http://2013.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Jakobmatthes&year=&month=2013.igem.org - User contributions [en]2024-03-28T13:37:18ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:Tuebingen/Results/OverviewTeam:Tuebingen/Results/Overview2013-10-05T03:45:25Z<p>Jakobmatthes: </p>
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Results<br />
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<h3>Cloning</h3><br />
<p>This year, we succeeded in cloning all missing parts required for our measuring system. While mPR Xl, mPR Dr, mig1, Padh1 and rox1 were available in pUC-IDT vector or pGEM T-easy vector from <a href="https://2012.igem.org/Team:Tuebingen">last year's iGEM-Team Tuebingen</a>, Pfet3, Panb1, Psuc2 were cloned directly from yeast genome. Furthermore we used the fluorescent reporter protein mOrange (<a href="http://parts.igem.org/Part:BBa_E2050">BBa_E2050</a>) and the transcriptional terminator Tadh (<a href="http://parts.igem.org/Part:BBa_K801012">BBa_K801012</a>) from the parts registry. A galactose inducible promotor (<a href="http://parts.igem.org/Part:BBa_J63006">BBa_J63006</a>) from the registry was used to create some plasmids which we want to use for the characterization of some of our parts. The cloning of luciferase succeeded only shortly before wiki freeze. As a result, the assemblies requiring luciferase were not performed anymore.</p><br />
<br />
<p>&nbsp;</p><br />
<br />
<h3>Assembly</h3><br />
<p>Our general assembly strategy is illustrated in fig. 1. The pRS vectors do not fit the RFC10 criteria and can therefore not be used the for 3A-Assembly. Instead we used the BioBrick RFC10 vector pTUM100 (<a href="http://parts.igem.org/Part:BBa_K801000">BBa_K801000</a>) which was kindly provided by the iGEM Team of <a href="https://2013.igem.org/Team:TU-Munich">TU Munich</a> in order to fuse promoter and coding sequence. pTUM100 is a high copy shuttle plasmid which is based on the commercially available pYES2 vector and contains the cyc1 transcription terminator after the BioBrick suffix. Thus we could use some of the pTUM constructs directly for characterization of our repressible promoters (Pfet3-mOrange-pTUM100, Panb1-mOrange-pTUM100, Psuc2-mOrange-pTUM100, Padh1-mOrange-pTUM100). The parts required for the next steps of characterization were equipped with a the transcriptional terminator Tadh by ligation into a pSB1C3 plasmid containing Tadh. The resulting assemblies were again BioBricks. These inserts were then ligated into the pRS shuttle vectors losing parts of their prefix and/or suffix. In total we have created 29 new plasmids!</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/1/19/TueAssemblyStrategy.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 1</b>: General assembly scheme: First, promoter and coding sequence were assembled in pTUM100 using the 3A-Assembly protocol. Constructs with the reporter mOrange were used for characterization directly. Subsequently, the promotor and the fused coding sequence are ligated into a pSB1C3 plasmid with the transcriptional terminator Tadh1. The resulting assemblies are again BioBricks. These inserts are then ligated into the pRS shuttle vectors losing parts of their prefix and/or suffix.</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<h3>Readout</h3><br />
<p>For the characterization of our repressible promoters Pfet3, Panb1 and Psuc2 we transformed pTUM100 constructs with mOrange under the control of the promoter of interest in the laboratory <a href="http://wiki.yeastgenome.org/index.php/Commonly_used_strains">yeast strain w303</a>. We used the constitutive promoter Padh1 (sequence identical with <a href="http://parts.igem.org/Part:BBa_J63005">BBa_J63005</a>) as positive control. The promoter activity was analyzed qualitatively using fluorescence microscopy (fig. 2).</p><br />
<br />
<div style="padding: 10px; height: auto; width: 510px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/7/71/TueNurKontrollen.png" style="position: relative; width: 500px; margin: 5px 5px 10px 5px;"><b>Fig. 2</b>: Detection of mOrange expression in the fluorescence microscope. Non-transformed yeast was used as negative control. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).</div><br />
<br />
<h3>&nbsp;</h3><br />
<br />
<p>Next, we established a protocol for quantitative read-out of mOrange fluorescence using the plate reader. We recorded an excitation spectrum of non-transformed yeast and yeast expressing mOrange measuring the emission at 581nm (+/- 25nm). Fig. 3 compares the excitation spectra with the ideal excitation spectrum of mOrange. While the fluorescence decreases with higher excitation wavelength for non-transformed yeast, the spectrum of yeast expressing mOrange has a similar shape than the ideal excitation spectrum.</p><br />
<br />
<h3>&nbsp;</h3><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/2/27/TueSpektren.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 3</b>: Excitation spectra of non-transformed w303 yeast (neg. control) and yeast expressing mOrange. Both spectra were recorded in the plate reader (Ex: 400nm-550nm +/-9nm, Em: 581nm +/-20nm). The excitation spectrum of mOrange is indicated by a dashed line (source: <a href="http://www.tsienlab.ucsd.edu/Documents/REF%20-%20Fluorophore%20Spectra.xls">http://www.tsienlab.ucsd.edu/Documents/REF%20-%20Fluorophore%20Spectra.xls</a>).<br />
</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<h3>Repressible Promotors</h3><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/6/6f/TueWt_Staemme.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 4</b>: Basal expression level of mOrange under the control of the promotors Pfet3, Panb1 and Psuc2. Non-transformed yeast was used as negative control. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).<br />
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<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/4/40/TueAll_strains.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 5</b>: Basal expression level of mOrange under the control of the promotors Pfet3, Panb1 and Psuc2. Non-transformed yeast was used as negative control. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as triplicates.<br />
</div><br />
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<p>&nbsp;</p><br />
<h3>Pfet3</h3><br />
<br />
<p>Before we could observe the regulation of Pfet3 through the co-expressed membrane-bound progestin receptors, we needed to investigated the native function of Pfet3 which is the response to intracellular iron concentration. Therefore we grew yeast containing the Pfet-mOrange-pTUM100 construct in a low-ion environment (Low-iron medium (LIM) contained 1 mM EDTA). The promoter activity of Pfet3 had increased after 4 h of incubation in low-iron medium compared to cells which were grown in normal synthetic complete medium (fig. 6). After 8 h of incubation in LIM the promoter activity had increased 3-fold. The dependence of Pfet3 activity on iron allows modifying the expression level of the repressors mig1 and rox1. This is desirable as sensitivity analysis has shown that the maximal repressor concentration is crucial for the functionality of the measurement system. Nevertheless, this finding brings along new challenges as we need to maintain Pfet3 activity constant to avoid an undesired influence on the measurement.</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/d/dc/TueLIM_Induction.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 6</b>: Alterations of mOrange expression in low-iron environment of w303 containing Pfet3-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates. </div><br />
<br />
<p>&nbsp;</p><br />
<h3>Panb1 and Psuc2</h3><br />
<br />
<p>First, we transformed the constructs Psuc2-mOrange-pTUM100 and Panb1-mOrange-pTUM100 in w303. The promoter activity was analysed in fluorescence microscopy and using the plate reader. The activity equals 3.8% of Padh1 activity for Psuc2 and 1.9% of Padh1 activity for Panb1. The maximal activity of these promotors also limits the maximal reporter expression and therefore also the sensitivity of the measurement system. Thus, a higher promoter activity was desirable.</p><br />
<br />
<p>Next, we investigated which other factors influence the promoter activity, especially the intrinsic repressor level. Therefore, we transformed the same constructs in mutant yeast strains with deficient rox1 (YPR065W) or mig1 (systematic gene name: YGL035C), see fig. 7 + 8. The promoter activity was enhanced by a factor of 2.6 for Psuc2 and 13.8 for Panb1. We therefore intend to implement our system in these deficient strains allowing higher reporter expression levels. Furthermore, the function of MIG1 in glucose repression is well described (Santangelo, 2006). We observed an increase in promotor activity upon changing the medium from SC-Ura (2% glucose) to SC-Ura (2% galactose) for wt as well as the Mig deficient strain (fig. 9 + 10). This effect has to be considered if we intend to use Psuc2 together with a GAL-promotoer in further characterization.<br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/d/d5/TueDefiziente_staemme.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 7</b>: Increased promoter activity of Psuc2 and Panb1 in repressor deficient strains. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/8/83/TuePanb_Psuc_deficient_strains.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 8</b>: Promoter activity of Psuc2 and Panb1 in repressor deficient strains. The promoter activity was enhanced by a factor of 2.6 for Psuc2 and 13.8 for Panb1. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as triplicates.</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/e/ee/TuePsuc_mO_GALinduction.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 9</b>: Alterations of mOrange expression upon galactose induction of <b>w303</b> containing Psuc2-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates.</div><br />
<br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/7/74/TueGALinduction_Psuc_mig.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 10</b>: Alterations of mOrange expression upon galactose induction of <b>mig1 deficient yeast</b> containing Psuc2-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates.</div><br />
<br />
<br />
<p>&nbsp;</p><br />
<h3>References</h3><br />
<p>SANTANGELO, G. M. 2006. Glucose signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev, 70, 253-82.</p><br />
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{{:Team:Tuebingen/Template/equalHeight}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Results/OverviewTeam:Tuebingen/Results/Overview2013-10-05T03:20:04Z<p>Jakobmatthes: </p>
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Results<br />
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<h3>Cloning</h3><br />
<p>This year, we succeeded in cloning all missing parts required for our measuring system. While mPR Xl, mPR Dr, mig1, Padh1 and rox1 were available in pUC-IDT vector or pGEM T-easy vector from <a href="https://2012.igem.org/Team:Tuebingen">last year's iGEM-Team Tuebingen</a>, Pfet3, Panb1, Psuc2 were cloned directly from yeast genome. Furthermore we used the fluorescent reporter protein mOrange (<a href="http://parts.igem.org/Part:BBa_E2050">BBa_E2050</a>) and the transcriptional terminator Tadh (<a href="http://parts.igem.org/Part:BBa_K801012">BBa_K801012</a>) from the parts registry. A galactose inducible promotor (<a href="http://parts.igem.org/Part:BBa_J63006">BBa_J63006</a>) from the registry was used to create some plasmids which we want to use for the characterization of some of our parts. The cloning of luciferase succeeded only shortly before wiki freeze. As a result, the assemblies requiring luciferase were not performed anymore.</p><br />
<br />
<p>&nbsp;</p><br />
<br />
<h3>Assembly</h3><br />
<p>Our general assembly strategy is illustrated in fig. 1. The pRS vectors do not fit the RFC10 criteria and can therefore not be used the for 3A-Assembly. Instead we used the BioBrick RFC10 vector pTUM100 (<a href="http://parts.igem.org/Part:BBa_K801000">BBa_K801000</a>) which was kindly provided by the iGEM Team of <a href="https://2013.igem.org/Team:TU-Munich">TU Munich</a> in order to fuse promoter and coding sequence. pTUM100 is a high copy shuttle plasmid which is based on the commercially available pYES2 vector and contains the cyc1 transcription terminator after the BioBrick suffix. Thus we could use some of the pTUM constructs directly for characterization of our repressible promoters (Pfet3-mOrange-pTUM100, Panb1-mOrange-pTUM100, Psuc2-mOrange-pTUM100, Padh1-mOrange-pTUM100). The parts required for the next steps of characterization were equipped with a the transcriptional terminator Tadh by ligation into a pSB1C3 plasmid containing Tadh. The resulting assemblies were again BioBricks. These inserts were then ligated into the pRS shuttle vectors losing parts of their prefix and/or suffix. In total we have created 29 new plasmids!</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/1/19/TueAssemblyStrategy.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 1</b>: General assembly scheme: First, promoter and coding sequence were assembled in pTUM100 using the 3A-Assembly protocol. Constructs with the reporter mOrange were used for characterization directly. Subsequently, the promotor and the fused coding sequence are ligated into a pSB1C3 plasmid with the transcriptional terminator Tadh1. The resulting assemblies are again BioBricks. These inserts are then ligated into the pRS shuttle vectors losing parts of their prefix and/or suffix.</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<h3>Readout</h3><br />
<p>For the characterization of our repressible promoters Pfet3, Panb1 and Psuc2 we transformed pTUM100 constructs with mOrange under the control of the promoter of interest in the laboratory <a href="http://wiki.yeastgenome.org/index.php/Commonly_used_strains">yeast strain w303</a>. We used the constitutive promoter Padh1 (sequence identical with <a href="http://parts.igem.org/Part:BBa_J63005">BBa_J63005</a>) as positive control. The promoter activity was analyzed qualitatively using fluorescence microscopy (fig. 2).</p><br />
<br />
<div style="padding: 10px; height: auto; width: 510px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/7/71/TueNurKontrollen.png" style="position: relative; width: 500px; margin: 5px 5px 10px 5px;"><b>Fig. 2</b>: Detection of mOrange expression in the fluorescence microscope. Non-transformed yeast was used as negative control. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).</div><br />
<br />
<h3>&nbsp;</h3><br />
<br />
<p>Next, we established a protocol for quantitative read-out of mOrange fluorescence using the plate reader. We recorded an excitation spectrum of non-transformed yeast and yeast expressing mOrange measuring the emission at 581nm (+/- 25nm). Fig. 3 compares the excitation spectra with the ideal excitation spectrum of mOrange. While the fluorescence decreases with higher excitation wavelength for non-transformed yeast, the spectrum of yeast expressing mOrange has a similar shape than the ideal excitation spectrum.</p><br />
<br />
<h3>&nbsp;</h3><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/2/27/TueSpektren.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 3</b>: Excitation spectra of non-transformed w303 yeast (neg. control) and yeast expressing mOrange. Both spectra were recorded in the plate reader (Ex: 400nm-550nm +/-9nm, Em: 581nm +/-20nm). The excitation spectrum of mOrange is indicated by a dashed line (source: <a href="http://www.tsienlab.ucsd.edu/Documents/REF%20-%20Fluorophore%20Spectra.xls">http://www.tsienlab.ucsd.edu/Documents/REF%20-%20Fluorophore%20Spectra.xls</a>).<br />
</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<h3>Repressible Promotors</h3><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/6/6f/TueWt_Staemme.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 4</b>: Basal expression level of mOrange under the control of the promotors Pfet3, Panb1 and Psuc2. Non-transformed yeast was used as negative control. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).<br />
</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/4/40/TueAll_strains.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 5</b>: Basal expression level of mOrange under the control of the promotors Pfet3, Panb1 and Psuc2. Non-transformed yeast was used as negative control. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as triplicates.<br />
</div><br />
<br />
<p>&nbsp;</p><br />
<h3>Pfet3</h3><br />
<br />
<p>Before we could observe the regulation of Pfet3 through the co-expressed membrane-bound progestin receptors, we needed to investigated the native function of Pfet3 which is the response to intracellular iron concentration. Therefore we grew yeast containing the Pfet-mOrange-pTUM100 construct in a low-ion environment (Low-iron medium (LIM) contained 1 mM EDTA). The promoter activity of Pfet3 had increased after 4 h of incubation in low-iron medium compared to cells which were grown in normal synthetic complete medium (fig. 6). After 8 h of incubation in LIM the promoter activity had increased 3-fold. The dependence of Pfet3 activity on iron allows modifying the expression level of the repressors mig1 and rox1. This is desirable as sensitivity analysis has shown that the maximal repressor concentration is crucial for the functionality of the measurement system. Nevertheless, this finding brings along new challenges as we need to maintain Pfet3 activity constant to avoid an undesired influence on the measurement.</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/d/dc/TueLIM_Induction.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 6</b>: Alterations of mOrange expression in low-iron environment of w303 containing Pfet3-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates. </div><br />
<br />
<p>&nbsp;</p><br />
<h3>Panb1 and Psuc2</h3><br />
<br />
<p>First, we transformed the constructs Psuc2-mOrange-pTUM100 and Panb1-mOrange-pTUM100 in w303. The promoter activity was analysed in fluorescence microscopy and using the plate reader. The activity equals 3.8% of Padh1 activity for Psuc2 and 1.9% of Padh1 activity for Panb1. The maximal activity of these promotors also limits the maximal reporter expression and therefore also the sensitivity of the measurement system. Thus, a higher promoter activity was desirable.</p><br />
<br />
<p>Next, we investigated which other factors influence the promoter activity, especially the intrinsic repressor level. Therefore, we transformed the same constructs in mutant yeast strains with deficient rox1 (YPR065W) or mig1 (systematic gene name: YGL035C), see fig. 7 + 8. The promoter activity was enhanced by a factor of 2.6 for Psuc2 and 13.8 for Panb1. We therefore intend to implement our system in these deficient strains allowing higher reporter expression levels. Furthermore, the function of MIG1 in glucose repression is well described (Santangelo, 2006). We observed an increase in promotor activity upon changing the medium from SC-Ura (2% glucose) to SC-Ura (2% galactose) for wt as well as the Mig deficient strain (fig. 9 + 10). This effect has to be considered if we intend to use Psuc2 together with a GAL-promotoer in further characterization.<br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/d/d5/TueDefiziente_staemme.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 7</b>: Increased promoter activity of Psuc and Panb in repressor deficient strains. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/8/83/TuePanb_Psuc_deficient_strains.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 8</b>: Promoter activity of Psuc and Panb in repressor deficient strains. The promoter activity was enhanced by a factor of 2.6 for Psuc and 13.8 for Panb. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as triplicates.</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/e/ee/TuePsuc_mO_GALinduction.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 9</b>: Alterations of mOrange expression upon galactose induction of <b>w303</b> containing Psuc2-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates.</div><br />
<br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/7/74/TueGALinduction_Psuc_mig.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 10</b>: Alterations of mOrange expression upon galactose induction of <b>mig1 deficient yeast</b> containing Psuc2-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates.</div><br />
<br />
<br />
<p>&nbsp;</p><br />
<h3>References</h3><br />
<p>SANTANGELO, G. M. 2006. Glucose signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev, 70, 253-82.</p><br />
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{{:Team:Tuebingen/Template/equalHeight}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Results/OverviewTeam:Tuebingen/Results/Overview2013-10-05T02:42:42Z<p>Jakobmatthes: </p>
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Results<br />
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<h3>Cloning</h3><br />
<p>This year, we succeeded in cloning all missing parts required for our measuring system. While mPR Xl, mPR Dr, mig1, Padh1 and rox1 were available in pUC-IDT vector or pGEM T-easy vector from <a href="https://2012.igem.org/Team:Tuebingen">last year's iGEM-Team Tuebingen</a>, Pfet3, Panb1, Psuc2 were cloned directly from yeast genome. Furthermore we used the fluorescent reporter protein mOrange (<a href="http://parts.igem.org/Part:BBa_E2050">BBa_E2050</a>) and the transcriptional terminator Tadh (<a href="http://parts.igem.org/Part:BBa_K801012">BBa_K801012</a>) from the parts registry. A galactose inducible promotor (<a href="http://parts.igem.org/Part:BBa_J63006">BBa_J63006</a>) from the registry was used to create some plasmids which we want to use for the characterization of some of our parts. The cloning of luciferase succeeded only shortly before wiki freeze. As a result, the assemblies requiring luciferase were not performed anymore.</p><br />
<br />
<p>&nbsp;</p><br />
<br />
<h3>Assembly</h3><br />
<p>Our general assembly strategy is illustrated in fig. 1. The pRS vectors do not fit the RFC10 criteria and can therefore not be used the for 3A-Assembly. Instead we used the BioBrick RFC10 vector pTUM100 (<a href="http://parts.igem.org/Part:BBa_K801000">BBa_K801000</a>) which was kindly provided by the iGEM Team of the <a href="https://2013.igem.org/Team:TU-Munich">TU Munich</a> to fuse promotor and coding sequence. pTUM100 is a high copy shuttle plasmid which is based on the commercially available pYES2 vector and contains the cyc1 transcription terminator after the BioBrick suffix. Thus we could use some of the pTUM constructs directly for characterization of our repressible promotors (Pfet3-mOrange-pTUM100, Panb1-mOrange-pTUM100, Psuc2-mOrange-pTUM100, Padh1-mOrange-pTUM100). The parts required for the next steps of characterization were equipped with a the transcriptional terminator Tadh by ligation into a pSB1C3 plasmid containing Tadh. The resulting assemblies were again BioBricks. These inserts were then ligated into the pRS shuttle vectors losing parts of their prefix and/or suffix. In total we have created 29 new plasmids!</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/1/19/TueAssemblyStrategy.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 1</b>: General assembly scheme: First, promoter and coding sequence were assembled in pTUM100 using the 3A-Assembly protocol. Constructs with the reporter mOrange were used for characterization directly. Subsequently, the promotor and the fused coding sequence are ligated into a pSB1C3 plasmid with the transcriptional terminator Tadh1. The resulting assemblies are again BioBricks. These inserts are then ligated into the pRS shuttle vectors losing parts of their prefix and/or suffix.</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<h3>Readout</h3><br />
<p>For the characterization of our repressible promotors Pfet3, Panb1 and Psuc2 we transformed pTUM100 constructs with mOrange under the control of the promotor of interest in the laboratory <a href="http://wiki.yeastgenome.org/index.php/Commonly_used_strains">yeast strain w303</a>. We used the constitutive promotor Padh1 (sequence identical with <a href="http://parts.igem.org/Part:BBa_J63005">BBa_J63005</a>) as positive control. The promoter activity was analysed qualitatively using fluorescence microscopy (fig. 2).</p><br />
<br />
<div style="padding: 10px; height: auto; width: 510px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/7/71/TueNurKontrollen.png" style="position: relative; width: 500px; margin: 5px 5px 10px 5px;"><b>Fig. 2</b>: Detection of mOrange expression in the fluorescence microscope. Non-transformed yeast was used as negative control. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).</div><br />
<br />
<h3>&nbsp;</h3><br />
<br />
<p>Next, we established a protocol for quantitative read-out of mOrange fluorescence using the plate reader. We recorded an excitation spectrum of non-transformed yeast and yeast expressing mOrange measuring the emission at 581nm (+/- 25nm). Fig. 3 compares the excitation spectra with the ideal excitation spectrum of mOrange. While the fluorescence decreases with higher excitation wavelength for non-transformed yeast, the spectrum of yeast expressing mOrange has a similar shape than the ideal excitation spectrum.</p><br />
<br />
<h3>&nbsp;</h3><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/2/27/TueSpektren.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 3</b>: Excitation spectra of non-transformed w303 yeast (neg. control) and yeast expressing mOrange. Both spectra were recorded in the plate reader (Ex: 400nm-550nm +/-9nm, Em: 581nm +/-20nm). The excitation spectrum of mOrange is indicated by a dashed line (source: <a href="http://www.tsienlab.ucsd.edu/Documents/REF%20-%20Fluorophore%20Spectra.xls">http://www.tsienlab.ucsd.edu/Documents/REF%20-%20Fluorophore%20Spectra.xls</a>).<br />
</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<h3>Repressible Promotors</h3><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/6/6f/TueWt_Staemme.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 4</b>: Basal expression level of mOrange under the control of the promotors Pfet3, Panb1 and Psuc2. Non-transformed yeast was used as negative control. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).<br />
</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/4/40/TueAll_strains.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 5</b>: Basal expression level of mOrange under the control of the promotors Pfet3, Panb1 and Psuc2. Non-transformed yeast was used as negative control. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as triplicates.<br />
</div><br />
<br />
<p>&nbsp;</p><br />
<h3>Pfet3</h3><br />
<br />
<p>Before we could observe the regulation of Pfet3 through the co-expressed membrane-bound progestin receptors, we needed to investigated the native function of Pfet3 which is the response to intracellular iron concentration. Therefore we grew yeast containing the Pfet-mOrange-pTUM100 construct in a low-ion environment (Low-iron medium (LIM) contained 1 mM EDTA). The promotor activity of Pfet3 had increased after 4 h of incubation in low-iron medium compared to cells which were grown in normal synthetic complete medium (fig. 6). After 8 h of incubation in LIM the promoter activity had increased 3-fold. The dependence of Pfet3 activity on iron allows modifying the expression level of the repressors mig1 and rox1. This is desirable as sensitivity analysis has shown that the maximal repressor concentration is crucial for the functionality of the measurement system. Nevertheless, this finding brings along new challenges as we need to maintain Pfet3 activity constant to avoid an undesired influence on the measurement.</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/d/dc/TueLIM_Induction.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 6</b>: Alterations of mOrange expression in low-iron environment of w303 containing Pfet3-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates. </div><br />
<br />
<p>&nbsp;</p><br />
<h3>Panb1 and Psuc2</h3><br />
<br />
<p>First we transformed the constructs Psuc2-mOrange-pTUM100 and Panb-mOrange-pTUM100 in w303. The promotor activity was analysed in fluorescence microscopy and using the plate reader. The activity equals 3.8% of Padh activity for Psuc and 1.9% of Padh activity for Panb. The maximal activity of these promotors also limits the maximal reporter expression and therefore also the sensitivity of the measurement system. Thus, a higher promotor activity was desirable.</p><br />
<br />
<p>Next we investigated which other factors influence the promotor activity, especially the intrinsic repressor level. Therefore we transformed the same constructs in mutant yeast strains with deficient rox1 (YPR065W) or mig1 (systematic gene name: YGL035C), see fig. 7 + 8. The promotor activity was enhanced by a factor of 2.6 for Psuc2 and 13.8 for Panb1. We therefore intend to implement our system in these deficient strains allowing higher reporter expression levels. Furthermore the function of MIG1 in glucose repression is well described (Santangelo, 2006). We observed an increase in promotor activity upon changing the medium from SC-Ura (2% glucose) to SC-Ura (2% galactose) for wt as well as the Mig deficient strain (fig. 9 + 10). This effect has to be considered if we intend to use Psuc together with GAL-Promotor in further characterization.<br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/d/d5/TueDefiziente_staemme.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 7</b>: Increased promoter activity of Psuc and Panb in repressor deficient strains. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/8/83/TuePanb_Psuc_deficient_strains.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 8</b>: Promoter activity of Psuc and Panb in repressor deficient strains. The promoter activity was enhanced by a factor of 2.6 for Psuc and 13.8 for Panb. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as triplicates.</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/e/ee/TuePsuc_mO_GALinduction.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 9</b>: Alterations of mOrange expression upon galactose induction of <b>w303</b> containing Psuc2-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates.</div><br />
<br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/7/74/TueGALinduction_Psuc_mig.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 10</b>: Alterations of mOrange expression upon galactose induction of <b>mig1 deficient yeast</b> containing Psuc2-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates.</div><br />
<br />
<br />
<p>&nbsp;</p><br />
<h3>References</h3><br />
<p>SANTANGELO, G. M. 2006. Glucose signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev, 70, 253-82.</p><br />
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{{:Team:Tuebingen/Template/equalHeight}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Results/OverviewTeam:Tuebingen/Results/Overview2013-10-05T02:41:18Z<p>Jakobmatthes: </p>
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Results<br />
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<br />
<h3>Cloning</h3><br />
<p>This year, we succeeded in cloning all missing parts required for our measuring system. While mPR Xl, mPR Dr, mig1, Padh1 and rox1 were available in pUC-IDT vector or pGEM T-easy vector from <a href="https://2012.igem.org/Team:Tuebingen">last year's iGEM-Team Tuebingen</a>, Pfet3, Panb1, Psuc2 were cloned directly from yeast genome. Furthermore we used the fluorescent reporter protein mOrange (<a href="http://parts.igem.org/Part:BBa_E2050">BBa_E2050</a>) and the transcriptional terminator Tadh (<a href="http://parts.igem.org/Part:BBa_K801012">BBa_K801012</a>) from the parts registry. A galactose inducible promotor (<a href="http://parts.igem.org/Part:BBa_J63006">BBa_J63006</a>) from the registry was used to create some plasmids which we want to use for the characterization of some of our parts. The cloning of luciferase succeeded only shortly before wiki freeze. As a result, the assemblies requiring luciferase were not performed anymore.</p><br />
<br />
<p>&nbsp;</p><br />
<br />
<h3>Assembly</h3><br />
<p>Our general assembly strategy is illustrated in fig. 1. The pRS vectors do not fit the RFC10 criteria and can therefore not be used the for 3A-Assembly. Instead we used the BioBrick RFC10 vector pTUM100 (<a href="http://parts.igem.org/Part:BBa_K801000">BBa_K801000</a>) which was kindly provided by the iGEM Team of the <a href="https://2013.igem.org/Team:TU-Munich">TU Munich</a> to fuse promotor and coding sequence. pTUM100 is a high copy shuttle plasmid which is based on the commercially available pYES2 vector and contains the cyc1 transcription terminator after the BioBrick suffix. Thus we could use some of the pTUM constructs directly for characterization of our repressible promotors (Pfet3-mOrange-pTUM100, Panb-mOrange-pTUM100, Psuc-mOrange-pTUM100, Padh-mOrange-pTUM100). The parts required for the next steps of characterization were equipped with a the transcriptional terminator Tadh by ligation into a pSB1C3 plasmid containing Tadh. The resulting assemblies were again BioBricks. These inserts were then ligated into the pRS shuttle vectors losing parts of their prefix and/or suffix. In total we have created 29 new plasmids!</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/1/19/TueAssemblyStrategy.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 1</b>: General assembly scheme: First, promoter and coding sequence were assembled in pTUM100 using the 3A-Assembly protocol. Constructs with the reporter mOrange were used for characterization directly. Subsequently, the promotor and the fused coding sequence are ligated into a pSB1C3 plasmid with the transcriptional terminator Tadh1. The resulting assemblies are again BioBricks. These inserts are then ligated into the pRS shuttle vectors losing parts of their prefix and/or suffix.</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<h3>Readout</h3><br />
<p>For the characterization of our repressible promotors Pfet3, Panb1 and Psuc2 we transformed pTUM100 constructs with mOrange under the control of the promotor of interest in the laboratory <a href="http://wiki.yeastgenome.org/index.php/Commonly_used_strains">yeast strain w303</a>. We used the constitutive promotor Padh1 (sequence identical with <a href="http://parts.igem.org/Part:BBa_J63005">BBa_J63005</a>) as positive control. The promoter activity was analysed qualitatively using fluorescence microscopy (fig. 2).</p><br />
<br />
<div style="padding: 10px; height: auto; width: 510px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/7/71/TueNurKontrollen.png" style="position: relative; width: 500px; margin: 5px 5px 10px 5px;"><b>Fig. 2</b>: Detection of mOrange expression in the fluorescence microscope. Non-transformed yeast was used as negative control. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).</div><br />
<br />
<h3>&nbsp;</h3><br />
<br />
<p>Next, we established a protocol for quantitative read-out of mOrange fluorescence using the plate reader. We recorded an excitation spectrum of non-transformed yeast and yeast expressing mOrange measuring the emission at 581nm (+/- 25nm). Fig. 3 compares the excitation spectra with the ideal excitation spectrum of mOrange. While the fluorescence decreases with higher excitation wavelength for non-transformed yeast, the spectrum of yeast expressing mOrange has a similar shape than the ideal excitation spectrum.</p><br />
<br />
<h3>&nbsp;</h3><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/2/27/TueSpektren.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 3</b>: Excitation spectra of non-transformed w303 yeast (neg. control) and yeast expressing mOrange. Both spectra were recorded in the plate reader (Ex: 400nm-550nm +/-9nm, Em: 581nm +/-20nm). The excitation spectrum of mOrange is indicated by a dashed line (source: <a href="http://www.tsienlab.ucsd.edu/Documents/REF%20-%20Fluorophore%20Spectra.xls">http://www.tsienlab.ucsd.edu/Documents/REF%20-%20Fluorophore%20Spectra.xls</a>).<br />
</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<h3>Repressible Promotors</h3><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/6/6f/TueWt_Staemme.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 4</b>: Basal expression level of mOrange under the control of the promotors Pfet3, Panb1 and Psuc2. Non-transformed yeast was used as negative control. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).<br />
</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/4/40/TueAll_strains.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 5</b>: Basal expression level of mOrange under the control of the promotors Pfet3, Panb1 and Psuc2. Non-transformed yeast was used as negative control. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as triplicates.<br />
</div><br />
<br />
<p>&nbsp;</p><br />
<h3>Pfet3</h3><br />
<br />
<p>Before we could observe the regulation of Pfet3 through the co-expressed membrane-bound progestin receptors, we needed to investigated the native function of Pfet3 which is the response to intracellular iron concentration. Therefore we grew yeast containing the Pfet-mOrange-pTUM100 construct in a low-ion environment (Low-iron medium (LIM) contained 1 mM EDTA). The promotor activity of Pfet3 had increased after 4 h of incubation in low-iron medium compared to cells which were grown in normal synthetic complete medium (fig. 6). After 8 h of incubation in LIM the promoter activity had increased 3-fold. The dependence of Pfet3 activity on iron allows modifying the expression level of the repressors mig1 and rox1. This is desirable as sensitivity analysis has shown that the maximal repressor concentration is crucial for the functionality of the measurement system. Nevertheless, this finding brings along new challenges as we need to maintain Pfet3 activity constant to avoid an undesired influence on the measurement.</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/d/dc/TueLIM_Induction.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 6</b>: Alterations of mOrange expression in low-iron environment of w303 containing Pfet3-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates. </div><br />
<br />
<p>&nbsp;</p><br />
<h3>Panb1 and Psuc2</h3><br />
<br />
<p>First we transformed the constructs Psuc2-mOrange-pTUM100 and Panb-mOrange-pTUM100 in w303. The promotor activity was analysed in fluorescence microscopy and using the plate reader. The activity equals 3.8% of Padh activity for Psuc and 1.9% of Padh activity for Panb. The maximal activity of these promotors also limits the maximal reporter expression and therefore also the sensitivity of the measurement system. Thus, a higher promotor activity was desirable.</p><br />
<br />
<p>Next we investigated which other factors influence the promotor activity, especially the intrinsic repressor level. Therefore we transformed the same constructs in mutant yeast strains with deficient rox1 (YPR065W) or mig1 (systematic gene name: YGL035C), see fig. 7 + 8. The promotor activity was enhanced by a factor of 2.6 for Psuc2 and 13.8 for Panb1. We therefore intend to implement our system in these deficient strains allowing higher reporter expression levels. Furthermore the function of MIG1 in glucose repression is well described (Santangelo, 2006). We observed an increase in promotor activity upon changing the medium from SC-Ura (2% glucose) to SC-Ura (2% galactose) for wt as well as the Mig deficient strain (fig. 9 + 10). This effect has to be considered if we intend to use Psuc together with GAL-Promotor in further characterization.<br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/d/d5/TueDefiziente_staemme.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 7</b>: Increased promoter activity of Psuc and Panb in repressor deficient strains. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/8/83/TuePanb_Psuc_deficient_strains.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 8</b>: Promoter activity of Psuc and Panb in repressor deficient strains. The promoter activity was enhanced by a factor of 2.6 for Psuc and 13.8 for Panb. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as triplicates.</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/e/ee/TuePsuc_mO_GALinduction.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 9</b>: Alterations of mOrange expression upon galactose induction of <b>w303</b> containing Psuc2-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates.</div><br />
<br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/7/74/TueGALinduction_Psuc_mig.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 10</b>: Alterations of mOrange expression upon galactose induction of <b>mig1 deficient yeast</b> containing Psuc2-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates.</div><br />
<br />
<br />
<p>&nbsp;</p><br />
<h3>References</h3><br />
<p>SANTANGELO, G. M. 2006. Glucose signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev, 70, 253-82.</p><br />
<br />
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{{:Team:Tuebingen/Template/equalHeight}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Results/OverviewTeam:Tuebingen/Results/Overview2013-10-05T02:40:35Z<p>Jakobmatthes: </p>
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<h3>Cloning</h3><br />
<p>This year, we succeeded in cloning all missing parts required for our measuring system. While mPR Xl, mPR Dr, mig1, Padh1 and rox1 were available in pUC-IDT vector or pGEM T-easy vector from <a href="https://2012.igem.org/Team:Tuebingen">last year's iGEM-Team Tuebingen</a>, Pfet3, Panb1, Psuc2 were cloned directly from yeast genome. Furthermore we used the fluorescent reporter protein mOrange (<a href="http://parts.igem.org/Part:BBa_E2050">BBa_E2050</a>) and the transcriptional terminator Tadh (<a href="http://parts.igem.org/Part:BBa_K801012">BBa_K801012</a>) from the parts registry. A galactose inducible promotor (<a href="http://parts.igem.org/Part:BBa_J63006">BBa_J63006</a>) from the registry was used to create some plasmids which we want to use for the characterization of some of our parts. The cloning of luciferase succeeded only shortly before wiki freeze. As a result, the assemblies requiring luciferase were not performed anymore.</p><br />
<br />
<p>&nbsp;</p><br />
<br />
<h3>Assembly</h3><br />
<p>Our general assembly strategy is illustrated in fig. 1. The pRS vectors do not fit the RFC10 criteria and can therefore not be used the for 3A-Assembly. Instead we used the BioBrick RFC10 vector pTUM100 (<a href="http://parts.igem.org/Part:BBa_K801000">BBa_K801000</a>) which was kindly provided by the iGEM Team of the <a href="https://2013.igem.org/Team:TU-Munich">TU Munich</a> to fuse promotor and coding sequence. pTUM100 is a high copy shuttle plasmid which is based on the commercially available pYES2 vector and contains the cyc1 transcription terminator after the BioBrick suffix. Thus we could use some of the pTUM constructs directly for characterization of our repressible promotors (Pfet3-mOrange-pTUM100, Panb-mOrange-pTUM100, Psuc-mOrange-pTUM100, Padh-mOrange-pTUM100). The parts required for the next steps of characterization were equipped with a the transcriptional terminator Tadh by ligation into a pSB1C3 plasmid containing Tadh. The resulting assemblies were again BioBricks. These inserts were then ligated into the pRS shuttle vectors losing parts of their prefix and/or suffix. In total we have created 29 new plasmids!</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/1/19/TueAssemblyStrategy.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 1</b>: General assembly scheme: First, promoter and coding sequence were assembled in pTUM100 using the 3A-Assembly protocol. Constructs with the reporter mOrange were used for characterization directly. Subsequently, the promotor and the fused coding sequence are ligated into a pSB1C3 plasmid with the transcriptional terminator Tadh1. The resulting assemblies are again BioBricks. These inserts are then ligated into the pRS shuttle vectors losing parts of their prefix and/or suffix.</div><br />
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<p>&nbsp;</p><br />
<br />
<h3>Readout</h3><br />
<p>For the characterization of our repressible promotors Pfet3, Panb1 and Psuc2 we transformed pTUM100 constructs with mOrange under the control of the promotor of interest in the laboratory yeast strain w303. We used the constitutive promotor Padh1 (sequence identical with <a href="http://parts.igem.org/Part:BBa_J63005">BBa_J63005</a>) as positive control. The promoter activity was analysed qualitatively using fluorescence microscopy (fig. 2).</p><br />
<br />
<div style="padding: 10px; height: auto; width: 510px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/7/71/TueNurKontrollen.png" style="position: relative; width: 500px; margin: 5px 5px 10px 5px;"><b>Fig. 2</b>: Detection of mOrange expression in the fluorescence microscope. Non-transformed yeast was used as negative control. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).</div><br />
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<h3>&nbsp;</h3><br />
<br />
<p>Next, we established a protocol for quantitative read-out of mOrange fluorescence using the plate reader. We recorded an excitation spectrum of non-transformed yeast and yeast expressing mOrange measuring the emission at 581nm (+/- 25nm). Fig. 3 compares the excitation spectra with the ideal excitation spectrum of mOrange. While the fluorescence decreases with higher excitation wavelength for non-transformed yeast, the spectrum of yeast expressing mOrange has a similar shape than the ideal excitation spectrum.</p><br />
<br />
<h3>&nbsp;</h3><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/2/27/TueSpektren.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 3</b>: Excitation spectra of non-transformed w303 yeast (neg. control) and yeast expressing mOrange. Both spectra were recorded in the plate reader (Ex: 400nm-550nm +/-9nm, Em: 581nm +/-20nm). The excitation spectrum of mOrange is indicated by a dashed line (source: <a href="http://www.tsienlab.ucsd.edu/Documents/REF%20-%20Fluorophore%20Spectra.xls">http://www.tsienlab.ucsd.edu/Documents/REF%20-%20Fluorophore%20Spectra.xls</a>).<br />
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<p>&nbsp;</p><br />
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<h3>Repressible Promotors</h3><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/6/6f/TueWt_Staemme.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 4</b>: Basal expression level of mOrange under the control of the promotors Pfet3, Panb1 and Psuc2. Non-transformed yeast was used as negative control. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).<br />
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<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/4/40/TueAll_strains.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 5</b>: Basal expression level of mOrange under the control of the promotors Pfet3, Panb1 and Psuc2. Non-transformed yeast was used as negative control. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as triplicates.<br />
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<p>&nbsp;</p><br />
<h3>Pfet3</h3><br />
<br />
<p>Before we could observe the regulation of Pfet3 through the co-expressed membrane-bound progestin receptors, we needed to investigated the native function of Pfet3 which is the response to intracellular iron concentration. Therefore we grew yeast containing the Pfet-mOrange-pTUM100 construct in a low-ion environment (Low-iron medium (LIM) contained 1 mM EDTA). The promotor activity of Pfet3 had increased after 4 h of incubation in low-iron medium compared to cells which were grown in normal synthetic complete medium (fig. 6). After 8 h of incubation in LIM the promoter activity had increased 3-fold. The dependence of Pfet3 activity on iron allows modifying the expression level of the repressors mig1 and rox1. This is desirable as sensitivity analysis has shown that the maximal repressor concentration is crucial for the functionality of the measurement system. Nevertheless, this finding brings along new challenges as we need to maintain Pfet3 activity constant to avoid an undesired influence on the measurement.</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/d/dc/TueLIM_Induction.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 6</b>: Alterations of mOrange expression in low-iron environment of w303 containing Pfet3-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates. </div><br />
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<p>&nbsp;</p><br />
<h3>Panb1 and Psuc2</h3><br />
<br />
<p>First we transformed the constructs Psuc2-mOrange-pTUM100 and Panb-mOrange-pTUM100 in w303. The promotor activity was analysed in fluorescence microscopy and using the plate reader. The activity equals 3.8% of Padh activity for Psuc and 1.9% of Padh activity for Panb. The maximal activity of these promotors also limits the maximal reporter expression and therefore also the sensitivity of the measurement system. Thus, a higher promotor activity was desirable.</p><br />
<br />
<p>Next we investigated which other factors influence the promotor activity, especially the intrinsic repressor level. Therefore we transformed the same constructs in mutant yeast strains with deficient rox1 (YPR065W) or mig1 (systematic gene name: YGL035C), see fig. 7 + 8. The promotor activity was enhanced by a factor of 2.6 for Psuc2 and 13.8 for Panb1. We therefore intend to implement our system in these deficient strains allowing higher reporter expression levels. Furthermore the function of MIG1 in glucose repression is well described (Santangelo, 2006). We observed an increase in promotor activity upon changing the medium from SC-Ura (2% glucose) to SC-Ura (2% galactose) for wt as well as the Mig deficient strain (fig. 9 + 10). This effect has to be considered if we intend to use Psuc together with GAL-Promotor in further characterization.<br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/d/d5/TueDefiziente_staemme.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 7</b>: Increased promoter activity of Psuc and Panb in repressor deficient strains. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).</div><br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/8/83/TuePanb_Psuc_deficient_strains.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 8</b>: Promoter activity of Psuc and Panb in repressor deficient strains. The promoter activity was enhanced by a factor of 2.6 for Psuc and 13.8 for Panb. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as triplicates.</div><br />
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<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/e/ee/TuePsuc_mO_GALinduction.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 9</b>: Alterations of mOrange expression upon galactose induction of <b>w303</b> containing Psuc2-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates.</div><br />
<br />
<br />
<p>&nbsp;</p><br />
<br />
<div style="padding: 10px; height: auto; width: 610px; background-color: #E0E0E0; margin: auto;"><img src="https://static.igem.org/mediawiki/2013/7/74/TueGALinduction_Psuc_mig.png" style="position: relative; width: 600px; margin: 5px 5px 10px 5px;"><b>Fig. 10</b>: Alterations of mOrange expression upon galactose induction of <b>mig1 deficient yeast</b> containing Psuc2-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates.</div><br />
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<p>&nbsp;</p><br />
<h3>References</h3><br />
<p>SANTANGELO, G. M. 2006. Glucose signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev, 70, 253-82.</p><br />
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{{:Team:Tuebingen/Template/equalHeight}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/ModelingTeam:Tuebingen/Modeling2013-10-04T12:01:47Z<p>Jakobmatthes: </p>
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Modeling<br />
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<h3>Motivation</h3><br />
<br />
<p>The aim of our modeling approach is the formal description of the biosensor system. We look from an engineering viewpoint and want to create a technical specification for our device similar to the specifications often found in electrical engineering.</p><br />
<br />
<p>The basis for the mathematical formulation has to be an abstract representation. The underlying network we used for our modeling tasks is structured as follows:</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/18/Model_Overview.png" title="model overview"/><br />
<br />
<p>Another aspect we want to consider with a computational model are the benefits for both the model and the biological system. Through several iterations of analyses of the computational model and data generation with the biological system, we can verify the model and find interesting characteristics of the biological system.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/09/Workflow_cycle.png" title="workflow cycle"><br />
<br />
<br />
<h3>Model equations</h3><br />
<br />
<p>In essence, the computational model consists of the following three equations. The all specify the concentrations for the key components of the system: (1) for the active receptor (ligand-receptor-complex), (2) for the repressor protein and (3) for the reporter protein.</p><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/Model_equations.png" title="model equations"><br />
<br />
<p>This approach combines the transcription and translation processes into a single production process. There is also only one degradation process. Through this simplification, the number of parameters is reduced to 8. Some parameters are available from literature, others are estimated based on similar, known interactions.</p><br />
<br />
<p>The binding characteristic of the membrane progesterone receptor and the hormone progesterone has been fitted using the non-linear least squares method approach and yields the half-maximal concentration.</p><br />
<img src="https://static.igem.org/mediawiki/2013/d/d0/Experimental_fit.png" title="fitting"><br />
<br />
<br />
<h3>Results</h3><br />
<p>Two important simulations using the model have been conducted. The first one describes the dependence of the output signal (the reporter concentration) on the input signal (the hormone concentration). The second one tries to identify sensitive parts of the system.</p><br />
<br />
<p>The general behavior of the system is shown in the following plot. Two simulations are shown which differ slithly because of random parameter variations.</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a9/Time_course.png" title="time course"><br />
<p>The detection range is clearly in the range of nanomolar concentrations of progesterone. This renders the biosensor suitable because the physiological active levels of progesterone are covered.</p><br />
<br />
<p>A sensitivity analysis has uncovered that only two of the eight parameters have a significant effect on strength of the output signal: (1) the maximal concentraion of the repressor protein and (2) the binding affinity of the fet3 promoter.</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/35/Sensitivity.png" title="sensitivitiy analysis"><br />
<br />
<h3>Outlook</h3><br />
<br />
<p>Using the model and the specific results above, we hope to provide a suitable theoretical description of the biosensor system. We also want to use to model to find parts of the system which can be modified to improve the sensitivity, the detection range and the output signal strength.</p><br />
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{{:Team:Tuebingen/Template/equalHeight}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/ModelingTeam:Tuebingen/Modeling2013-10-04T11:46:46Z<p>Jakobmatthes: </p>
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Modeling<br />
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<h3>Motivation</h3><br />
<br />
<p>The aim of our modeling approach is the formal description of the biosensor system. We look from an engineering viewpoint and want to create a technical specification for our device similar to the specifications often found in electrical engineering.</p><br />
<br />
<p>The basis for the mathematical formulation has to be an abstract representation. The underlying network we used for our modeling tasks is structured as follows:</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/18/Model_Overview.png" title="model overview"/><br />
<br />
<p>Another aspect we want to consider with a computational model are the benefits for both the model and the biological system. Through several iterations of analyses of the computational model and data generation with the biological system, we can verify the model and find interesting characteristics of the biological system.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/09/Workflow_cycle.png" title="workflow cycle"><br />
<br />
<br />
<h3>Model equations</h3><br />
<br />
<p>In essence, the computational model consists of the following three equations. The all specify the concentrations for the key components of the system: (1) for the active receptor (ligand-receptor-complex), (2) for the repressor protein and (3) for the reporter protein.</p><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/Model_equations.png" title="model equations"><br />
<br />
<p>This approach combines the transcription and translation processes into a single production process. There is also only one degradation process. Through this simplification, the number of parameters is reduced to 8.</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/d/d0/Experimental_fit.png" title="fitting"><br />
<br />
<br />
<h3>Results</h3><br />
<p>General behavior</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a9/Time_course.png" title="time course"><br />
<br />
<p>sensitivity analysis</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/35/Sensitivity.png" title="sensitivitiy analysis"><br />
<br />
<h3>Outlook</h3><br />
<br />
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{{:Team:Tuebingen/Template/equalHeight}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/ModelingTeam:Tuebingen/Modeling2013-10-04T11:42:51Z<p>Jakobmatthes: </p>
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<h3>Motivation</h3><br />
<br />
<p>The aim of our modeling approach is the formal description of the biosensor system. We look from an engineering viewpoint and want to create a technical specification for our device similar to the specifications often found in electrical engineering.</p><br />
<br />
<p>The basis for the mathematical formulation has to be an abstract representation. The underlying network we used for our modeling tasks is structured as follows:</p><br />
<img src="https://static.igem.org/mediawiki/2013/1/18/Model_Overview.png" title="model overview"/><br />
<br />
<p>Another aspect we want to consider with a computational model are the benefits for both the model and the biological system. Through several iterations of analyses of the computational model and data generation with the biological system, we can verify the model and find interesting characteristics of the biological system.</p><br />
<img src="https://static.igem.org/mediawiki/2013/0/09/Workflow_cycle.png" title="workflow cycle"><br />
<br />
<br />
<h3>Model equations</h3><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/Model_equations.png" title="model equations"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d0/Experimental_fit.png" title="fitting"><br />
<br />
<br />
<h3>Results</h3><br />
<p>General behavior</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/a9/Time_course.png" title="time course"><br />
<br />
<p>sensitivity analysis</p><br />
<img src="https://static.igem.org/mediawiki/2013/3/35/Sensitivity.png" title="sensitivitiy analysis"><br />
<br />
<h3>Outlook</h3><br />
<br />
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{{:Team:Tuebingen/Template/equalHeight}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/ProjectTeam:Tuebingen/Project2013-07-21T19:24:06Z<p>Jakobmatthes: Undo revision 704 by IGEM HQ (talk)</p>
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{{:Team:Tuebingen/Template/Twitter}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/SandboxTeam:Tuebingen/Sandbox2013-03-26T17:28:50Z<p>Jakobmatthes: /* Test 123 */</p>
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{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Seitentitel =<br />
<br />
...</nowiki><br />
<br />
=== Textformatierung ===<br />
<br />
Die wichtigesten Textformatierung werden auf http://www.mediawiki.org/wiki/Help:Formatting/de beschrieben.<br />
<br />
=== Bilder ===<br />
<br />
Zunächst auf einer Seite einfach das Bild mit gewünschtem Dateinamen einbinden, das Bild kann danach über ein Klick auf das fehlende Bild in der Seite hochgeladen werden:<br />
<nowiki>[[Image:Dateiname.xyz|center|400px]]<br />
[[Image:Dateiname.xyz|right|thumb|200px|Mit Bildunterschrift]]</nowiki><br />
<br />
=== Verlinkung ===<br />
<br />
* Interne Verlinkungen erfolgen mit <nowiki>[[ ... ]]</nowiki>:<br />
<br />
<nowiki>[[Team:Tuebingen/Seite|Text des Links]]</nowiki><br />
<br />
* Alle unsere Seiten befinden sich unter <code>Team:Tuebingen/...</code>.<br />
<br />
* Externe Verlinkungen funktionieren mit <code><nowiki>[http://...]</nowiki></code> (erscheinen nur als Nummern-Referenz, bspw. [4]) oder einfach nur mit <code>http://...</code> (erscheint als volle Adresse, bspw. http://...).</div>Jakobmattheshttp://2013.igem.org/File:Tuebingen-logo.pngFile:Tuebingen-logo.png2013-03-26T17:28:15Z<p>Jakobmatthes: </p>
<hr />
<div></div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/SandboxTeam:Tuebingen/Sandbox2013-03-26T17:27:58Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Sandbox =<br />
<br />
== Test 123 ==<br />
<br />
[[Team:Tuebingen/Sandbox2|neue Seite]]<br />
<br />
[[Image:tuebingen-logo.png|center|400px]]<br />
<br />
== Quick Start ==<br />
<br />
=== Seiten anlegen ===<br />
<br />
Zum Anlegen einer neuen Seite reicht es aus, wenn man von einer bestehenden Seite einen Link anlegt (s.u.). Folgt man dem Link, erhält man ein Feld zum Bearbeiten (Anlegen) der Seite.<br />
<br />
Jede Seite braucht das Tuebingen-Template (erste Zeile). In der zweiten Zeile muss der Seitentitel stehen:<br />
<nowiki><br />
{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Seitentitel =<br />
<br />
...</nowiki><br />
<br />
=== Textformatierung ===<br />
<br />
Die wichtigesten Textformatierung werden auf http://www.mediawiki.org/wiki/Help:Formatting/de beschrieben.<br />
<br />
=== Bilder ===<br />
<br />
Zunächst auf einer Seite einfach das Bild mit gewünschtem Dateinamen einbinden, das Bild kann danach über ein Klick auf das fehlende Bild in der Seite hochgeladen werden:<br />
<nowiki>[[Image:Dateiname.xyz|center|400px]]<br />
[[Image:Dateiname.xyz|right|thumb|200px|Mit Bildunterschrift]]</nowiki><br />
<br />
=== Verlinkung ===<br />
<br />
* Interne Verlinkungen erfolgen mit <nowiki>[[ ... ]]</nowiki>:<br />
<br />
<nowiki>[[Team:Tuebingen/Seite|Text des Links]]</nowiki><br />
<br />
* Alle unsere Seiten befinden sich unter <code>Team:Tuebingen/...</code>.<br />
<br />
* Externe Verlinkungen funktionieren mit <code><nowiki>[http://...]</nowiki></code> (erscheinen nur als Nummern-Referenz, bspw. [4]) oder einfach nur mit <code>http://...</code> (erscheint als volle Adresse, bspw. http://...).</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/SandboxTeam:Tuebingen/Sandbox2013-03-26T17:26:04Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Sandbox =<br />
<br />
== Test 123 ==<br />
<br />
[[Team:Tuebingen/Sandbox2|neue Seite]]<br />
<br />
== Quick Start ==<br />
<br />
=== Seiten anlegen ===<br />
<br />
Zum Anlegen einer neuen Seite reicht es aus, wenn man von einer bestehenden Seite einen Link anlegt (s.u.). Folgt man dem Link, erhält man ein Feld zum Bearbeiten (Anlegen) der Seite.<br />
<br />
Jede Seite braucht das Tuebingen-Template (erste Zeile). In der zweiten Zeile muss der Seitentitel stehen:<br />
<nowiki><br />
{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Seitentitel =<br />
<br />
...</nowiki><br />
<br />
=== Textformatierung ===<br />
<br />
Die wichtigesten Textformatierung werden auf http://www.mediawiki.org/wiki/Help:Formatting/de beschrieben.<br />
<br />
=== Bilder ===<br />
<br />
Zunächst auf einer Seite einfach das Bild mit gewünschtem Dateinamen einbinden, das Bild kann danach über ein Klick auf das fehlende Bild in der Seite hochgeladen werden:<br />
<nowiki>[[Image:Dateiname.xyz|center|400px]]<br />
[[Image:Dateiname.xyz|right|thumb|200px|Mit Bildunterschrift]]</nowiki><br />
<br />
=== Verlinkung ===<br />
<br />
* Interne Verlinkungen erfolgen mit <nowiki>[[ ... ]]</nowiki>:<br />
<br />
<nowiki>[[Team:Tuebingen/Seite|Text des Links]]</nowiki><br />
<br />
* Alle unsere Seiten befinden sich unter <code>Team:Tuebingen/...</code>.<br />
<br />
* Externe Verlinkungen funktionieren mit <code><nowiki>[http://...]</nowiki></code> (erscheinen nur als Nummern-Referenz, bspw. [4]) oder einfach nur mit <code>http://...</code> (erscheint als volle Adresse, bspw. http://...).</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/SandboxTeam:Tuebingen/Sandbox2013-03-26T15:53:20Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Sandbox =<br />
<br />
== Test 123 ==<br />
<br />
<br />
== Quick Start ==<br />
<br />
=== Seiten anlegen ===<br />
<br />
Zum Anlegen einer neuen Seite reicht es aus, wenn man von einer bestehenden Seite einen Link anlegt (s.u.). Folgt man dem Link, erhält man ein Feld zum Bearbeiten (Anlegen) der Seite.<br />
<br />
Jede Seite braucht das Tuebingen-Template (erste Zeile). In der zweiten Zeile muss der Seitentitel stehen:<br />
<nowiki><br />
{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Seitentitel =<br />
<br />
...</nowiki><br />
<br />
=== Textformatierung ===<br />
<br />
Die wichtigesten Textformatierung werden auf http://www.mediawiki.org/wiki/Help:Formatting/de beschrieben.<br />
<br />
=== Bilder ===<br />
<br />
Zunächst auf einer Seite einfach das Bild mit gewünschtem Dateinamen einbinden, das Bild kann danach über ein Klick auf das fehlende Bild in der Seite hochgeladen werden:<br />
<nowiki>[[Image:Dateiname.xyz|center|400px]]<br />
[[Image:Dateiname.xyz|right|thumb|200px|Mit Bildunterschrift]]</nowiki><br />
<br />
=== Verlinkung ===<br />
<br />
* Interne Verlinkungen erfolgen mit <nowiki>[[ ... ]]</nowiki>:<br />
<br />
<nowiki>[[Team:Tuebingen/Seite|Text des Links]]</nowiki><br />
<br />
* Alle unsere Seiten befinden sich unter <code>Team:Tuebingen/...</code>.<br />
<br />
* Externe Verlinkungen funktionieren mit <code><nowiki>[http://...]</nowiki></code> (erscheinen nur als Nummern-Referenz, bspw. [4]) oder einfach nur mit <code>http://...</code> (erscheint als volle Adresse, bspw. http://...).</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Template/TuebingenTeam:Tuebingen/Template/Tuebingen2013-03-26T15:52:26Z<p>Jakobmatthes: </p>
<hr />
<div><html><br />
<!-- template base for all sites --><br />
<!-- styles --><br />
</html><br />
<html><br />
<style type="text/css"><br />
/* Droid Sans from Google Web fonts */<br />
@import url(http://fonts.googleapis.com/css?family=Droid+Sans:400,700);<br />
<br />
/* hide elements */<br />
h1.firstHeading {<br />
display: none;<br />
}<br />
<br />
body {<br />
background-color: #f5f5f5;<br />
}<br />
<br />
table#toc {<br />
float: right;<br />
margin: 0 0 1em 1em;<br />
}<br />
<br />
div#bodyContent {<br />
margin: 1em;<br />
}<br />
<br />
pre {<br />
font-size: 1.5em;<br />
}<br />
<br />
<br />
/* navigation */<br />
#nav {<br />
margin: auto;<br />
}<br />
<br />
#nav ul {<br />
list-style-type: none;<br />
list-style-image: none;<br />
margin: 0;<br />
padding: 0;<br />
height: 2.5em;<br />
}<br />
<br />
#nav li {<br />
display: block;<br />
position: relative;<br />
padding: 0;<br />
margin: 0 .5em 0 .5em;<br />
float: left;<br />
height: 2.7em;<br />
}<br />
<br />
/* main heading */<br />
h1 {<br />
border: 0;<br />
}<br />
<br />
/* edit links on headings */<br />
span.editsection {<br />
font-size: .5em;<br />
color: lightgrey;<br />
}<br />
span.editsection a {<br />
color: lightgrey;<br />
}<br />
</style><br />
</html><br />
<!-- google analytics --><br />
<html><br />
<script type="text/javascript"><br />
<br />
var _gaq = _gaq || [];<br />
_gaq.push(['_setAccount', 'UA-39523076-1']);<br />
_gaq.push(['_trackPageview']);<br />
<br />
(function() {<br />
var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true;<br />
ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js';<br />
var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s);<br />
})();<br />
<br />
</script><br />
</html><br />
<br />
<!-- include navigation --><br />
{{:Team:Tuebingen/Templates/Navigation}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/SandboxTeam:Tuebingen/Sandbox2013-03-26T15:51:42Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Sandbox =<br />
<br />
== Quick Start ==<br />
<br />
=== Seiten anlegen ===<br />
<br />
Zum Anlegen einer neuen Seite reicht es aus, wenn man von einer bestehenden Seite einen Link anlegt (s.u.). Folgt man dem Link, erhält man ein Feld zum Bearbeiten (Anlegen) der Seite.<br />
<br />
Jede Seite braucht das Tuebingen-Template (erste Zeile). In der zweiten Zeile muss der Seitentitel stehen:<br />
<nowiki><br />
{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Seitentitel =<br />
<br />
...</nowiki><br />
<br />
=== Textformatierung ===<br />
<br />
Die wichtigesten Textformatierung werden auf http://www.mediawiki.org/wiki/Help:Formatting/de beschrieben.<br />
<br />
=== Bilder ===<br />
<br />
Zunächst auf einer Seite einfach das Bild mit gewünschtem Dateinamen einbinden, das Bild kann danach über ein Klick auf das fehlende Bild in der Seite hochgeladen werden:<br />
<nowiki>[[Image:Dateiname.xyz|center|400px]]<br />
[[Image:Dateiname.xyz|right|thumb|200px|Mit Bildunterschrift]]</nowiki><br />
<br />
=== Verlinkung ===<br />
<br />
* Interne Verlinkungen erfolgen mit <nowiki>[[ ... ]]</nowiki>:<br />
<br />
<nowiki>[[Team:Tuebingen/Seite|Text des Links]]</nowiki><br />
<br />
* Alle unsere Seiten befinden sich unter <code>Team:Tuebingen/...</code>.<br />
<br />
* Externe Verlinkungen funktionieren mit <code><nowiki>[http://...]</nowiki></code> (erscheinen nur als Nummern-Referenz, bspw. [4]) oder einfach nur mit <code>http://...</code> (erscheint als volle Adresse, bspw. http://...).</div>Jakobmattheshttp://2013.igem.org/File:TueSponsorsAmedrix.pngFile:TueSponsorsAmedrix.png2013-03-25T21:34:28Z<p>Jakobmatthes: </p>
<hr />
<div></div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/SponsorsTeam:Tuebingen/Sponsors2013-03-25T21:25:05Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
__NOTOC__<br />
= Sponsors =<br />
<br />
We warmly thank the following companies which are supporting our scientific undertaking in 2013:<br />
<br />
<!-- {{:Team:Tuebingen/Templates/SponsorEntry|Agilent Technologies|TueSponsorsAgilent.png||http://www.agilent.de}} --><br />
{{:Team:Tuebingen/Templates/SponsorEntry|Amedrix GmbH|TueSponsorsAmedrix.png||http://www.amedrix.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Bio-Rad|TueSponsorsBioRad.png||http://www.bio-rad.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Eppendorf|TueSponsorsEppendorf.png||http://www.eppendorf.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Kreissparkasse Tübingen|TueSponsorsSparkasse.png||http://www.ksk-tuebingen.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|New England Biolabs GmbH|TueSponsorsNEB.png||http://www.neb-online.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|SERVA Electrophoresis GmbH|TueSponsorsSERVA.png||http://www.serva.de}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/SponsorsTeam:Tuebingen/Sponsors2013-03-25T15:50:41Z<p>Jakobmatthes: /* Sponsors */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
__NOTOC__<br />
= Sponsors =<br />
<br />
We warmly thank the following companies which are supporting our scientific undertaking in 2013:<br />
<br />
<!-- {{:Team:Tuebingen/Templates/SponsorEntry|Agilent Technologies|TueSponsorsAgilent.png||http://www.agilent.de}} --><br />
{{:Team:Tuebingen/Templates/SponsorEntry|Bio-Rad|TueSponsorsBioRad.png||http://www.bio-rad.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Eppendorf|TueSponsorsEppendorf.png||http://www.eppendorf.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Kreissparkasse Tübingen|TueSponsorsSparkasse.png||http://www.ksk-tuebingen.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|New England Biolabs GmbH|TueSponsorsNEB.png||http://www.neb-online.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|SERVA Electrophoresis GmbH|TueSponsorsSERVA.png||http://www.serva.de}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/TeamTeam:Tuebingen/Team2013-03-25T12:59:04Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Team =<br />
<br />
Our [https://igem.org/Team.cgi?id=1029 official team roster] can be found on the igem.org team page.<br />
<br />
<br />
<!--<br />
description of team<br />
<br />
member names<br />
<br />
advisor names<br />
<br />
team photo<br />
--></div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/TeamTeam:Tuebingen/Team2013-03-25T12:58:06Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Team =<br />
<br />
Our [https://igem.org/Team.cgi?id=1029 official team roster] can be found on the igem.org team page.<br />
<br />
<br />
<!--<br />
description of team<br />
--></div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/TeamTeam:Tuebingen/Team2013-03-25T12:57:47Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Team =<br />
<br />
Our [https://igem.org/Team.cgi?id=1029 official team roster] can be found on the igem.org team page.</div>Jakobmattheshttp://2013.igem.org/Category:Team:Tuebingen/LabCategory:Team:Tuebingen/Lab2013-03-25T12:54:17Z<p>Jakobmatthes: Created page with "{{:Team:Tuebingen/Template/Tuebingen}}"</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Template/TuebingenTeam:Tuebingen/Template/Tuebingen2013-03-25T12:53:23Z<p>Jakobmatthes: </p>
<hr />
<div><html><br />
<!-- template base for all sites --><br />
<!-- styles --><br />
</html><br />
<html><br />
<style type="text/css"><br />
/* Droid Sans from Google Web fonts */<br />
@import url(http://fonts.googleapis.com/css?family=Droid+Sans:400,700);<br />
<br />
/* hide elements */<br />
h1.firstHeading {<br />
display: none;<br />
}<br />
<br />
body {<br />
background-color: #f5f5f5;<br />
}<br />
<br />
table#toc {<br />
float: right;<br />
margin: 0 0 1em 1em;<br />
}<br />
<br />
div#bodyContent {<br />
margin: 1em;<br />
}<br />
<br />
<br />
/* navigation */<br />
#nav {<br />
margin: auto;<br />
}<br />
<br />
#nav ul {<br />
list-style-type: none;<br />
list-style-image: none;<br />
margin: 0;<br />
padding: 0;<br />
height: 2.5em;<br />
}<br />
<br />
#nav li {<br />
display: block;<br />
position: relative;<br />
padding: 0;<br />
margin: 0 .5em 0 .5em;<br />
float: left;<br />
height: 2.7em;<br />
}<br />
<br />
/* main heading */<br />
h1 {<br />
border: 0;<br />
}<br />
<br />
/* edit links on headings */<br />
span.editsection {<br />
font-size: .5em;<br />
color: lightgrey;<br />
}<br />
span.editsection a {<br />
color: lightgrey;<br />
}<br />
</style><br />
</html><br />
<!-- google analytics --><br />
<html><br />
<script type="text/javascript"><br />
<br />
var _gaq = _gaq || [];<br />
_gaq.push(['_setAccount', 'UA-39523076-1']);<br />
_gaq.push(['_trackPageview']);<br />
<br />
(function() {<br />
var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true;<br />
ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js';<br />
var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s);<br />
})();<br />
<br />
</script><br />
</html><br />
<br />
<!-- include navigation --><br />
{{:Team:Tuebingen/Templates/Navigation}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/SponsorsTeam:Tuebingen/Sponsors2013-03-24T20:17:06Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
__NOTOC__<br />
= Sponsors =<br />
<br />
We warmly thank the following companies which are supporting our scientific undertaking in 2013:<br />
<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Agilent Technologies|TueSponsorsAgilent.png||http://www.agilent.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Bio-Rad|TueSponsorsBioRad.png||http://www.bio-rad.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Eppendorf|TueSponsorsEppendorf.png||http://www.eppendorf.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Kreissparkasse Tübingen|TueSponsorsSparkasse.png||http://www.ksk-tuebingen.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|New England Biolabs GmbH|TueSponsorsNEB.png||http://www.neb-online.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|SERVA Electrophoresis GmbH|TueSponsorsSERVA.png||http://www.serva.de}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/LabNotebookTeam:Tuebingen/LabNotebook2013-03-24T14:46:29Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Lab Notebook =<br />
<br />
== March 18: Our First Week ==<br />
<!--<br />
* introduction to new members<br />
* enthusiasm in the lab etc.<br />
* GelRed adventures<br />
--><br />
<br />
[[Category:Team:Tuebingen/Lab]]</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Template/TuebingenTeam:Tuebingen/Template/Tuebingen2013-03-24T14:41:38Z<p>Jakobmatthes: </p>
<hr />
<div><html><br />
<!-- template base for all sites --><br />
<!-- styles --><br />
</html><br />
<html><br />
<style type="text/css"><br />
/* Droid Sans from Google Web fonts */<br />
@import url(http://fonts.googleapis.com/css?family=Droid+Sans:400,700);<br />
<br />
/* hide elements */<br />
h1.firstHeading {<br />
display: none;<br />
}<br />
<br />
body {<br />
background-color: #f5f5f5;<br />
}<br />
<br />
table#toc {<br />
float: right;<br />
margin: 0 0 1em 1em;<br />
}<br />
<br />
<br />
/* navigation */<br />
#nav {<br />
margin: auto;<br />
}<br />
<br />
#nav ul {<br />
list-style-type: none;<br />
list-style-image: none;<br />
margin: 0;<br />
padding: 0;<br />
height: 2.5em;<br />
}<br />
<br />
#nav li {<br />
display: block;<br />
position: relative;<br />
padding: 0;<br />
margin: 0 .5em 0 .5em;<br />
float: left;<br />
height: 2.7em;<br />
}<br />
<br />
/* main heading */<br />
h1 {<br />
border: 0;<br />
}<br />
<br />
/* edit links on headings */<br />
span.editsection {<br />
font-size: .5em;<br />
color: lightgrey;<br />
}<br />
span.editsection a {<br />
color: lightgrey;<br />
}<br />
</style><br />
</html><br />
<!-- google analytics --><br />
<html><br />
<script type="text/javascript"><br />
<br />
var _gaq = _gaq || [];<br />
_gaq.push(['_setAccount', 'UA-39523076-1']);<br />
_gaq.push(['_trackPageview']);<br />
<br />
(function() {<br />
var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true;<br />
ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js';<br />
var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s);<br />
})();<br />
<br />
</script><br />
</html><br />
<br />
<!-- include navigation --><br />
{{:Team:Tuebingen/Templates/Navigation}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/SandboxTeam:Tuebingen/Sandbox2013-03-24T14:40:54Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Sandbox =<br />
<br />
== Quick Start ==<br />
<br />
=== Seiten anlegen ===<br />
<br />
Zum Anlegen einer neuen Seite reicht es aus, wenn man von einer bestehenden Seite einen Link anlegt (s.u.). Folgt man dem Link, erhält man ein Feld zum Bearbeiten (Anlegen) der Seite.<br />
<br />
Jede Seite braucht das Tuebingen-Template (erste Zeile). In der zweiten Zeile muss der Seitentitel stehen:<br />
<nowiki><br />
{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Seitentitel =<br />
<br />
...</nowiki><br />
<br />
=== Textformatierung ===<br />
<br />
Die wichtigesten Textformatierung werden auf http://www.mediawiki.org/wiki/Help:Formatting/de beschrieben.<br />
<br />
=== Verlinkung ===<br />
<br />
* Interne Verlinkungen erfolgen mit <nowiki>[[ ... ]]</nowiki>:<br />
<br />
<nowiki>[[Team:Tuebingen/Seite|Text des Links]]</nowiki><br />
<br />
* Alle unsere Seiten befinden sich unter <code>Team:Tuebingen/...</code>.<br />
<br />
* Externe Verlinkungen funktionieren mit <code><nowiki>[http://...]</nowiki></code> (erscheinen nur als Nummern-Referenz, bspw. [4]) oder einfach nur mit <code>http://...</code> (erscheint als volle Adresse, bspw. http://...).</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/SponsorsTeam:Tuebingen/Sponsors2013-03-24T14:32:03Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
__NOTOC__<br />
= Sponsors =<br />
<br />
We warmly thank the following companies which are supporting our scientific undertaking in 2013:<br />
<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Agilent Technologies|TueSponsorsAgilent.png||http://www.agilent.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Bio-Rad|TueSponsorsBioRad.png||http://www.bio-rad.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Eppendorf|TueSponsorsEppendorf.png||http://www.eppendorf.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Kreissparkasse Tübingen|TueSponsorsSparkasse.png||http://www.ksk-tuebingen.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|New England Biolabs GmbH|TueSponsorsNEB.png||http://www.neb-online.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|SERVA Electrophoresis GmBH|TueSponsorsSERVA.png||http://www.serva.de}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/SponsorsTeam:Tuebingen/Sponsors2013-03-24T14:30:00Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
__NOTOC__<br />
= Sponsors =<br />
<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Agilent Technologies|TueSponsorsAgilent.png||http://www.agilent.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Bio-Rad|TueSponsorsBioRad.png||http://www.bio-rad.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Eppendorf|TueSponsorsEppendorf.png||http://www.eppendorf.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Kreissparkasse Tübingen|TueSponsorsSparkasse.png||http://www.ksk-tuebingen.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|New England Biolabs GmbH|TueSponsorsNEB.png||http://www.neb-online.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|SERVA Electrophoresis GmBH|TueSponsorsSERVA.png||http://www.serva.de}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/SponsorsTeam:Tuebingen/Sponsors2013-03-24T14:29:33Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
__NOTOC__<br />
= Sponsors =<br />
<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Agilent Technologies|TueSponsorsAgilent.png|description|http://www.agilent.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Bio-Rad|TueSponsorsBioRad.png||http://www.bio-rad.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Eppendorf|TueSponsorsEppendorf.png||http://www.eppendorf.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Kreissparkasse Tübingen|TueSponsorsSparkasse.png||http://www.ksk-tuebingen.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|New England Biolabs GmbH|TueSponsorsNEB.png||http://www.neb-online.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|SERVA Electrophoresis GmBH|TueSponsorsSERVA.png||http://www.serva.de}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Templates/SponsorEntryTeam:Tuebingen/Templates/SponsorEntry2013-03-24T14:29:24Z<p>Jakobmatthes: </p>
<hr />
<div><h3 class="sponsorname">{{{1}}}</h3><br />
[[Image:{{{2}}}|200px|left]]<br />
{{{3}}}<br />
<br clear="all" /><br />
Website: [{{{4}}} {{{1}}}]</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Templates/SponsorEntryTeam:Tuebingen/Templates/SponsorEntry2013-03-24T14:29:02Z<p>Jakobmatthes: </p>
<hr />
<div><h3 class="sponsorname">{{{1}}}</h3><br />
[[Image:{{{2}}}|200px|left]]<br />
<br clear="all" /><br />
Website: [{{{4}}} {{{1}}}]</div>Jakobmattheshttp://2013.igem.org/File:TueSponsorsEppendorf.pngFile:TueSponsorsEppendorf.png2013-03-24T14:28:36Z<p>Jakobmatthes: </p>
<hr />
<div></div>Jakobmattheshttp://2013.igem.org/File:TueSponsorsSERVA.pngFile:TueSponsorsSERVA.png2013-03-24T14:27:56Z<p>Jakobmatthes: </p>
<hr />
<div></div>Jakobmattheshttp://2013.igem.org/File:TueSponsorsNEB.pngFile:TueSponsorsNEB.png2013-03-24T14:27:10Z<p>Jakobmatthes: </p>
<hr />
<div></div>Jakobmattheshttp://2013.igem.org/File:TueSponsorsBioRad.pngFile:TueSponsorsBioRad.png2013-03-24T14:25:29Z<p>Jakobmatthes: </p>
<hr />
<div></div>Jakobmattheshttp://2013.igem.org/File:TueSponsorsAgilent.pngFile:TueSponsorsAgilent.png2013-03-24T14:24:25Z<p>Jakobmatthes: </p>
<hr />
<div></div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Templates/SponsorEntryTeam:Tuebingen/Templates/SponsorEntry2013-03-24T14:23:32Z<p>Jakobmatthes: </p>
<hr />
<div><h3 class="sponsorname">{{{1}}}</h3><br />
[[Image:{{{2}}}|200px|left]]<br />
<br clear="all" /><br />
<br />
{{{3}}}<br />
<br />
Website: [{{{4}}} {{{1}}}]</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Templates/SponsorEntryTeam:Tuebingen/Templates/SponsorEntry2013-03-24T14:23:15Z<p>Jakobmatthes: </p>
<hr />
<div><h3 class="sponsorname">{{{1}}}</h3><br />
[[Image:{{{2}}}|left]]<br />
<br />
{{{3}}}<br />
<br />
Website: [{{{4}}} {{{1}}}]<br />
<br clear="all" /></div>Jakobmattheshttp://2013.igem.org/File:TueSponsorsSparkasse.pngFile:TueSponsorsSparkasse.png2013-03-24T14:22:34Z<p>Jakobmatthes: </p>
<hr />
<div></div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Template/TuebingenTeam:Tuebingen/Template/Tuebingen2013-03-24T12:29:06Z<p>Jakobmatthes: </p>
<hr />
<div><html><br />
<!-- template base for all sites --><br />
<!-- styles --><br />
</html><br />
<html><br />
<style type="text/css"><br />
/* Droid Sans from Google Web fonts */<br />
@import url(http://fonts.googleapis.com/css?family=Droid+Sans:400,700);<br />
<br />
/* hide elements */<br />
h1.firstHeading {<br />
display: none;<br />
}<br />
<br />
body {<br />
background-color: #f5f5f5;<br />
}<br />
<br />
/* navigation */<br />
#nav {<br />
margin: auto;<br />
}<br />
<br />
#nav ul {<br />
list-style-type: none;<br />
list-style-image: none;<br />
margin: 0;<br />
padding: 0;<br />
height: 2.5em;<br />
}<br />
<br />
#nav li {<br />
display: block;<br />
position: relative;<br />
padding: 0;<br />
margin: 0 .5em 0 .5em;<br />
float: left;<br />
height: 2.7em;<br />
}<br />
<br />
/* main heading */<br />
h1 {<br />
border: 0;<br />
}<br />
<br />
/* edit links on headings */<br />
span.editsection {<br />
font-size: .5em;<br />
color: lightgrey;<br />
}<br />
span.editsection a {<br />
color: lightgrey;<br />
}<br />
</style><br />
</html><br />
<!-- google analytics --><br />
<html><br />
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var _gaq = _gaq || [];<br />
_gaq.push(['_setAccount', 'UA-39523076-1']);<br />
_gaq.push(['_trackPageview']);<br />
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var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true;<br />
ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js';<br />
var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s);<br />
})();<br />
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</html><br />
<br />
<!-- include navigation --><br />
{{:Team:Tuebingen/Templates/Navigation}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Template/TuebingenTeam:Tuebingen/Template/Tuebingen2013-03-24T12:28:49Z<p>Jakobmatthes: </p>
<hr />
<div><html><br />
<!-- template base for all sites --><br />
<!-- styles --><br />
</html><br />
<html><br />
<style type="text/css"><br />
/* Droid Sans from Google Web fonts */<br />
@import url(http://fonts.googleapis.com/css?family=Droid+Sans:400,700);<br />
<br />
/* hide elements */<br />
h1.firstHeading {<br />
display: none;<br />
}<br />
<br />
body {<br />
background-color: #f5f5f5;<br />
}<br />
<br />
body, html, #globalWrapper, #content, * {<br />
font-family: "Droid Sans", sans-serif !important;<br />
}<br />
<br />
/* navigation */<br />
#nav {<br />
margin: auto;<br />
}<br />
<br />
#nav ul {<br />
list-style-type: none;<br />
list-style-image: none;<br />
margin: 0;<br />
padding: 0;<br />
height: 2.5em;<br />
}<br />
<br />
#nav li {<br />
display: block;<br />
position: relative;<br />
padding: 0;<br />
margin: 0 .5em 0 .5em;<br />
float: left;<br />
height: 2.7em;<br />
}<br />
<br />
/* main heading */<br />
h1 {<br />
border: 0;<br />
}<br />
<br />
/* edit links on headings */<br />
span.editsection {<br />
font-size: .5em;<br />
color: lightgrey;<br />
}<br />
span.editsection a {<br />
color: lightgrey;<br />
}<br />
</style><br />
</html><br />
<!-- google analytics --><br />
<html><br />
<script type="text/javascript"><br />
<br />
var _gaq = _gaq || [];<br />
_gaq.push(['_setAccount', 'UA-39523076-1']);<br />
_gaq.push(['_trackPageview']);<br />
<br />
(function() {<br />
var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true;<br />
ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js';<br />
var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s);<br />
})();<br />
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<br />
<!-- include navigation --><br />
{{:Team:Tuebingen/Templates/Navigation}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Template/TuebingenTeam:Tuebingen/Template/Tuebingen2013-03-24T12:28:19Z<p>Jakobmatthes: </p>
<hr />
<div><html><br />
<!-- template base for all sites --><br />
<!-- styles --><br />
</html><br />
<html><br />
<style type="text/css"><br />
/* Droid Sans from Google Web fonts */<br />
@import url(http://fonts.googleapis.com/css?family=Droid+Sans:400,700);<br />
<br />
/* hide elements */<br />
h1.firstHeading {<br />
display: none;<br />
}<br />
<br />
body {<br />
background-color: #f5f5f5;<br />
}<br />
<br />
body, html, #globalWrapper, #content, * {<br />
font-family: "Droid Sans", sans-serif;<br />
}<br />
<br />
/* navigation */<br />
#nav {<br />
margin: auto;<br />
}<br />
<br />
#nav ul {<br />
list-style-type: none;<br />
list-style-image: none;<br />
margin: 0;<br />
padding: 0;<br />
height: 2.5em;<br />
}<br />
<br />
#nav li {<br />
display: block;<br />
position: relative;<br />
padding: 0;<br />
margin: 0 .5em 0 .5em;<br />
float: left;<br />
height: 2.7em;<br />
}<br />
<br />
/* main heading */<br />
h1 {<br />
border: 0;<br />
}<br />
<br />
/* edit links on headings */<br />
span.editsection {<br />
font-size: .5em;<br />
color: lightgrey;<br />
}<br />
span.editsection a {<br />
color: lightgrey;<br />
}<br />
</style><br />
</html><br />
<!-- google analytics --><br />
<html><br />
<script type="text/javascript"><br />
<br />
var _gaq = _gaq || [];<br />
_gaq.push(['_setAccount', 'UA-39523076-1']);<br />
_gaq.push(['_trackPageview']);<br />
<br />
(function() {<br />
var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true;<br />
ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js';<br />
var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s);<br />
})();<br />
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</script><br />
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<br />
<!-- include navigation --><br />
{{:Team:Tuebingen/Templates/Navigation}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Template/TuebingenTeam:Tuebingen/Template/Tuebingen2013-03-24T12:27:48Z<p>Jakobmatthes: </p>
<hr />
<div><html><br />
<!-- template base for all sites --><br />
<!-- styles --><br />
</html><br />
<html><br />
<style type="text/css"><br />
/* Droid Sans from Google Web fonts */<br />
@import url(http://fonts.googleapis.com/css?family=Droid+Sans:400,700);<br />
<br />
/* hide elements */<br />
h1.firstHeading {<br />
display: none;<br />
}<br />
<br />
body {<br />
background-color: #f5f5f5;<br />
}<br />
<br />
body, html, #globalWrapper, #content {<br />
font-family: "Droid Sans", sans-serif;<br />
}<br />
<br />
/* navigation */<br />
#nav {<br />
margin: auto;<br />
}<br />
<br />
#nav ul {<br />
list-style-type: none;<br />
list-style-image: none;<br />
margin: 0;<br />
padding: 0;<br />
height: 2.5em;<br />
}<br />
<br />
#nav li {<br />
display: block;<br />
position: relative;<br />
padding: 0;<br />
margin: 0 .5em 0 .5em;<br />
float: left;<br />
height: 2.7em;<br />
}<br />
<br />
/* main heading */<br />
h1 {<br />
border: 0;<br />
}<br />
<br />
/* edit links on headings */<br />
span.editsection {<br />
font-size: .5em;<br />
color: lightgrey;<br />
}<br />
span.editsection a {<br />
color: lightgrey;<br />
}<br />
</style><br />
</html><br />
<!-- google analytics --><br />
<html><br />
<script type="text/javascript"><br />
<br />
var _gaq = _gaq || [];<br />
_gaq.push(['_setAccount', 'UA-39523076-1']);<br />
_gaq.push(['_trackPageview']);<br />
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var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true;<br />
ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js';<br />
var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s);<br />
})();<br />
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<br />
<!-- include navigation --><br />
{{:Team:Tuebingen/Templates/Navigation}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/SponsorsTeam:Tuebingen/Sponsors2013-03-24T12:26:57Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
__NOTOC__<br />
= Sponsors =<br />
<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Agilent Technologies|TueSponsorsAgilent.png||http://www.agilent.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Bio-Rad|TueSponsorsBioRad.png||http://www.bio-rad.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Eppendorf|TueSponsorsEppendorf.png||http://www.eppendorf.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|Kreissparkasse Tübingen|TueSponsorsSparkasse.png||http://www.ksk-tuebingen.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|New England Biolabs GmbH|TueSponsorsNEB.png||http://www.neb-online.de}}<br />
{{:Team:Tuebingen/Templates/SponsorEntry|SERVA Electrophoresis GmBH|TueSponsorsSERVA.png||http://www.serva.de}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Template/TuebingenTeam:Tuebingen/Template/Tuebingen2013-03-24T12:18:32Z<p>Jakobmatthes: </p>
<hr />
<div><html><br />
<!-- template base for all sites --><br />
<!-- styles --><br />
</html><br />
<html><br />
<style type="text/css"><br />
/* Droid Sans from Google Web fonts */<br />
@import url(http://fonts.googleapis.com/css?family=Droid+Sans:400,700);<br />
<br />
/* hide elements */<br />
h1.firstHeading {<br />
display: none;<br />
}<br />
<br />
body {<br />
background-color: #f5f5f5;<br />
font-family: "Droid Sans", sans-serif;<br />
}<br />
<br />
/* navigation */<br />
#nav {<br />
margin: auto;<br />
}<br />
<br />
#nav ul {<br />
list-style-type: none;<br />
list-style-image: none;<br />
margin: 0;<br />
padding: 0;<br />
height: 2.5em;<br />
}<br />
<br />
#nav li {<br />
display: block;<br />
position: relative;<br />
padding: 0;<br />
margin: 0 .5em 0 .5em;<br />
float: left;<br />
height: 2.7em;<br />
}<br />
<br />
/* main heading */<br />
h1 {<br />
border: 0;<br />
}<br />
<br />
/* edit links on headings */<br />
span.editsection {<br />
font-size: .5em;<br />
color: lightgrey;<br />
}<br />
span.editsection a {<br />
color: lightgrey;<br />
}<br />
</style><br />
</html><br />
<!-- google analytics --><br />
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var _gaq = _gaq || [];<br />
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_gaq.push(['_trackPageview']);<br />
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var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true;<br />
ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js';<br />
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})();<br />
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<br />
<!-- include navigation --><br />
{{:Team:Tuebingen/Templates/Navigation}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/Template/TuebingenTeam:Tuebingen/Template/Tuebingen2013-03-24T12:18:00Z<p>Jakobmatthes: </p>
<hr />
<div><html><br />
<!-- template base for all sites --><br />
<!-- styles --><br />
</html><br />
<html><br />
<style type="text/css"><br />
/* Droid Sans from Google Web fonts */<br />
@import url(http://fonts.googleapis.com/css?family=Droid+Sans:400,700);<br />
<br />
/* hide elements */<br />
h1.firstHeading {<br />
display: none;<br />
}<br />
<br />
body {<br />
background-color: #f5f5f5;<br />
}<br />
<br />
/* navigation */<br />
#nav {<br />
margin: auto;<br />
}<br />
<br />
#nav ul {<br />
list-style-type: none;<br />
list-style-image: none;<br />
margin: 0;<br />
padding: 0;<br />
height: 2.5em;<br />
}<br />
<br />
#nav li {<br />
display: block;<br />
position: relative;<br />
padding: 0;<br />
margin: 0 .5em 0 .5em;<br />
float: left;<br />
height: 2.7em;<br />
}<br />
<br />
/* main heading */<br />
h1 {<br />
border: 0;<br />
}<br />
<br />
/* edit links on headings */<br />
span.editsection {<br />
font-size: .5em;<br />
color: lightgrey;<br />
}<br />
span.editsection a {<br />
color: lightgrey;<br />
}<br />
</style><br />
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<!-- google analytics --><br />
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{{:Team:Tuebingen/Templates/Navigation}}</div>Jakobmattheshttp://2013.igem.org/Team:Tuebingen/LabNotebookTeam:Tuebingen/LabNotebook2013-03-24T12:14:41Z<p>Jakobmatthes: </p>
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= Lab Notebook =<br />
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[[Category:Team:Tuebingen/Lab]]</div>Jakobmatthes