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| - | <a name="c0051"></a><p class="header"><b>Characterization of BBa_C0051, one of the most widely used and studied parts</b></a><p>
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| - | <p class="indent">Bba_C0051 cI-Lamba, the promoter in disguise
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| - | During the construction of one of our larger pieces of the spatial oscillator, the ON switch, we stumbled upon an unexpected phenotype.</a><br />
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| - | <img src="https://static.igem.org/mediawiki/2010/1/17/Uhoh1.jpg"><p>
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| - | <a name="problem"></a><p class="header"><b>The Problem</b></a><p>
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| - | <table border=0 margin="0" width="600px" padding:0 margin:0>
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| - | <tr>
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| - | <td><img src="https://static.igem.org/mediawiki/2010/5/55/Uhohtest1.jpg"></td>
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| - | <td>
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| - | <ul>
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| - | <li>Our intermediate ligation of RBS-C0051 to RBS-RFP produced a red phenotype but it has no promoter! </li>
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| - | <li>Since Bba_C0051 is a highly studied and widely used part in the registry, it presents a huge problem not only in the context of the spatial oscillator but also in the 76 parts that utilize it throughout the registry.</li>
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| - | <li>This exciting new find causes many problems in our design and must cause malfunctions in a huge number of parts entered and used in the registry. </li>
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| - | </ul>
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| - | </td>
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| - | </tr>
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| - | <tr>
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| - | <td><img src="https://static.igem.org/mediawiki/2010/7/77/RBS_C0051_RBS_RFP_F1610.png"></td>
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| - | <td>
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| - | </td>
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| - | </tr>
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| - | </table><p><br />
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| - | <a name="investigation"></a><p class="header"><b>The Investigation</b></a><p>
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| - | <table border=0 margin="0" width="600px" padding:0 margin:0>
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| - | <tr>
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| - | <td>
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| - | <ul>
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| - | <li>We began by sequencing the DNA of the red colonies from the ligation of RBS-C0051 to RBS-RFP in order to rule out the presence of any promoter or mutation. The sequence analysis revealed that we did indeed have the correct sequence.</li>
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| - | <li>Further investigation into Bba_C0051 showed that the part not only contained the coding region, cI, but it is also followed by an LVA degradation tag and a barcode. When looking further into the RBS (Bba_E1010), we found that it also contained an LVA degradation tag and a barcode after the RFP coding region. </li>
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| - | <li>We then conducted a number of experiments in order to determine which part or combination of parts contained in the construct caused this unwanted promoter-like behavior.</li>
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| - | <li>After determining the cause of the unwanted transcription, we continued to conduct a number of experiments in order to measure the results.</li>
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| - | </ul>
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| - | </td>
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| - | </tr>
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| - | <tr>
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| - | <td>
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| - | <img src="https://static.igem.org/mediawiki/2010/3/3c/C0051zoomed.jpg">
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| - | </td>
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| - | </tr>
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| - | <tr>
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| - | <td>
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| - | <img src="https://static.igem.org/mediawiki/2010/a/ad/RFPzoomed.jpg">
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| - | </td>
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| - | </tr>
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| - | <tr>
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| - | </table><p><br />
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| - | <a name="test1"></a><p class="header"><b>Test #1</b></a><p>
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| - | <table border=0 margin="0" width="600px" padding:0 margin:0>
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| - | <tr>
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| - | <td>
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| - | <img src="https://static.igem.org/mediawiki/2010/0/04/Uhohtest1x.jpg">
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| - | </td>
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| - | <td>
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| - | <ul>
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| - | <li>We ligated RBS-RFP to RBS-RFP to see if transcription occurred. </li>
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| - | <li>The construct exhibited a white phenotype. Thus, we concluded that transcription didn't start in any part of the RFP</li>
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| - | </ul>
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| - | </tr>
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| - | <tr>
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| - | <td>
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| - | <img src="https://static.igem.org/mediawiki/2010/2/2f/Whitecolonies.jpg">
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| - | </td>
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| - | </tr>
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| - | </table>
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| - | <a name="test2"></a><p class="header"><b>Test #2</b></a><p>
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| - | <table border=0 margin="0" width="600px" padding:0 margin:0>
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| - | <tr>
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| - | <td>
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| - | <img src="https://static.igem.org/mediawiki/2010/6/68/Uhohtest2x.jpg">
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| - | </td>
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| - | <td>
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| - | <ul>
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| - | <li>We ligated RBS-RFP to Chris Anderson's Promoter Screening Plasmid. </li>
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| - | <li>We also obtained white colonies.</li>
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| - | </ul>
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| - | </td>
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| - | </tr>
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| - | <tr>
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| - | <td>
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| - | <img src="https://static.igem.org/mediawiki/2010/b/b5/RFP_PSP.png">
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| - | </td>
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| - | </tr>
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| - | </table><p><br />
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| - | <a name="test3"></a><p class="header"><b>Test #3</b></a><p>
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| - | <table border=0 margin="0" width="600px" padding:0 margin:0>
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| - | <tr>
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| - | <td>
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| - | <img src="https://static.igem.org/mediawiki/2010/4/41/Uhohtest3x.jpg">
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| - | </td>
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| - | <td>
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| - | <ul>
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| - | <li>We ligated RBS-C0051 to Chris Anderson's Promoter Screening Plasmid.</li>
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| - | <li>And we have obtained red colonies! We know that the transcription initiation is somewhere in the BBa_C0051 region.</li>
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| - | </ul>
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| - | </td>
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| - | </tr>
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| - | <tr>
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| - | <td>
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| - | <img src="https://static.igem.org/mediawiki/2010/9/9b/C0051_PSP.png">
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| - | </td>
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| - | </tr>
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| - | </table>
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| - | <a name="5race"></a><p class="header"><b>5' Race PCR</b></a><p>
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| - | <table border=0 margin="0" width="600px" padding:0 margin:0>
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| - | <tr>
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| - | <td><img src="https://static.igem.org/mediawiki/2010/3/3f/5race.jpg"</td>
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| - | </tr>
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| - | <tr>
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| - | <td><ul><li>To determine where transcription is initiating, we have decided to perform 5' Race PCR on select regions of c0051</li></ul></td>
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| - | </tr>
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| - | </table><p><br />
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| - | <a name="test4"></a><p class="header"><b>Test #4</b></a><p>
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| - | <table border=0 margin="0" width="600px" padding:0 margin:0>
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| - | <tr>
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| - | <td><img src="https://static.igem.org/mediawiki/2010/c/c0/Uhohtest4zoomedx.jpg"></td>
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| - | </tr>
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| - | <tr>
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| - | <td><img src="https://static.igem.org/mediawiki/2010/f/fe/C0051_w_LVA_in_PSP.png"></td>
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| - | </tr>
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| - | <tr>
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| - | <td>
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| - | <ul><li>Surely enough, after ligating the C0051 without the barcode into the Chris Anderson promoter screening plasmid, we have obtained white colonies. </li></ul>
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| - | </td>
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| - | </tr>
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| - | </table><p><br />
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| - | <p>
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| | </div> | | </div> |
| | </td> | | </td> |
"
