Team:TU Darmstadt/labbook

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<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a>
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/solution">
 
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<img alt="solution" src="/wiki/images/8/87/Darmstadt_green_Solution.jpg" width="100" height="30"></a>
 
<a href="https://2013.igem.org/Team:TU_Darmstadt/result">
<a href="https://2013.igem.org/Team:TU_Darmstadt/result">
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook">
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook">
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<img alt="team" src="/wiki/images/f/f3/Darmstadt_green_Labbook.jpg" width="90" height="30"></a>
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{| style="color:green;background:none;"
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<br><br><br><br>
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!align="center"|[[Team:TU_Darmstadt/labbook|<span style="color:white;font-size:160%;"> Labbook |</span>]]
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<center>
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!align="center"|[[Team:TU_Darmstadt/protocols|<span style="color:white;font-size:160%;"> Protocols |</span> ]]
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook">
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!align="center"|[[Team:TU_Darmstadt/materials|<span style="color:white;font-size:160%;"> Materials |</span>]]
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<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font>
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|}
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</a>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/materials">
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<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font>
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</a>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols">
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<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font>
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</a>
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<html>
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<head>
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<br><br>
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<meta name="author" content="Anne Schieferdecker">
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<h2><font size="6" color="#F0F8FF" face="Arial regular">Detection construct</font></h2> <br>
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<font size="3" color="#F0F8FF" face="Arial regular">
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<p text-aligne:left style="margin-left:50px; margin-right:50px">
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The assembly of our detection construct proved to be tricky. Not only the traditional cloning approach failed but also the assembly using synthesized gBlocks delivered no results. We finally turned to an In-Fusion cloning approach to assemble our construct. Visit our lab book to read more about our work flow.
 +
 
 +
</p></font>
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 +
<br><br>
 +
 
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook/DetectionConstruct">
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<img alt="Labbookdetection" border="5" width="57,5" height="50" src="/wiki/images/8/81/Book2.gif">
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<font size="6" color="#F0F8FF" face="Arial regular">Visit our lab book for the detection construct</font><br>
 +
</a>
 +
 
 +
 
 +
<br><br>
 +
 
 +
<h2><font size="6" color="#F0F8FF" face="Arial regular">Fluorescence proteins</font></h2> <br>
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<font size="3" color="#F0F8FF" face="Arial regular">
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<p text-aligne:left style="margin-left:50px; margin-right:50px">
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The genes for our FRET pair, mKate and LssmOrange, were isolated by PCR, cloned into the biobrick vector and sequenced afterwards. The genes were also cloned into a commercially available expression vector in order to characterize the fluorescent proteins. Visit our lab book to read more about our work flow.
 +
<center>
<body>
<body>
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<br>
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        <tr>
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<br>
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                <td>13.08</td>
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                <td>gBLOCK assembly of CMK and TLO</td>
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                <td></td>
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<img alt="mkate" src="/wiki/images/3/3a/Mkate-active_light1.png" width="50%" height="50%" align="right">
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        </tr>
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        <tr>
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<img alt="mkateactive" src="/wiki/images/5/58/Mkate-active_surface_activesite4_light1.png" width="50%" height="50%" align="right">
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                <td></td>
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                <td><ul><li>PCR mixture
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<br>
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                                <ul><li>1 µL of each gBLOCK
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<br>
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                                <ul>
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</p></font>
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                                        <li>CMK: B_01 - B_04</li>
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                                        <li>TLO: A_01 - A_06</li>
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<br><br>
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                                </ul></li>
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                                <li>10 µL 5x Q5 Reaction Buffer</li>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook/Biobricks">
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                                <li>2 µL dNTPs</li>
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<img alt="Labbookbiobricks" border="5" width="57,5" height="50" src="/wiki/images/8/81/Book2.gif">
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                                <li>1 µL Q5 Hot Start Polymerase</li>
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<font size="6" color="#F0F8FF" face="Arial regular">Visit our lab book for the fluorescence proteins</font><br>
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                                <li>10 µL 5x Q5 High GC Enhancer</li>
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</a>
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                                <li>1 µL primer suffix-R (10 mM)</li>
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<br>
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                                <li>1 µL primer prefix_R (10 mM)</li>
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-
                        </ul></ul></li>
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<h2><font size="6" color="#F0F8FF" face="Arial regular">Safety</font></h2> <br>
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                        <ul><li>PCR program (40 cycles)
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                                <ul><li>initial denaturation 94°C, 100s</li>
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                                <li>denaturation 94°C, 55s</li>
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<font size="3" color="#F0F8FF" face="Arial regular">
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                                <li>annealing 64°C, 55s</li>
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<p text-aligne:left style="margin-left:50px; margin-right:50px">
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                                <li>elongation 72°C, 120s</li>
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                                <li>final elongation 72°C, 300s</li>
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The assembly of the blue-light sensitive pDawn construct from gBlocks failed. Although the construction of the pezT construct from gBlocks was successful, multiple transformations of pSB1C3-pezT failed. Visit our lab book to read more about our work flow.
-
                        </li></ul></ul>
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                        <ul><li>preparative 1% agarose gel
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</p></font>
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                                <ul><li>gel displays bands of expected size ' assembly was successful</li>
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<br><br>
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                        </ul></li></ul>
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                </td>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/safety/Labjournal">
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                <td><img src="https://static.igem.org/mediawiki/2013/6/6c/Darmstadt13_InFusion_130813.png" alt=""</td>
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<img alt="Labbookbiobricks" border="5" width="57,5" height="50" src="/wiki/images/8/81/Book2.gif">
-
        </tr>
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<font size="6" color="#F0F8FF" face="Arial regular">Visit our lab book for the safety construct</font><br />
-
      </table>
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</a>
 +
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;
 +
 
 +
<h2><font size="6" color="#F0F8FF" face="Arial regular">Cutinase</font></h2> <br>
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 +
 
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<font size="3" color="#F0F8FF" face="Arial regular">
 +
<p text-aligne:left style="margin-left:50px; margin-right:50px">
 +
 
 +
Here, we present the improvement approach of the <i>Fusarium Solani Cutinase</i> (FsC, PID: BBa K808025).
 +
The FsC was one of the PET cleavage enzymes from the iGEM Team TU-Darmstadt 2012. We improved this part
 +
with a pelB leader and a reporter(mRFP1-His6) system with a TEV cleavage site. This construct is regulated by pBAD
 +
(PID: BBa I0500) and cloned into the pSB1C3 backbone.
 +
 
 +
</p></font>
 +
<center>
 +
<img alt="fsc" src="/wiki/images/0/0d/Fscpet2.png" width="50%" height="50%">
 +
<br><br>
 +
 
 +
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook/Cutinase">
 +
<img alt="Labbookdetection" border="5" width="57,5" height="50" src="/wiki/images/8/81/Book2.gif">
 +
<font size="6" color="#F0F8FF" face="Arial regular">Visit our lab book for the cutinase</font><br />
 +
</a>
 +
 
 +
 
</body>
</body>
 +
</html>
</html>

Latest revision as of 03:13, 5 October 2013







Lab book | Materials | Protocols


Detection construct


The assembly of our detection construct proved to be tricky. Not only the traditional cloning approach failed but also the assembly using synthesized gBlocks delivered no results. We finally turned to an In-Fusion cloning approach to assemble our construct. Visit our lab book to read more about our work flow.



Labbookdetection Visit our lab book for the detection construct

Fluorescence proteins


The genes for our FRET pair, mKate and LssmOrange, were isolated by PCR, cloned into the biobrick vector and sequenced afterwards. The genes were also cloned into a commercially available expression vector in order to characterize the fluorescent proteins. Visit our lab book to read more about our work flow.



mkate mkateactive



Labbookbiobricks Visit our lab book for the fluorescence proteins

Safety


The assembly of the blue-light sensitive pDawn construct from gBlocks failed. Although the construction of the pezT construct from gBlocks was successful, multiple transformations of pSB1C3-pezT failed. Visit our lab book to read more about our work flow.



Labbookbiobricks Visit our lab book for the safety construct
                

Cutinase


Here, we present the improvement approach of the Fusarium Solani Cutinase (FsC, PID: BBa K808025). The FsC was one of the PET cleavage enzymes from the iGEM Team TU-Darmstadt 2012. We improved this part with a pelB leader and a reporter(mRFP1-His6) system with a TEV cleavage site. This construct is regulated by pBAD (PID: BBa I0500) and cloned into the pSB1C3 backbone.

fsc

Labbookdetection Visit our lab book for the cutinase

Retrieved from "http://2013.igem.org/Team:TU_Darmstadt/labbook"