Team:Bielefeld-Germany/Test

From 2012.igem.org

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==Week 25 (10/15 - 10/21/12)==
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{{Team:Bielefeld/Head}}
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<a href="https://2012.igem.org/Team:Bielefeld-Germany/Public_relations_Overview#8"><img src="http://2012.igem-bielefeld.de/includes/wiki/images/Pfeil_links2.png"></a>
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        <div id=page-title>
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            <span id=page-title-text>
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                How our project reaches the world
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  <div id="grey_bg">
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<table>
 
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<!-- Flexslider CSS and JS -->
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<link rel="stylesheet" href="http://2012.igem-bielefeld.de/includes/wiki/flex/css/flexslider.css" type="text/css">
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<script src="https://ajax.googleapis.com/ajax/libs/jquery/1.6.2/jquery.min.js"></script>
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<script src="http://2012.igem-bielefeld.de/includes/wiki/flex/js/jquery.flexslider.js"></script>
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===Monday October 15th===
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<!-- Load the flexslider -->
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* '''Team Shuttle Vector:'''
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<script type="text/javascript" charset="utf-8">
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** There are no colonies of pBS1C3::BBa_K863202 KRX transformantion.
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  $(window).load(function() {
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** The P. pastoris GS115 cells integrated with BBa_K863204::GFP were harvested and the supernatant was examined on GFP by fluorescence. But there was no GFP. The next steps are: disrupt the cells and test the supernatant on GFP. Maybe the GFP could not be secreted.
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    $('.flexslider').flexslider({
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* '''Team Fungal and Plant Laccases:'''
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          animation: "fade",  // slide or fade
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** Yeah, the yeast cells are growing.
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          slideshow: false,
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* '''Team Cellulose Binding Domain:'''
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          controlsContainer: ".flex-container" // the container that holds the flexslider
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** Tried to isolate the eight plated, but did get low concentrations again (except one J61101+GFP_Freiburg+CBDcex_Freiburg with conc >100 ng/µL)
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    });
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** cleaned up some more PCR-products (GFP_Freiburg; CBDcex_Freiburg; CBDclos_Freiburg)
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  });
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*'''Team Substrate Analysis''': We stopped the reaction from the 12th October with Methanol and gave it to Dr. Marcus Persicke to measure the degradation on LC-MS
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</script>
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===Tuesday October 16th===
 
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* '''Team Activity Tests:'''
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<!-- Slider Container and Images -->
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** Today we received the samples from Team Cultivation & Purification. We are really motivated to measure the activity of our new produced enzymes. But before that we have to prepare the samples. This implied the determination of the protein concentration in each fraction, which we did today. The results are listed in the following table:
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<div style="padding:5px; z-index:1;">
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[[File:Bielefeld2012_Prot_vor_Umpuffern.jpg|300px|thumb|center]]
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        <div class="flexslider" style="margin-top:10px;">
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            <ul class="slides">
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                <li>
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                    <div class="frontpage-slide">
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                        <img src=""></a>
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                        <div class="slide-right-col">
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* '''Team Cellulose Binding Domain:'''
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** Transformed B0034 into KRX
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** Transformed J23100 into KRX
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** Plated both on AMP-select-agar
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** Plated one more Colony of J61101 + GFP_Freiburg and J61101 + GFP_Freiburg + CBDclos
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region=DE Laccase] || 150 µL ||
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|-
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|}
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*'''Team Substrate Analysis:''' The Massspectrometry data showed two potential products after the Laccase treatment of Estradiol and Ethinyl estradiol but we unfortunatly could not identify them so we decided to do MS-MS on thoose. The products showed that after Laccase treatment both Estradiol and Ethinyl estradiol losses two '''H''' atoms which was new for us.
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===Wednesday October 17th===
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                        </div>
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* '''Team Activity Tests:'''
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                        <div class=clear></div>
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** Before starting our activity assay we re-buffered the fractions we got from Team Cultivation and Purification. It took some time, that's why we had no time for activity analysis yet. But we prepared everything for the next day, including determining the protein concentration of the fractions after they have been re-buffered. This is what we got:
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                    </div>
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[[File:Bielefeld2012_Prot_nach_Umpuffern.jpg|300px|thumb|center]]
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                </li>
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* '''Team Shuttle Vector:'''
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                <li>
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** Genomic DNA isolation of yeast cells with integrated BBa_K863204::GFP construct was done with the Kit. For genotype characterisation an PCR with the praimer pair 5AOX-Genotyp-FW and TT-Genotyp-RV was done and the fragment size was determined.
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                    <div class="frontpage-slide">
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* '''Team Fungal and Plant Laccases:'''
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                        <img src=""></a>
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** YPD liquid culture was inoculated with one yeast colony (BBa_K863204::TV5) and incubated at 30°C and 150 rpm.
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                        <div class="slide-right-col">
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* '''Team Cellulose Binding Domain:'''
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** Picked colonies of B0034 and J23100 and plated them on AMP selection agar for plasmid isolation
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* '''Team Site Directed Mutagenesis:'''
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** SDM-PCR of the Shuttle vector
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** Added ''Dpn''I over night
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===Thursday October 18th===
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                        </div>
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* '''Team Activity Tests:'''
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                        <div class=clear></div>
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** Finally the day had come to measure the activity of our own produced laccases. This activity assay should give information about the enzyme content in every fraction. To make the measurements comparable we applied the same protein amount in every sample. Again, we used our [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#General_setup_of_enzyme_activity_measurements/ standard activity assay protocol], but this time we used the [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Britton-Robinson_Buffer/ Britton-Robinson Buffer] at pH 5. Also we applied 0.1 mM ABTS and measured over night.
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                    </div>
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[[File:Bielefeld2012_new_ECOL_activity.jpg|360px|thumb|left|Activity assay of each purified fraction of our new cultivation with ECOL. Samples were re-buffered into H<sub>2</sub> and the protein amount in each fraction has been adjusted. The Measurement was done using the [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#General_setup_of_enzyme_activity_measurements/ standard activity assay protocol] over night.]][[File:Bielefeld2012_new_BPUL_acitivity.jpg|360px|thumb|right|Activity assay of each purified fraction of our new cultivation with BPUL. Samples were re-buffered into H<sub>2</sub> and the protein amount in each fraction has been adjusted. The Measurement was done using the [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#General_setup_of_enzyme_activity_measurements/ standard activity assay protocol] over night.]]
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                </li>
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[[File:Bielefeld2012_new_BHAL_activity.jpg|360px|thumb|left|Activity assay of each purified fraction of our new cultivation with BHAL. Samples were re-buffered into H<sub>2</sub> and the protein amount in each fraction has been adjusted. The Measurement was done using the [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#General_setup_of_enzyme_activity_measurements/ standard activity assay protocol] over night.]][[File:Bielefeld2012_new_TTHL_activity.jpg|360px|thumb|right|Activity assay of each purified fraction of our new cultivation with TTHL. Samples were re-buffered into H<sub>2</sub> and the protein amount in each fraction has been adjusted. The Measurement was done using the [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#General_setup_of_enzyme_activity_measurements/ standard activity assay protocol] over night.]]
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                <li>
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
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                    <div class="frontpage-slide">
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                        <img src=""></a>
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                        <div class="slide-right-col">
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                        </div>
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                        <div class=clear></div>
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                    </div>
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                </li>
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                <li>
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                    <div class="frontpage-slide">
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                        <img src=""></a>
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                        <div class="slide-right-col">
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* '''Team Cellulose Binding Domain:'''
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                        </div>
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** Isolated J23100 plasmid
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                        <div class=clear></div>
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** Digested J23100 with ''Eco''RI and ''Pst''I
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                    </div>
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** Digested pSB1C3-Backbone with ''Eco''RI and ''Pst''I
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                </li>
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** Clean up of pSB1C3-BB and J23100 + RFP - Insert via Gel
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                <li>
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** Ligation of J23100 and pSB1C3-BB
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                    <div class="frontpage-slide">
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** Isolated B0034 plasmid
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                        <img src=""></a>
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** Digested B0034 with ''Spe''I and ''Pst''I
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                        <div class="slide-right-col">
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** Digested GFP_Freiburg-PCR-product with ''Xba''I and ''Pst''I
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** Digested an other fraction of the GFP_Freiburg-PCR-product with ''Xba''I and ''Age''I
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** Assembled (Ligation):
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*** B0034 with GFP_Freiburg
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*** B0034 with GFP_Freiburg and CBDcex_Freiburg
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*** B0034 with GFP_Freiburg and CBDclos_Freiburg
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** Transformed these three into KRX
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* '''Team Site Directed Mutagenesis:'''
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** Bands of 19C-PRC-product in Gel at 7 kbp (which is correct)
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</div>
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** Clean-Up of 19C-PRC-product
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                        <div class=clear></div>
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** Transformation of the 19C-PRC-product in XL1 Blue
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</div>
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                </li>
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            </ul>
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        </div>
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===Friday October 19th===
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    </div>
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* '''Team Activity Tests:'''
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** After getting our interesting activity measurement results, we had to figure out which fraction is going to be used in the following experiments and how much laccase it contains besides other proteins. We agreed, that the most active fraction contains 90 % laccase, which is commonly used in the literature. Since we applied the same protein amount of each fraction for the activity measurements, we made sure that the results really correspond to the amount of laccase. These are the most active fractions we have chosen with their contained laccase amount:
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<ul>
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*** ECOL: Fraction 50% 2 with a laccase concentration of 63,9 µg mL<sup>-1</sup>.
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*** BPUL: Fraction 50% 2 with a laccase concentration of 25,1 µg mL<sup>-1</sup>.
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<li><b>We did it! About our qualification for the World Championship in Boston</b><br><br>
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*** BHAL: Fraction  5% 3 with a laccase concentration of 10,9 µg mL<sup>-1</sup>.
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*** TTHL: The best fraction of our TTHL laccase was fraction 50% 1 with a laccase concentration of 4,03 µg mL<sup>-1</sup>. In regard to Team Immobilization and Team Substrate Analysis we had to reach a higher amount of TTHL laccase. So we thought of combining two fractions. We chose the second best fraction of TTHL, which is 5% 3. To calculate the amount of contained laccase we had to compare the activity and reconsidered the slope. Since we stated the fraction with its highest activity as 90 % contained laccase, we calculated the amount of laccase regarding to that one. The following figure shows the slopes we got out of the activity from fraction 50% 1 and fraction 5% 3 (which is the second best). Accordingly to this, fraction 5% 3 contains 68,9% of laccase, which is 4,8 µg mL<sup>-1</sup>. In total, the composed fraction contains 4,4 µg mL<sup>-1</sup>.
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&#10158; <a href="http://www.laborwelt.de/aktuelles/nachrichten/2012-q4/igem-fuenf-teams-nach-boston.html">iGEM: Fünf Teams nach Boston, Laborwelt - 10/09/12</a><br>
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[[File:Bielefeld2012_Regression.png|center|450px|thumb|Slope of the two most active fractions of purified TTHL laccase. By comparing them, the amount of contained laccase in the fractions had been calculated.]]
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 +
&#10158; <a href="http://www.biotechnologie.de/BIO/Navigation/DE/root,did=154774.html?listBlId=74462&">iGEM: Fünf Teams lösen Ticket für Boston, biotechnologie.de - 10/10/12</a></li><br>
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<li><b>Neue Züricher Zeitung</b><br><br>
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&#10158; <a href="http://www.nzz.ch/wissen/wissenschaft/arzneimittel-in-gewaessern-minimieren-1.17368069">Arzneimittel in Gewässern minimieren - 07/18/12</a></li>
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<br>
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<li><b>Neue Westfälische</b><br><br>
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&#10158; <a href="https://static.igem.org/mediawiki/2012/7/7e/Article_NW.png">Das Wasser wird weiblich - Article in our local newspaper Neue Westfälische, Bielefelder Tageblatt, No. 160, Author: Uwe Pollmeier - 07/12/12</a></li>
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<br>
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<li><b>LIVE radio interview at Radio Hertz</b><br><br>
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&#10158; <a href="http://www.radiohertz.de/beta-site/livestream/">07/04/2012 at 6pm</a></li>
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<br>
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<li><b>WDR5 Leonardo</b><br><br>
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&#10158; <a href="http://www.wdr5.de/sendungen/leonardo/s/d/06.09.2012-16.05/b/hormone-und-betablocker-im-gewaesser.html">09/06/2012 at 4pm</a></li>
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<br>
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<li><b>Our project reaches the world </b><br><br>
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&#10158; <a href="http://www.wired.co.uk/news/archive/2012-06/25/tree-fungus-oestrogen-filter">Great Britain - Wired.co.uk - 06/25/2012</a><br>
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&#10158; <a href="http://scienceblog.com/55186/filtering-estrogen-from-drinking-water/?utm_source=feedburner&utm_medium=feed&utm_campaign=Feed%3A+scienceblogrssfeed+%28ScienceBlog.com%29">Scienceblog - 06/25/2012</a><br>
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&#10158; <a href="http://www.news-medical.net/news/20120626/Biological-filter-to-remove-estrogens-from-waste-water-and-drinking-water.aspx">Australia - News Medical - 06/26/2012</a><br>
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&#10158; <a href="http://savethewater.org/tag/bielefeld/">Los Angeles, USA - Save the Water<sup>TM</sup> - 06/26/2012</a><br>
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&#10158; <a href="http://www.eurekalert.org/pub_releases/2012-06/uob-ref062512.php">Washington, DC - EurekAlert! - 06/25/2012</a><br>
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&#10158; <a href="http://savethewater.org/tag/bielefeld/">Hollywood, USA - Save the water - 06/26/2012</a></li><br>
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&#10158; <a href="http://www.biofuelsdigest.com/biobased/2012/06/27/bielefeld-university-students-participating-in-igem-competition/">Miami, USA - BioBasedDigest - 06/27/2012</a></li><br>
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&#10158; <a href="http://phys.org/news/2012-06-estrogen.html"> Phys.org - 06/25/2012</a><br>
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&#10158; <a href="http://www.redorbit.com/news/science/1112645127/drinking-water-minus-the-estrogen/">Texas - RedOrbit - 06/25/2012</a><br>
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&#10158; <a href="http://www.provital-blog.com/en/nutrition/drinks/biological-filter-to-remove-estrogens-from-waste-water-and-drinking-water/">Provital-blog.com - 06/30/2012</a><br>
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&#10158; <a href="http://www.innovations-report.com/html/reports/awards_funding/removing_estrogen_drinking_water_197866.html"> Schmitten, Germany - innovations report - 06/25/2012</a><br>
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&#10158; <a href="http://www.pressrelations.de/new%5C/standard/result_main.cfm?r=499469&sid=&aktion=jour_pm&quelle=0&n_firmanr_=102898&pfach=1&detail=1&sektor=pm&popup_vorschau=0"> Düsseldorf, Germany - pressrelations - 06/25/2012</a><br>
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&#10158; <a href="http://www.sciencecodex.com/removing_estrogen_from_drinking_water-93912">Science Codex - 06/25/2012</a><br>
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&#10158; <a href="http://healthresearchreport.me/2012/07/01/132nd-issue-health-technology-research-synopsis-29-jun-2012/">Health Research Report - 06/29/2012</a><br>
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&#10158; <a href="http://www.scoop.it/t/syntheticbio/p/2037868685/removing-estrogen-from-drinking-water">Scoop.it! - 06/26/2012</a><br>
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&#10158; <a href="http://www.medindia.net/news/estrogen-removal-from-drinking-water-using-biological-filters-103306-1.htm?utm_source=feedburner&utm_medium=feed&utm_campaign=Feed%3A+allhealthnews+%28Medindia+Health+News%29">Chennai, India - MedINDIA Network for Health - 06/27/2012</a><br>
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&#10158; <a href="http://www.analytic-news.com/press/2012/69.html">Ober-Ramstadt, Germany - News Analytic - 06/26/2012</a><br>
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 +
<li><b>Removing estrogen from drinking water</b><br><br>
 +
&#10158; <a href="http://idw-online.de/pages/de/news484982">Informationsdienst Wissenschaft - 06/25/2012</a><br>
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 +
&#10158; <a href="http://www.bio.nrw.de/nachrichten">BIO.NRW - 06/25/2012</a><br>
 +
 
 +
&#10158; <a href="http://www.laborpraxis.vogel.de/forschung-und-entwicklung/analytik/articles/369125/">Laborpraxis.de - 06/25/2012</a>
 +
<br>
 +
&#10158; <a href="http://www.uni-protokolle.de/nachrichten/id/240229/">uni-protokolle.de - 06/25/12</a><br>
 +
 
 +
&#10158; <a href="http://mag3.i-magazine.de/magPage/gwf_Wasser_Abwasser_Anlagen_und_Verfahren_zur_Wasseraufbereitung/Seite_28/#/28"> gwf Wasser Abwasser - 7-8/2012 p. 766-767</a></li><br>
 +
 
 +
&#10158; <a href="http://www.bio.nrw.de/en/latest_news"> BioNRW </a></li><br>
 +
 
 +
<li><b>Students from Bielefeld University are participating in the international iGEM competition</b><br><br>
 +
 
 +
&#10158; <a href="http://ekvv.uni-bielefeld.de/blog/pressemitteilungen/entry/%C3%B6strogen_aus_trinkwasser_entfernen_nr">Bielefeld University - 06/25/2012 - German</a><br>
 +
&#10158; <a href="http://ekvv.uni-bielefeld.de/blog/uninews/entry/removing_estrogen_from_drinking_water">Bielefeld University - 06/25/2012 - English</a><br>
 +
&#10158; <a href="http://www.laborjournal.de/rubric/aktuell/index.lasso">Synthetische Biologie: Das Bielefelder iGEM-Team, Laborjournal - 10/2012</a></li><br>
 +
 
 +
 
 +
<li><b>Street Science</b><br><br>
 +
&#10158; <a href="http://www.bielefeld.de/de/pressedienst/detail.html?id=2012-08-21-12.20.37.146281"> Press release for our Street Science Day - 06/25/2012</a><br>
 +
 
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&#10158; <a href="https://static.igem.org/mediawiki/2012/b/bf/Street_Science_NW_2708.jpg">DNA isolieren? Das kann doch jeder! (Street Science) - Article in our local newspaper Westfalen-Blatt - 08/27/12</a><br>
 +
 
 +
&#10158; <a href="https://static.igem.org/mediawiki/2012/f/fe/Street_Science_NW_2708_2.jpg">Spielerische Wissenschaft (Street Science) - Article in our local newspaper Neue Westfälische, Bielefelder Tageblatt, No. 199, Author: Jonny Dähne - 08/27/12</a></li><br>
 +
 
 +
</ul>
 +
 
 +
</p>
 +
</div>
 +
</html>
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* '''Team Cellulose Binding Domain:'''
 
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** Colony-PCR of B0034 + GFP_Freiburg; B0034 + GFP_Freiburg + CBDcex; B0034 + GFP_Freiburg + CBDcex_Freiburg; B0034 + GFP_Freiburg + CBDclos_Freiburg.
 
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*** All negative
 
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** Restriction of Ecol_Freiburg with ''Xba''I, ''Age''I and ''Dpn''I
 
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* '''Team Site Directed Mutagenesis:'''
 
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===Saturday October 20th===
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{{Team:Bielefeld/Sponsoren}}
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===Sunday October 21st===
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* '''Team Cellulose Binding Domain:'''
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** Colonies PCRs of B0034 + GFP_Freiburg; B0034 + GFP_Freiburg + CBDcex_Freiburg; B0034 + GFP_Freiburg + CBDclos_Freiburg
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*** All negativ
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** Transformed once again (with an mix of old an new GFP_Freiburg)
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** Isolated plated colonies of transformed colonies (two GFP; two GFP+Cex; two GFP+Clos
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** Restriction analysis showed only bands at 2K bp (pSB1C3+BBa_B0034 vector without insert)
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* '''Team Site Directed Mutagenesis:'''
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** Isolated the plasmids of the six SDM-dishes
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** Over-night digestion with ''Not''I
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Latest revision as of 20:20, 26 October 2012

How our project reaches the world


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About our Wiki

Fifteen students from different fields of study from the Bielefeld University aspired to complete a project called TOXIC COMPOUNDS IN NATURAL WATER - A CASE FOR LACCASE. In his Wiki we document our achievements and our approach.

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Universität Bielefeld
Universitätsstraße 25
D-33615 Bielefeld

info(at)igem-bielefeld.de

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