />
</p><br />
<br />
</div><br />
<br />
<div> <br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/strategic-process"><img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/2020+-big.png" /></a><br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/strategic-process">Strategic Process</a></h3><br />
<br />
<p class="more"><br />
The German Federal Ministry of Education and Research manages the Strategic Process Biotechnology 2020+. <br />
</p><br />
<br />
<p><br />
Together with research organizations and universities the vision is to develop the next generation of biotechnological processes as well as initiate their realization. Apart from funding of research projects, expert talks on the topics medicine, energy, environment and industry are arranged. Every year a highlight congress is organized, where representatives from academia, industry and politics meet to discuss the future of biotechnology. This year, parts of the iGEM Team Bielefeld were invited to actively participate and present posters about the 2010 and 2011 projects. <a href="https://2011.igem.org/Team:Bielefeld-Germany/strategic-process">read more</a><br />
</p> <br />
<br />
</div><br />
<br />
<div> <br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/Black-box" ><img src="https://static.igem.org/mediawiki/2011/e/e6/Bielefeld-Germany2011-circuitboard-240px.png"></a><br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Black-box">Beacon Box</a></h3><br />
<br />
<p class="more"><br />
A prototype to show how the fluorescence would look like when using our cell-free bisphenol A biosensor system.<br />
</p><br />
<br />
<p><br />
We build this "Beacon Box" to show the audience at the Science Cafe presentation the fluorescence light which would be emitted by our cell-free bishphenol A biosensor system. It is a simple prototype which can easyly be build by everyone for home-use. To see the specifications and construction manual for our "Beacon Box" <a href="https://2011.igem.org/Team:Bielefeld-Germany/Black-box">read more</a><br />
</p> <br />
<br />
</div><br />
<div> <br />
<a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Press"><img src="https://static.igem.org/mediawiki/2011/7/75/Bielefeld-Germany-2011-Timo-2020gross.jpg" /></a><br />
<br />
<h3><a style="text-decoration:none; color:black;" href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Press">Press</a></h3><br />
<br />
<p class="more"><br />
A comprehensive list of all press articles about our iGEM team.<br />
</p><br />
<br />
<p><br />
This page gives a list of all news paper articles, internet news spots, radio feature, etc. about our iGEM team project. <a href="https://2011.igem.org/Team:Bielefeld-Germany/Human_Practices/Press">read more</a><br />
</p> <br />
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<a href="#1"><br />
<img src="https://static.igem.org/mediawiki/2011/7/72/Bielefeld-Germany-2011-Pana-kittel-klein.jpg"/><br />
<strong>Summary</strong><br />
<br />
Bielefeld, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#2"><br />
<img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/symposium-small.png"/><br />
<strong>Cebitec Symposium</strong><br />
<br />
Bielefeld, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#3"><br />
<img src="https://static.igem.org/mediawiki/2011/c/c9/Bielefeld-Germany2011-labcourse15.jpg"/><br />
<strong>Open Lab Course</strong><br />
<br />
Bielefeld, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#4"><br />
<img src="https://static.igem.org/mediawiki/2011/d/d0/Bielefeld-Germany2011-science-cafe10.jpg"/><br />
<strong>Science Cafe</strong><br />
<br />
Bielefeld, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#5"><br />
<img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/convention-small.png"/><br />
<strong>Student Convention</strong><br />
Leverkusen, Germany<br />
</a><br />
<br />
</li><br />
<li><br />
<a href="#6"><br />
<img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/school-small.png" /><br />
<strong>High School</strong><br />
Herford, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#7"><br />
<img src="http://2011.igem-bielefeld.de/includes/wiki/overview-img/2020+-small.png" /><br />
<strong>Strategic Process</strong><br />
Berlin, Germany<br />
</a><br />
</li><br />
<li><br />
<a href="#8"><br />
<img src="https://static.igem.org/mediawiki/2011/b/be/Bielefeld-Germany-2011-Beacon-Box-75.JPG" /><br />
<strong>Beacon Box</strong><br />
Bielefeld, Germany<br />
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<a href="#9"><br />
<img src="https://static.igem.org/mediawiki/2011/0/03/Bielefeld-Germany-2011-Timo-Interview.geaendert.jpg" /><br />
<strong>Press</strong><br />
Bielefeld, Germany<br />
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<hr />
<div></div>Panahttp://2011.igem.org/Team:Bielefeld-Germany/StudentsTeam:Bielefeld-Germany/Students2011-09-22T03:37:42Z<p>Pana: </p>
<hr />
<div>{{Bielefeld_2011_Header}}<br />
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<img id="Image-Maps_9201109211806015" src="https://static.igem.org/mediawiki/2011/4/4d/Bielefeld-Germany2011-team-900px.jpeg" usemap="#Image-Maps_9201109211806015" border="0" width="900" height="575" alt="" /><br />
<map id="_Image-Maps_9201109211806015" name="Image-Maps_9201109211806015"><br />
<area shape="rect" coords="50,177,130,265" href="#" alt="Simon Schäper" title="Simon Schäper" /><br />
<area shape="rect" coords="160,182,240,270" href="#" alt="Panagiotis Papavasiliou" title="Panagiotis Papavasiliou" /><br />
<area shape="rect" coords="252,155,310,229" href="#" alt="Michael Limberg" title="Michael Limberg" /><br />
<area shape="rect" coords="298,231,364,300" href="#" alt="Katharina Thiedig" title="Katharina Thiedig" /><br />
<area shape="rect" coords="382,175,429,256" href="#" alt="Timo Wolf" title="Timo Wolf" /><br />
<area shape="rect" coords="430,233,476,314" href="#" alt="Anna Drong" title="Anna Drong" /><br />
<area shape="rect" coords="510,193,583,274" href="#" alt="Manuel Wittchen" title="Manuel Wittchen" /><br />
<area shape="rect" coords="596,187,662,273" href="#" alt="Jonas Aretz" title="Jonas Aretz" /><br />
<area shape="rect" coords="667,186,723,261" href="#" alt="Robert Braun" title="Robert Braun" /><br />
<area shape="rect" coords="744,171,796,246" href="#" alt="Matthias Eder" title="Matthias Eder" /><br />
<area shape="rect" coords="801,179,853,254" href="#" alt="Jan Schwarzhans" title="Jan Schwarzhans" /><br />
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<br />
In 2011 a student command unit locked themselves in the Bielefeld University for the construction of cell-free biosensors. They promptly identified S-layer proteins as nanobiotechnological building blocks. Today still characterizing their Bisphenol A Biosensor they are the iGEM team Bielefeld. If your cells are a problem, if no one else can help and if you can find them, maybe you can hire the Bisphenol A-Team. <br />
<br />
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<img src="https://static.igem.org/mediawiki/2011/e/ed/Bielefeld-Germany-2011-Jonas.jpg" alt="Jonas Aretz" /><br />
<h3>Jonas Aretz<br/></h3><br />
Studies: Molecular Biotechnology<br />
</div><br />
<div class="supervisor"><br />
<img src="https://static.igem.org/mediawiki/2011/0/04/Bielefeld-Germany-2011-Robert.jpg" alt="Robert Braun" /><br />
<h3>Robert Braun<br/></h3><br />
Studies: Molecular Biotechnology<br />
</div><br />
<br />
<div class="supervisor"><br />
<img src="https://static.igem.org/mediawiki/2011/e/eb/Bielefeld-Germany-2011-Anna.jpg" alt="Anna Drong" /><br />
<h3>Anna Drong<br/></h3><br />
Studies: Molecular Biotechnology<br />
</div><br />
<br />
<div class="supervisor"><br />
<img src="https://static.igem.org/mediawiki/2011/f/f1/Bielefeld-Germany-2011-Matthias.jpg" alt="Matthias Eder" /><br />
<h3>Matthias Eder<br/></h3><br />
Studies: Molecular Biotechnology<br />
</div><br />
<br />
<div class="supervisor"><br />
<img src="https://static.igem.org/mediawiki/2011/a/ae/Bielefeld-Germany-2011-Michael.jpg" alt="Michael Limberg" /><br />
<h3>Michael Limberg<br/></h3><br />
Studies: Molecular Biotechnology<br />
</div><br />
<br />
<div class="supervisor"><br />
<img src="https://static.igem.org/mediawiki/2011/4/4d/Bielefeld-Germany-2011-Pana.jpg" alt="Panagiotis Papavasiliou" /><br />
<h3>Panagiotis Papavasiliou<br/></h3><br />
Studies: Genome Based Systems Biology<br />
</div><br />
<br />
<br />
<div class="supervisor"><br />
<img src="https://static.igem.org/mediawiki/2011/a/a2/Bielefeld-Germany-2011-Simon.jpg" alt="Simon Sch&auml;per" /><br />
<h3>Simon Sch&auml;per<br/></h3><br />
Studies: Genome Based Systems Biology<br />
</div><br />
<br />
<div class="supervisor"><br />
<img src="https://static.igem.org/mediawiki/2011/9/99/Bielefeld-Germany-2011-Jan.jpg" alt="Jan Schwarzhans" /><br />
<h3>Jan Schwarzhans<br/></h3><br />
Studies: Molecular Biotechnology<br />
<br />
<br />
</div><br />
<br />
<div class="supervisor"><br />
<img src="https://static.igem.org/mediawiki/2011/2/2c/Bielefeld-Germany-2011-Kathi.jpg" alt="Katharina Thiedig" /><br />
<h3>Katharina Thiedig<br/></h3><br />
Studies: Genome Based Systems Biology<br />
</div><br />
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<div class="supervisor"><br />
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<h3>Manuel Wittchen<br/></h3><br />
Studies: Molecular Biotechnology<br />
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<h3>Timo Wolf<br/></h3><br />
Studies: Molecular Biotechnology<br />
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<a style="font-size:15px; color:#3baeff;" href="https://2011.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Photos/Symposium">CeBiTec Symposium</a><br />
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<a style="font-size:15px; color:#3baeff;" href="https://2011.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Photos/School">School presentation</a><br />
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<a style="font-size:15px; color:#3baeff;" href="https://2011.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Photos/Strategieprozess">Strategieprozess 2020+</a><br />
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<a style="font-size:15px; color:#3baeff;" href="https://2011.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Photos/Lab-Tour">Geniale Laboratory Tour</a><br />
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<a style="font-size:15px; color:#3baeff;" href="https://2011.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Photos/Symposium">CeBiTec Symposium</a><br />
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<a style="font-size:15px; color:#3baeff;" href="https://2011.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Photos/School">School presentation</a><br />
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<a style="font-size:15px; color:#3baeff;" href="https://2011.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Photos/Strategieprozess">Strategieprozess 2020+</a><br />
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<a style="font-size:15px; color:#3baeff;" href="https://2011.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Photos/Pizza">Making Pizza</a><br />
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<a style="font-size:15px; color:#3baeff;" href="https://2011.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Photos/Studentconvention">PhD Studentconvention</a><br />
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<a style="font-size:15px; color:#3baeff;" href="https://2011.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Photos/BBQ">CeBiTec BBQ</a><br />
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<a style="font-size:15px; color:#3baeff;" href="https://2011.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Photos/Fire">Fire Drill</a><br />
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