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Team:UCL/Science/Experiment
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| + | |||
| + | <!--- | ||
| + | <a data-tip="true" class="top large" data-tip-content="TOOLTIP TEXT" href="javascript:void(0)"><b>VISIBLE TEXT</b></a> | ||
| + | ---> | ||
| + | <div id="bodyContent"> | ||
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<div id="TopGapO"></div> | <div id="TopGapO"></div> | ||
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<img src="https://static.igem.org/mediawiki/2014/2/21/OExperiments_Bannero.jpg" width="100%" height="100%" alt="Experiments" /> | <img src="https://static.igem.org/mediawiki/2014/2/21/OExperiments_Bannero.jpg" width="100%" height="100%" alt="Experiments" /> | ||
</div> | </div> | ||
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| - | + | <div class="textArena"> | |
| - | + | <!--- This is the coding for the tabs (ask sanjay before altering this) ---> | |
| - | + | <ul class="tabs"> | |
| + | <li><a href="#view1">Stage 1</a></li> | ||
| + | <li><a href="#view2">Stage 2</a></li> | ||
| + | <li><a href="#view3">Stage 3</a></li> | ||
| + | <li><a href="#view4">Stage 4</a></li> | ||
| + | <li><a href="#view5">Stage 5</a></li> | ||
| + | <li><a href="#view6">Stage 6</a></li> | ||
| + | <li><a href="#view7">Stage 7</a></li> | ||
| + | <li><a href="#view8">Stage 8</a></li> | ||
| + | </ul> | ||
| + | <div class="tabcontents"> | ||
| - | + | <!--- This is the overview section ---> | |
| - | + | <div id="view1"><div class="textTitle"><h4>Stage 01: Extraction of useful BioBrick plasmids from iGEM 2014 Distribution Kit</h4></div><br> | |
| - | + | <!-- <a data-tip="true" class="top large" data-tip-content="Here's Tanel doing some pipetting in our lab!" href="javascript:void(0)" style="width: 25%;float: right;margin-left:2%"><img src="https://static.igem.org/mediawiki/2014/c/c9/UCLTANELPIPETTING.JPG" style="max-width: 100%;"></a> --> | |
| - | + | <!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--> | |
| - | < | + | <div><strong>Protocols </strong> |
| - | < | + | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">competent cells</span></a> |
| - | < | + | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">transformation</span></a> |
| - | < | + | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">miniprep</span></a> |
| - | < | + | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a> |
| - | < | + | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a></div> |
| - | < | + | <p>We began our project by identifying a range of BioBrick parts present in the iGEM 2014 distribution kit which we required as part of our cloning strategy. These parts primarily consisted of both constituitive and inducible promoter systems with ribosome binding sites which we could then use in conjunction with our azo-reductase BioBricks to assemble a functional azo dye degrading gene. We also decided that we would use the Red Florescent Protein expressing BioBrick as a control for any further transformation experiments. As the level of DNA present within each plate of the distribution kit is insufficient to perform digest and ligation reactions on it was necessary to transform each of these plasmids into our NEB5alpha competent cells. After growing our transformed cells overnight we then mini-prepped each of them to obtain BioBrick plasmids at suitable concentrations for future experiments.</p><br> |
| - | </ | + | <font size="2"> |
| - | < | + | <table border="1px" width="100%" height="auto"> |
| - | </div> | + | <thead> |
| + | <tr> | ||
| + | <th> </th> | ||
| + | <th> Registry ID </th> | ||
| + | <th> Name / Function </th> | ||
| + | <th> Antibiotic Resistance </th> | ||
| + | <th> Source </th> | ||
| + | <th> Size </th> | ||
| + | </tr> | ||
| + | </thead> | ||
| + | <tbody> | ||
| + | <tr> | ||
| + | <td> <center>U</center> </td> | ||
| + | <td> <a href="http://parts.igem.org/Part:BBa_K314103">BBa_K314103</a> </td> | ||
| + | <td> IPTG-inducible LacI Expression Cassette </td> | ||
| + | <td> Chloramphenicol</td> | ||
| + | <td> Spring 2014 BioBrick Distribution. Plate 1, Well 4D.</td> | ||
| + | <td> <a href="/Team:UCL/Science/Sequences#BBa_K314103">1638 bp</a> </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <center>T</center> </td> | ||
| + | <td> <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a> </td> | ||
| + | <td> RFP Coding Device </td> | ||
| + | <td> Chloramphenicol</td> | ||
| + | <td> Spring 2014 BioBrick Distribution. Plate 4, Well 4B. </td> | ||
| + | <td> <a href="/Team:UCL/Science/Sequences#BBa_J04450">1069 bp</a> </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <center>T</center> </td> | ||
| + | <td> <a href="http://parts.igem.org/Part:BBa_R0010">BBa_R0010</a> </td> | ||
| + | <td> IPTG-inducible LacI Promoter</td> | ||
| + | <td> Chloramphenicol</td> | ||
| + | <td> Spring 2014 BioBrick Distribution. Plate 3, Well 4G.</td> | ||
| + | <td> <a href="/Team:UCL/Science/Sequences#BBa_R0010">200 bp</a> </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <center>T</center> </td> | ||
| + | <td> <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> </td> | ||
| + | <td> Ribosomal Binding Site (RBS)</td> | ||
| + | <td> Chloramphenicol</td> | ||
| + | <td> Spring 2014 BioBrick Distribution. Plate 4, Well 1N.</td> | ||
| + | <td> <a href="/Team:UCL/Science/Sequences#BBa_B0034">12 bp</a> </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <center>T</center> </td> | ||
| + | <td> <a href="http://parts.igem.org/Part:BBa_K518012">BBa_K518012</a> </td> | ||
| + | <td> RBS + RFP + double Terminator</td> | ||
| + | <td> Chloramphenicol</td> | ||
| + | <td> Spring 2014 BioBrick Distribution. Plate 1, Well 18C.</td> | ||
| + | <td> <a href="/Team:UCL/Science/Sequences#BBa_K518012">828 bp</a> </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <center>N</center> </td> | ||
| + | <td> <a href="http://parts.igem.org/Part:BBa_K206000">BBa_K206000</a> </td> | ||
| + | <td> pBAD Strong Promoter</td> | ||
| + | <td> Chloramphenicol</td> | ||
| + | <td> Spring 2014 BioBrick Distribution. Plate 3, Well 14A.</td> | ||
| + | <td> <a href="/Team:UCL/Science/Sequences#BBa_K206000">130 bp</a> </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <center>! N</center> </td> | ||
| + | <td> <a href="http://parts.igem.org/Part:BBa_R0011">BBa_R0011</a> </td> | ||
| + | <td> LacI-Regulated, Lambda pL Hybrid Promoter</td> | ||
| + | <td> Chloramphenicol</td> | ||
| + | <td> Spring 2014 BioBrick Distribution. Plate 2, Well 6D.</td> | ||
| + | <td> <a href="/Team:UCL/Science/Sequences#BBa_R0011">55 bp</a> </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <center>! N</center> </td> | ||
| + | <td> <a href="http://parts.igem.org/Part:BBa_B0012">BBa_B0012</a> </td> | ||
| + | <td> Transcription Terminator for E. coli RNA Polymerase</td> | ||
| + | <td> Chloramphenicol</td> | ||
| + | <td> Spring 2014 BioBrick Distribution. Plate 2, Well 2B.</td> | ||
| + | <td> <a href="/Team:UCL/Science/Sequences#BBa_B0012">41 bp</a> </td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | </font> | ||
| + | <div><font size="1">Note: U = Used in experiments; T = Used for testing purposes but not for making BioBrick Devices; N = Transformed from Distribution Kits, but not used in experiments; ! = Problematic parts (see Parts Registry), were not used.</font></div> | ||
| - | + | </div> | |
| - | + | ||
| - | <!-- | + | <!--- This is the first biobrick ---> |
| - | + | <div id="view2"><div class="textTitle"><h4>Stage 02: Identification of useful genes for making new BioBricks</h4></div><br> | |
| - | + | <!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--> | |
| - | + | <strong>Extraction of Bacillus subtilis genomic DNA</strong> | |
| - | + | <div><strong>Protocols </strong> | |
| - | + | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">DNA extraction</span></a></div> | |
| - | + | <p>Our literature search identified a number of bacterial species that have been proven to degrade azo dye compounds including <i>B. subtilis</i> and <i>P. aeruginosa</i>. We were able to obtain a <i>B. subtilis</i> strain for use in our project from ?. We extracted the genomic DNA from this strain using a Promega Wizard Genomic DNA extraction kit so that we could subsequently amplify the azo-reducatase gene (AzoR1) and create our first azo-reductase BioBrick. After completing the genomic DNA extracton we ran a gel to show that we had successfully extracted the <i>B. subtilis</i> genomic DNA.</p><br> | |
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| - | + | </div> | |
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| - | + | <!--- This is the second biobrick ---> | |
| - | + | <div id="view3"><div class="textTitle"><h4>Stage 03: Transforming E. coli with azo-reductase plasmids</h4></div><br> | |
| - | + | <!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--> | |
| - | + | <div><strong>Protocols </strong> | |
| - | + | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">competent cells</span></a> | |
| - | + | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">transformation</span></a> | |
| - | + | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">miniprep</span></a></div> | |
| - | + | <p>We were gratefully provided with a set of five plasmids from the <a href="http://www.itqb.unl.pt/martins">Microbial & Enzyme Technology Lab</a> led by Dr Lígia O. Martins at the Universidade Nova de Lisboa. They are currently researching how azo-dye degrading enzymes function and are keen to collaborate with us. These plasmids contained a number of genes encoding azo-dye degrading enzymes from both <i>B. subtilis</i> and <i>P. putida</i> including mutated forms found to exhibit enhanced degradation activity. As the DNA concentration of the plasmids we were sent was insufficient to perform PCR amplification on we transformed each of these plasmids into our <i>E. coli</i> NEB5alpha derivative competent cells. After growing the cells overnight we then mini-prepped each of them to obtain plasmids at sufficient concentrations for future experimental work.</p><br> | |
| - | + | <font size="2"> | |
| - | + | <table border="1px" width="100%" height="auto"> | |
| - | + | <thead> | |
| - | + | <tr> | |
| - | + | <th> Gene ID</th> | |
| - | + | <th> Name / Function </th> | |
| - | + | <th> Source </th> | |
| - | + | <th> Size </th> | |
| - | + | <th> Plasmid </th> | |
| - | + | </tr> | |
| - | + | </thead> | |
| - | + | <tbody> | |
| - | + | <!--Lisbon plasmids--> | |
| - | + | <tr> | |
| - | + | <td> pAzoR </td> | |
| - | + | <td> FMN-dependent NADH-azoreductase 1 </td> | |
| - | + | <td> <em>Pseudomonas putida</em> </td> | |
| - | + | <td> <a href="http://www.ncbi.nlm.nih.gov/pubmed/21655981">612 bp</a> </td> | |
| - | + | <td> In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td> | |
| - | + | </tr> | |
| - | + | <tr> | |
| - | + | <td> p1B6 </td> | |
| + | <td> AzoR heat-stable mutant</td> | ||
| + | <td> <em>Pseudomonas putida</em> </td> | ||
| + | <td> <a href="http://www.ncbi.nlm.nih.gov/pubmed/24475252">612 bp</a> </td> | ||
| + | <td> In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> pCotA </td> | ||
| + | <td> Spore Coat Protein Laccase</td> | ||
| + | <td> <em>Bacillus subtilis</em> </td> | ||
| + | <td> <a href="http://www.itqb.unl.pt/martins/index_files/JBC2002.pdf">1542 bp</a> </td> | ||
| + | <td> In expression vector: pET-21a (+) (ampicillin resistant (ampR)) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <strong><em>NheI</em></strong> and <em>BamHI</em> restriction sites. </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> pBsDyP </td> | ||
| + | <td> Dye Decolourising Peroxidase BSU38260</td> | ||
| + | <td> <em>Bacillus subtilis</em> </td> | ||
| + | <td> <a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">1251 bp</a> </td> | ||
| + | <td> In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> pPpDyP </td> | ||
| + | <td> Dye Decolourising Peroxidase PP_3248 </td> | ||
| + | <td> <em>Pseudomonas putida</em> </td> | ||
| + | <td> <a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">864 bp</a> </td> | ||
| + | <td> In expression vector: pET-21a (+) (ampicillin resistant) <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">[2] <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">[3] </a>, <br>initially cloned between <em>NdeI</em> and <em>BamHI</em> restriction sites. </td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | </font> | ||
| + | </div> | ||
| - | + | <!--- This is the third biobrick ---> | |
| - | + | <div id="view4"><div class="textTitle"><h4>Stage 04: Diagnostic digest of azo-reductase plasmids</h4></div><br> | |
| - | + | <!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--> | |
| - | + | <div><strong>Protocols </strong> | |
| - | + | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a> | |
| - | + | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a></div> | |
| - | + | <p>After successfully transforming these plasmids into competent <i>E. coli</i> NEB5alpha cells we then performed a diagnostic digest and gel electrophoresis experiment to ascertain that these plasmids contained the gene we expected. Each plasmid was digested using two restriction enzymes chosen to digest DNA as specific points on the plasmids and create fragments of known length which we could then confirm using gel electrophoresis.</p> | |
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| - | + | <!--- This is the fourth biobrick ---> | |
| - | + | <div id="view5"><div class="textTitle"><h4>Stage 05: Creation of azo-reductase BioBrick parts from plasmids</h4></div><br> | |
| - | + | <!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--> | |
| - | + | <p>...</p><br> | |
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| - | <!-- | + | <!--- This is the fifth biobrick ---> |
| - | + | <div id="view6"><div class="textTitle"><h4>Stage 06: Diagnostic digest of azo-reductase BioBrick parts</h4></div><br> | |
| - | + | <!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--> | |
| - | + | <p>...</p><br> | |
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| - | <!-- | + | <!--- This is the fifth biobrick ---> |
| - | + | <div id="view7"><div class="textTitle"><h4>Stage 07: Assembling azo-reductase BioBrick Device(s)</h4></div><br> | |
| - | + | <!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--> | |
| - | + | <p>...</p><br> | |
| - | + | <font size="2"> | |
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<tr> | <tr> | ||
| - | + | <th> </th> | |
| - | + | <th> Registry ID </th> | |
| - | + | <th> Gene ID</th> | |
| - | + | <th> Name / Function </th> | |
| - | + | <th> Source </th> | |
| - | + | <th> Size </th> | |
| - | + | <th> Status </th> | |
</tr> | </tr> | ||
| - | + | </thead> | |
| - | + | <tbody> | |
| - | + | <!--Lisbon plasmids--> | |
<tr> | <tr> | ||
| - | + | <td> </td> | |
| - | + | <td> <a href="http://parts.igem.org/Part:BBa_K1336000">BBa_K1336000</a> </td> | |
| - | + | <td> AzoR </td> | |
| - | + | <td> FMN-dependent NADH-azoreductase 1 </td> | |
| - | < | + | <td> <em>Pseudomonas putida</em> </td> |
| - | + | <td> <a href="/Team:UCL/Science/Sequences#BBa_K1336000">612 bp</a> </td> | |
| - | + | <td> [<a href="/Team:UCL/Science/Primers">In Progress</a>]: primers designed </td> | |
| - | + | ||
| - | + | ||
</tr> | </tr> | ||
| - | |||
<tr> | <tr> | ||
| - | + | <td> </td> | |
| - | + | <td> <a href="http://parts.igem.org/Part:BBa_K1336001">BBa_K1336001</a> </td> | |
| - | + | <td> 1B6 </td> | |
| - | + | <td> AzoR heat-stable mutant</td> | |
| - | < | + | <td> <em>Pseudomonas putida</em> </td> |
| - | + | <td> <a href="/Team:UCL/Science/Sequences#BBa_K1336001">612 bp</a> </td> | |
| - | + | <td> [<a href="/Team:UCL/Science/Experiments">In Progress</a>]: to remove 2 illegal PstI sites </td> | |
| - | + | ||
| - | + | ||
</tr> | </tr> | ||
| - | + | <tr> | |
| - | <tr> | + | <td> </td> |
| - | + | <td> <a href="http://parts.igem.org/Part:BBa_K1336002">BBa_K1336002</a> </td> | |
| - | + | <td> CotA </td> | |
| - | + | <td> Spore Coat Protein Laccase</td> | |
| - | + | <td> <em>Bacillus subtilis</em> </td> | |
| - | < | + | <td> <a href="/Team:UCL/Science/Sequences#BBa_K1336002">1542 bp</a> </td> |
| - | + | <td> [<a href="/Team:UCL/Science/Primers">In Progress</a>]: primers designed </td> | |
| - | + | ||
| - | + | ||
| - | + | ||
</tr> | </tr> | ||
| - | |||
<tr> | <tr> | ||
| - | + | <td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td> | |
| - | + | <td> <a href="http://parts.igem.org/Part:BBa_K1336003">BBa_K1336003</a> </td> | |
| - | + | <td> BsDyP </td> | |
| - | + | <td> Dye Decolourising Peroxidase BSU38260</td> | |
| - | < | + | <td> <em>Bacillus subtilis</em> </td> |
| - | + | <td> <a href="/Team:UCL/Science/Sequences#BBa_K1336003">1251 bp</a> </td> | |
| - | + | <td> [<a href="/Team:UCL/Science/Experiments">New BioBrick Part</a>]: submitted </td> | |
| - | + | </tr> | |
| - | + | <tr> | |
| - | + | <td> </td> | |
| + | <td> <a href="http://parts.igem.org/Part:BBa_K1336004">BBa_K1336004</a> </td> | ||
| + | <td> PpDyP </td> | ||
| + | <td> Dye Decolourising Peroxidase PP_3248 </td> | ||
| + | <td> <em>Pseudomonas putida</em> </td> | ||
| + | <td> <a href="/Team:UCL/Science/Sequences#BBa_K1336004">864 bp</a> </td> | ||
| + | <td> [<a href="/Team:UCL/Science/Primers">In Progress</a>]: primers designed </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td> | ||
| + | <td> <a href="http://parts.igem.org/Part:BBa_K1336005">BBa_K1336005</a> </td> | ||
| + | <td> ispB RNAi </td> | ||
| + | <td> RNAi of Octaprenyl Diphosphate <br>Synthase fragment </td> | ||
| + | <td> <em>Escherichia coli, K12 strain</em> </td> | ||
| + | <td> <a href="/Team:UCL/Science/Sequences#BBa_K1336005">562 bp</a> </td> | ||
| + | <td> [<a href="/Team:UCL/Science/Experiment">New BioBrick Part</a>]: submitted </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td> | ||
| + | <td> <a href="http://parts.igem.org/Part:BBa_K1336006">BBa_K1336006</a> </td> | ||
| + | <td> LacIEC+ispB </td> | ||
| + | <td> IPTG inducible ispB RNAi </td> | ||
| + | <td> <em>Escherichia coli, K12 strain </em> </td> | ||
| + | <td> <a href="/Team:UCL/Science/Sequences#BBa_K1336006">2208 bp</a> </td> | ||
| + | <td> [<a href="/Team:UCL/Science/Experiment">New BioBrick Device</a>]: submitted </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <center><img src="https://static.igem.org/mediawiki/2014/e/e0/UCL_Bronze-metal-star.jpg" width="25px"></center> </td> | ||
| + | <td> <a href="http://parts.igem.org/Part:BBa_K1336007">BBa_K1336007</a> </td> | ||
| + | <td> LacIEC+BsDyP </td> | ||
| + | <td> IPTG inducible BsDyP </td> | ||
| + | <td> <em>Bacillus subtilis</em> </td> | ||
| + | <td> <a href="/Team:UCL/Science/Sequences#BBa_K1336007">2895 bp</a> </td> | ||
| + | <td> [<a href="/Team:UCL/Science/Experiment">New BioBrick Device</a>]: submitted </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> </td> | ||
| + | <td> <a href="http://parts.igem.org/Part:BBa_K729006">BBa_K729006</a> </td> | ||
| + | <td> CueO </td> | ||
| + | <td> Laccase </td> | ||
| + | <td> <em>Escherichia coli </em> </td> | ||
| + | <td> <a href="/Team:UCL/Science/Sequences#BBa_K729006">1612 bp</a> </td> | ||
| + | <td> [<a href="/Team:UCL/Science/Experiment">In Progress</a>]: ascertaining identity </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <center>(<img src="https://static.igem.org/mediawiki/2014/3/36/UCL_Gold-metal-star.jpg" width="25px">)</center> </td> | ||
| + | <td> <a href="http://parts.igem.org/Part:BBa_K500000">BBa_K500000</a> </td> | ||
| + | <td> LiP </td> | ||
| + | <td> Lignin Peroxidase </td> | ||
| + | <td> <em>Phanerochaete chrysosporium</em> </td> <!-- <br>(White-Rot Fungi) --> | ||
| + | <td> <a href="/Team:UCL/Science/Sequences#BBa_K500000">1116 bp</a> </td> <!--Check size!--> | ||
| + | <td> [<a href="/Team:UCL/Science/Results">Improved Characterisation</a>]: toxicity issues in gene synthesis. <br> [<a href="/Team:UCL/Science/Experiment">In Progress</a>]: to subclone into pSB1C3/pSB3C5. </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> <center><img src="https://static.igem.org/mediawiki/2014/3/36/UCL_Gold-metal-star.jpg" width="25px"></center> </td> | ||
| + | <td> <a href="http://parts.igem.org/Part:BBa_K729004">BBa_K729004</a> </td> | ||
| + | <td> nucB </td> | ||
| + | <td> Extracellular nuclease </td> | ||
| + | <td> <em>Staphylococcus aureus</em> </td> | ||
| + | <td> <a href="/Team:UCL/Science/Sequences#BBa_K729004">561 bp</a> </td> | ||
| + | <td> [<a href="/Team:UCL/Science/Results">Improved Function</a>] </td> | ||
| + | </tr> | ||
| + | </tbody> | ||
| + | </table> | ||
| + | </font> | ||
| + | </div> | ||
| - | + | <!--- This is the fifth biobrick ---> | |
| - | + | <div id="view8"><div class="textTitle"><h4>Stage 08: Characterisation of azo-reductase BioBrick devices</h4></div><br> | |
| - | + | <!-- This is the main text. Anything in a <p>TEXT</p> is a paragraph and will be spaced appropriately--> | |
| - | + | <p>...</p><br> | |
| - | + | <div> | |
| - | + | <h4><a name="Expt">Placeholder. Will be removed.</a></h4> | |
| - | + | <strong>Protocols </strong> | |
| - | + | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">PCR</span></a> | |
| - | + | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">analytical digest</span></a> | |
| - | + | ||
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| - | + | ||
| - | + | ||
| - | + | ||
| - | < | + | |
| - | <strong> | + | |
| - | <a href="/Team:UCL/Science/Proto"><span class="label label-warning"> | + | |
| - | <a href="/Team:UCL/Science/Proto"><span class="label label-warning"> | + | |
| - | + | ||
| - | + | ||
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| - | + | ||
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a> | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">gel</span></a> | ||
| - | </ | + | (<a href="/Team:UCL/Science/Proto"><span class="label label-warning">digest</span></a> |
| - | + | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">ligation</span></a> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">competent cells</span></a> | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">competent cells</span></a> | ||
<a href="/Team:UCL/Science/Proto"><span class="label label-warning">transformation</span></a> | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">transformation</span></a> | ||
| - | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">miniprep</span></a> | + | <a href="/Team:UCL/Science/Proto"><span class="label label-warning">miniprep</span></a>) |
| - | < | + | <br> |
| - | < | + | <p>[Insert table of Our Genes]</p> |
| - | <br | + | <br> |
| - | < | + | <div class="accordion"> |
| - | + | <h4><div class="byline"><i class="icon-user"></i> Adam Denyer, Tanel Ozdemir <i class="icon-time"></i> <abbr class="published" title="June 13, 2014">June 13, 2014</abbr></div></h4> | |
| - | + | <div> | |
| - | + | <p>...</p> | |
| - | + | </div> | |
| - | + | <h4><div class="byline"><i class="icon-user"></i> Adam Denyer, Tanel Ozdemir <i class="icon-time"></i> <abbr class="published" title="June 13, 2014">June 13, 2014</abbr></div></h4> | |
| - | + | <div> | |
| - | + | <p>...</p> | |
| - | + | </div> | |
| - | + | </div> | |
| - | + | <br> | |
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</div> | </div> | ||
| + | </div> | ||
| - | + | </div> | |
| + | </div> | ||
| + | <a href="#" class="back-to-top"><img src="https://static.igem.org/mediawiki/2014/5/5d/Scroll_Up_Button.png" width="64" height="64"></a> | ||
| + | <script type="text/javascript"> | ||
| + | jQuery(document).ready(function() { | ||
| + | var offset = 220; | ||
| + | var duration = 500; | ||
| + | jQuery(window).scroll(function() { | ||
| + | if (jQuery(this).scrollTop() > offset) { | ||
| + | jQuery('.back-to-top').fadeIn(duration); | ||
| + | } else { | ||
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| + | jQuery('.back-to-top').click(function(event) { | ||
| + | event.preventDefault(); | ||
| + | jQuery('html, body').animate({scrollTop: 0}, duration); | ||
| + | return false; | ||
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| + | }); | ||
| + | </script> | ||
| + | </div><!-- This div end tag should end the .textArena tag that defines light grey space. This is crucial--> | ||
| + | |||
| + | <!-- This is the css of the page. Dont change it unless you have consulted with Lewis or Adam about what your changing--> | ||
<style> | <style> | ||
/*=======PAGE HEADER=======*/ | /*=======PAGE HEADER=======*/ | ||
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padding: 5% 5% 5% 5%; | padding: 5% 5% 5% 5%; | ||
font-size:90%; | font-size:90%; | ||
| + | font-family: 'Open Sans', 'Helvetica Neue', Helvetica, Arial, sans-serif; | ||
| + | text-align: justify; | ||
| + | } | ||
| + | |||
} | } | ||
.widthCorrect { | .widthCorrect { | ||
| - | + | width:100%; | |
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.label-warning { | .label-warning { | ||
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table, th, td { | table, th, td { | ||
border: 1px solid black; | border: 1px solid black; | ||
| + | } | ||
| + | /*=========Object Alignment Classes=============*/ | ||
| + | .alignright { | ||
| + | float:right; | ||
| + | margin-left:3%; | ||
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Revision as of 17:52, 16 October 2014
Stage 01: Extraction of useful BioBrick plasmids from iGEM 2014 Distribution Kit
We began our project by identifying a range of BioBrick parts present in the iGEM 2014 distribution kit which we required as part of our cloning strategy. These parts primarily consisted of both constituitive and inducible promoter systems with ribosome binding sites which we could then use in conjunction with our azo-reductase BioBricks to assemble a functional azo dye degrading gene. We also decided that we would use the Red Florescent Protein expressing BioBrick as a control for any further transformation experiments. As the level of DNA present within each plate of the distribution kit is insufficient to perform digest and ligation reactions on it was necessary to transform each of these plasmids into our NEB5alpha competent cells. After growing our transformed cells overnight we then mini-prepped each of them to obtain BioBrick plasmids at suitable concentrations for future experiments.
| Registry ID | Name / Function | Antibiotic Resistance | Source | Size | |
|---|---|---|---|---|---|
| |
BBa_K314103 | IPTG-inducible LacI Expression Cassette | Chloramphenicol | Spring 2014 BioBrick Distribution. Plate 1, Well 4D. | 1638 bp |
| |
BBa_J04450 | RFP Coding Device | Chloramphenicol | Spring 2014 BioBrick Distribution. Plate 4, Well 4B. | 1069 bp |
| |
BBa_R0010 | IPTG-inducible LacI Promoter | Chloramphenicol | Spring 2014 BioBrick Distribution. Plate 3, Well 4G. | 200 bp |
| |
BBa_B0034 | Ribosomal Binding Site (RBS) | Chloramphenicol | Spring 2014 BioBrick Distribution. Plate 4, Well 1N. | 12 bp |
| |
BBa_K518012 | RBS + RFP + double Terminator | Chloramphenicol | Spring 2014 BioBrick Distribution. Plate 1, Well 18C. | 828 bp |
| |
BBa_K206000 | pBAD Strong Promoter | Chloramphenicol | Spring 2014 BioBrick Distribution. Plate 3, Well 14A. | 130 bp |
| |
BBa_R0011 | LacI-Regulated, Lambda pL Hybrid Promoter | Chloramphenicol | Spring 2014 BioBrick Distribution. Plate 2, Well 6D. | 55 bp |
| |
BBa_B0012 | Transcription Terminator for E. coli RNA Polymerase | Chloramphenicol | Spring 2014 BioBrick Distribution. Plate 2, Well 2B. | 41 bp |
Note: U = Used in experiments; T = Used for testing purposes but not for making BioBrick Devices; N = Transformed from Distribution Kits, but not used in experiments; ! = Problematic parts (see Parts Registry), were not used.
Stage 02: Identification of useful genes for making new BioBricks
Extraction of Bacillus subtilis genomic DNA
Protocols
DNA extraction
Our literature search identified a number of bacterial species that have been proven to degrade azo dye compounds including B. subtilis and P. aeruginosa. We were able to obtain a B. subtilis strain for use in our project from ?. We extracted the genomic DNA from this strain using a Promega Wizard Genomic DNA extraction kit so that we could subsequently amplify the azo-reducatase gene (AzoR1) and create our first azo-reductase BioBrick. After completing the genomic DNA extracton we ran a gel to show that we had successfully extracted the B. subtilis genomic DNA.
Stage 03: Transforming E. coli with azo-reductase plasmids
We were gratefully provided with a set of five plasmids from the Microbial & Enzyme Technology Lab led by Dr Lígia O. Martins at the Universidade Nova de Lisboa. They are currently researching how azo-dye degrading enzymes function and are keen to collaborate with us. These plasmids contained a number of genes encoding azo-dye degrading enzymes from both B. subtilis and P. putida including mutated forms found to exhibit enhanced degradation activity. As the DNA concentration of the plasmids we were sent was insufficient to perform PCR amplification on we transformed each of these plasmids into our E. coli NEB5alpha derivative competent cells. After growing the cells overnight we then mini-prepped each of them to obtain plasmids at sufficient concentrations for future experimental work.
| Gene ID | Name / Function | Source | Size | Plasmid |
|---|---|---|---|---|
| pAzoR | FMN-dependent NADH-azoreductase 1 | Pseudomonas putida | 612 bp | In expression vector: pET-21a (+) (ampicillin resistant) [2] [3] , initially cloned between NdeI and BamHI restriction sites. |
| p1B6 | AzoR heat-stable mutant | Pseudomonas putida | 612 bp | In expression vector: pET-21a (+) (ampicillin resistant) [2] [3] , initially cloned between NdeI and BamHI restriction sites. |
| pCotA | Spore Coat Protein Laccase | Bacillus subtilis | 1542 bp | In expression vector: pET-21a (+) (ampicillin resistant (ampR)) [2] [3] , initially cloned between NheI and BamHI restriction sites. |
| pBsDyP | Dye Decolourising Peroxidase BSU38260 | Bacillus subtilis | 1251 bp | In expression vector: pET-21a (+) (ampicillin resistant) [2] [3] , initially cloned between NdeI and BamHI restriction sites. |
| pPpDyP | Dye Decolourising Peroxidase PP_3248 | Pseudomonas putida | 864 bp | In expression vector: pET-21a (+) (ampicillin resistant) [2] [3] , initially cloned between NdeI and BamHI restriction sites. |
Stage 04: Diagnostic digest of azo-reductase plasmids
After successfully transforming these plasmids into competent E. coli NEB5alpha cells we then performed a diagnostic digest and gel electrophoresis experiment to ascertain that these plasmids contained the gene we expected. Each plasmid was digested using two restriction enzymes chosen to digest DNA as specific points on the plasmids and create fragments of known length which we could then confirm using gel electrophoresis.
Stage 05: Creation of azo-reductase BioBrick parts from plasmids
...
Stage 06: Diagnostic digest of azo-reductase BioBrick parts
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Stage 07: Assembling azo-reductase BioBrick Device(s)
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| Registry ID | Gene ID | Name / Function | Source | Size | Status | |
|---|---|---|---|---|---|---|
| BBa_K1336000 | AzoR | FMN-dependent NADH-azoreductase 1 | Pseudomonas putida | 612 bp | [In Progress]: primers designed | |
| BBa_K1336001 | 1B6 | AzoR heat-stable mutant | Pseudomonas putida | 612 bp | [In Progress]: to remove 2 illegal PstI sites | |
| BBa_K1336002 | CotA | Spore Coat Protein Laccase | Bacillus subtilis | 1542 bp | [In Progress]: primers designed | |
 |
BBa_K1336003 | BsDyP | Dye Decolourising Peroxidase BSU38260 | Bacillus subtilis | 1251 bp | [New BioBrick Part]: submitted |
| BBa_K1336004 | PpDyP | Dye Decolourising Peroxidase PP_3248 | Pseudomonas putida | 864 bp | [In Progress]: primers designed | |
| |
BBa_K1336005 | ispB RNAi | RNAi of Octaprenyl Diphosphate Synthase fragment |
Escherichia coli, K12 strain | 562 bp | [New BioBrick Part]: submitted |
| |
BBa_K1336006 | LacIEC+ispB | IPTG inducible ispB RNAi | Escherichia coli, K12 strain | 2208 bp | [New BioBrick Device]: submitted |
| |
BBa_K1336007 | LacIEC+BsDyP | IPTG inducible BsDyP | Bacillus subtilis | 2895 bp | [New BioBrick Device]: submitted |
| BBa_K729006 | CueO | Laccase | Escherichia coli | 1612 bp | [In Progress]: ascertaining identity | |
 ) |
BBa_K500000 | LiP | Lignin Peroxidase | Phanerochaete chrysosporium | 1116 bp | [Improved Characterisation]: toxicity issues in gene synthesis. [In Progress]: to subclone into pSB1C3/pSB3C5. |
| |
BBa_K729004 | nucB | Extracellular nuclease | Staphylococcus aureus | 561 bp | [Improved Function] |
Stage 08: Characterisation of azo-reductase BioBrick devices
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Placeholder. Will be removed.
Protocols PCR analytical digest gel (digest ligation competent cells transformation miniprep)[Insert table of Our Genes]
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