Team:UCL/Science/Experiment

From 2014.igem.org

(Difference between revisions)

Revision as of 12:59, 17 September 2014

Goodbye Azodye UCL iGEM 2014

Experiments

Laboratory Team

List of Experiments

Experiments

Extraction of Bacillus subtilis genomic DNA

Adam Denyer, Tanel Ozdemir June 13, 2014 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.

Transforming E. coli with Azo-reductase plasmids

Adam Denyer October 15, 2013 Protocols PCR competent cells transformation miniprep

We were gratefully provided with a set of five plasmids from a group of researchers working at the University of Lisbon, Portugal who are researching how azo-dye degrading enzymes function and who were 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 competent cells. After growing the cells overnight we then mini-prepped each of them to obtain plasmids at sufficient concentrations for future experimental work.

Name	Function	Source	Concentration	Sequence	Initial Plasmid / Vector	Comments
pAzoR	FMN-dependent NADH-azoreductase 1	Pseudomonas putida	Miniprep, 48 ng/uL,	597 bp [Check! Not 612 bp?]	Expression vector pET-21a (+) (ampicillin resistant (ampR)), initially cloned between NdeI and BamHI.	Plasmid provided by Lisbon
p1B6 (AzoR 1B6)	Mutant: Heat-stable; FMN-dependent NADH-azoreductase 1	Pseudomonas putida	Miniprep, 68 ng/uL,	597 bp [Check! Not 612 bp?]	Expression vector pET-21a (+) (ampR), initially cloned between NdeI and BamHI.	Plasmid provided by Lisbon.
pCotA	Spore Coat Protein Laccase	Bacillus subtilis	Miniprep, 103 ng/uL	1733 bp [Check! Not 1539 bp?]	Expression vector pET-21a (+) (ampR), initially cloned between NheI and BamHI.	Plasmid provided by Lisbon.
pBsDyP	Dye Decolourising Peroxidase BSU38260	Bacillus subtilis	Miniprep, 51 ng/uL,	1251 bp	Expression vector pET-21a (+) (ampR), initially cloned between NdeI and BamHI.	Plasmid provided by Lisbon.
pPpDyP	Dye Decolourising Peroxidase PP_3248	Pseudomonas putida	Miniprep, 55 ng/uL	861 bp [Check! Not 864 bp?]	Expression vector pET-21a (+) (ampR), initially cloned between NdeI and BamHI.	Plasmid provided by Lisbon.

Diagnostic digest of azo-reductase plasmids

Adam Denyer October 15, 2013 Protocols digest gel

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.

Creation of azo-reductase BioBrick parts from plasmids

Adam Denyer October 15, 2013 Protocols competent cells transformation miniprep

senectus et netus et malesuada

Diagnostic digest of azo-reductase BioBrick parts

Adam Denyer October 15, 2013 Protocols digest gel

senectus et netus et malesuada

Extraction of useful BioBrick plasmids from iGEM 2014 Distribution Kit

Adam Denyer October 15, 2013 Protocols competent cells transformation miniprep digest gel

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 expresing 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.

Assembling azo-reductase BioBrick Device(s)

Adam Denyer October 15, 2013 Protocols competent cells transformation miniprep digest gel

senectus et netus et malesuada

Characterisation of azo-reductase BioBrick devices

Adam Denyer October 15, 2013 Protocols competent cells transformation miniprep digest gel

senectus et netus et malesuada

Experiments

Extraction of Bacillus subtilis genomic DNA

Transforming E. coli with Azo-reductase plasmids

Diagnostic digest of azo-reductase plasmids

Creation of azo-reductase BioBrick parts from plasmids

Diagnostic digest of azo-reductase BioBrick parts

Extraction of useful BioBrick plasmids from iGEM 2014 Distribution Kit

Team:UCL/Science/Experiment

From 2014.igem.org

Revision as of 12:59, 17 September 2014

Experiments

Laboratory Team

Laboratory Team

List of Experiments

Experiments

Extraction of Bacillus subtilis genomic DNA

Transforming E. coli with Azo-reductase plasmids

Diagnostic digest of azo-reductase plasmids

Creation of azo-reductase BioBrick parts from plasmids

Diagnostic digest of azo-reductase BioBrick parts

Extraction of useful BioBrick plasmids from iGEM 2014 Distribution Kit

Assembling azo-reductase BioBrick Device(s)

Characterisation of azo-reductase BioBrick devices

Contents

List of Experiments

Experiments

Extraction of Bacillus subtilis genomic DNA

Transforming E. coli with Azo-reductase plasmids

Diagnostic digest of azo-reductase plasmids

Creation of azo-reductase BioBrick parts from plasmids

Diagnostic digest of azo-reductase BioBrick parts

Extraction of useful BioBrick plasmids from iGEM 2014 Distribution Kit

Assembling azo-reductase BioBrick Device(s)

Characterisation of azo-reductase BioBrick devices

Follow Us

@@ Line 1: / Line 1: @@
 {{:Team:UCL/Template:headerx}}
+<html>
+<div class="pageTitle">
+     <div><h3>Experiments</h3></div>
+     <div><!--- This is the Bit that describes the team and its logo DONT TOUCH-->
+         <div class="floater"><img src="https://static.igem.org/mediawiki/2014/c/c3/Team_Icons-01.png" height="50px" width="50px" style="margin-right:10px;"></img></div>
+         <div class="floater"><h4 class="minimyzr" style="margin:0px;">Laboratory Team</h4></div>
+     </div>
+</div>
+<div class="textArena"><!--- This is the were your text g{{:Team:UCL/Template:headerx}}
 <html>
 <div class="pageTitle">
@@ Line 9: / Line 18: @@
 </div>
 <div class="textArena"><!--- This is the were your text goes, play with it but dont change the class names-->
+<div class="textTitle"><h3 class="widthCorrect">List of Experiments</h3></div>
+<ol>
+<li>
+<a href="/Team:UCL/Science/Experiment#Expt01">Experiment 01: Extraction of useful BioBrick plasmids from iGEM 2014 Distribution Kit</a>
+</li>
+<li>
+<a href="/Team:UCL/Science/Experiment#Expt02">Experiment 02: Transforming <i>E. coli</i> with Azo-reductase plasmids</a>
+</li>
+<li>
+<a href="/Team:UCL/Science/Experiment#Expt03">Experiment 03: Diagnostic digest of azo-reductase plasmids</a>
+</li>
+<li>
+<a href="/Team:UCL/Science/Experiment#Expt04">Experiment 04: Creation of azo-reductase BioBrick parts from plasmids</a>
+</li>
+<li>
+<a href="/Team:UCL/Science/Experiment#Expt05">Experiment 05: Diagnostic digest of azo-reductase BioBrick parts</a>
+</li>
+<li>
+<a href="/Team:UCL/Science/Experiment#Expt06">Experiment  06: Assembling azo-reductase BioBrick Device(s)</a>
+</li>
+<li>
+<a href="/Team:UCL/Science/Experiment#Expt07">Experiment 07: Characterisation of azo-reductase BioBrick devices</a>
+</li>
+                </ol></p>
+<hr>
+                <h1>Experiments</h1>
+<h4>Extraction of <i>Bacillus subtilis</i> genomic DNA</h4>
+<div class="byline"><i class="icon-user"></i> Adam Denyer, Tanel Ozdemir &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published">June 13, 2014</abbr>
+<strong>&nbsp;&nbsp;Protocols&nbsp;&nbsp;</strong>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">DNA extraction</span></a></div>
+<br/>
+<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>
+<h4>Transforming <i>E. coli</i> with Azo-reductase plasmids</h4>
+<div class="byline"><i class="icon-user"></i> Adam Denyer &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="Monday, October 15, 2013,  8:21 PM">October 15, 2013</abbr>
+<strong>&nbsp;&nbsp;Protocols&nbsp;&nbsp;</strong>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">PCR</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">competent cells</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">transformation</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">miniprep</span></a></div>
+<br/>
+<p>We were gratefully provided with a set of five plasmids from a group of researchers working at the University of Lisbon, Portugal who are researching how azo-dye degrading enzymes function and who were 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 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>
+<table class="table table-striped table-bordered">
+  <thead>
+    <tr>
+      <th> Name </th>
+      <th> Function </th>
+      <th> Source </th>
+      <th> Concentration </th>
+      <th> Sequence </th>
+      <th> Initial Plasmid / Vector </th>
+      <th> Comments </th>
+    </tr>
+  </thead>
+  <tbody>
+    <tr>
+      <td> pAzoR </td>
+      <td> FMN-dependent NADH-azoreductase 1 </td>
+      <td> <em>Pseudomonas putida</em> </td>
+      <td> Miniprep,
+        <br>48 ng/uL,
+      </td>
+      <td><a href=" http://www.ncbi.nlm.nih.gov/nuccore/26986745?report=fasta&from=3267527&to=3268138">597 bp <strong>[Check! Not 612 bp?]</strong></a></td>
+      <td> Expression vector <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">pET-21a (+) <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">(ampicillin resistant (ampR))</a>, initially cloned between <em>NdeI</em> and <em>BamHI</em>.</td>
+      <td><a href="http://www.ncbi.nlm.nih.gov/pubmed/21655981">Plasmid provided by Lisbon</a></td>
+    </tr>
+    <tr>
+      <td> p1B6 (AzoR 1B6) </td>
+      <td> Mutant: Heat-stable; FMN-dependent NADH-azoreductase 1 </td>
+      <td> <em>Pseudomonas putida</em> </td>
+      <td> Miniprep,
+        <br>68 ng/uL,
+      </td>
+      <td><a href="">597 bp <strong>[Check! Not 612 bp?]</strong></strong> </td>
+      <td> Expression vector <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">pET-21a (+) <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">(ampR)</a>, initially cloned between <em>NdeI</em> and <em>BamHI</em>. </td>
+      <td> <a href=" http://www.ncbi.nlm.nih.gov/pubmed/24475252">Plasmid provided by Lisbon</a>.</td>
+    </tr>
+    <tr>
+      <td> pCotA </td>
+      <td> Spore Coat Protein Laccase </td>
+      <td> <em>Bacillus subtilis</em> </td>
+      <td> Miniprep,
+        <br>103 ng/uL
+      </td>
+      <td><a href="">1733 bp <strong>[Check! Not 1539 bp?]</strong></strong> </td>
+      <td> Expression vector <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">pET-21a (+) <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">(ampR)</a>, initially cloned between <em>NheI</em> and <em>BamHI</em>. </td>
+      <td> <a href=" http://www.itqb.unl.pt/martins/index_files/JBC2002.pdf">Plasmid provided by Lisbon</a>. </td>
+    </tr>
+    <tr>
+      <td> pBsDyP </td>
+      <td> Dye Decolourising Peroxidase BSU38260 </td>
+      <td> <em>Bacillus subtilis</em> </td>
+      <td> Miniprep,
+        <br>51 ng/uL,
+      </td>
+      <td><a href="">1251 bp</td>
+      <td> Expression vector <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">pET-21a (+) <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">(ampR)</a>, initially cloned between <em>NdeI</em> and <em>BamHI</em>. </td>
+      <td><a href="http://www.ncbi.nlm.nih.gov/pubmed/23820555">Plasmid provided by Lisbon</a>.</td>
+     </tr>
+     <tr>
+       <td> pPpDyP </td>
+       <td> Dye Decolourising Peroxidase PP_3248 </td>
+       <td> <em>Pseudomonas putida</em> </td>
+       <td> Miniprep,
+         <br>55 ng/uL</td>
+       <td><a href="">861 bp <strong>[Check! Not 864 bp?]</strong></strong> </td>
+       <td> Expression vector <a href="http://www.addgene.org/browse/sequence_vdb/2549/ ">pET-21a (+) <a href=" http://biochem.web.utah.edu/hill/links/pET21.pdf">(ampR)</a>, initially cloned between <em>NdeI</em> and <em>BamHI</em>. </td>
+        <td><a href="  http://www.ncbi.nlm.nih.gov/pubmed/23820555">Plasmid provided by Lisbon</a>.</td>
+      </tr>
+  </tbody>
+</table>
+<h4>Diagnostic digest of azo-reductase plasmids</h4>
+<div class="byline"><i class="icon-user"></i> Adam Denyer &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="Monday, October 15, 2013,  8:21 PM">October 15, 2013</abbr>
+<strong>&nbsp;&nbsp;Protocols&nbsp;&nbsp;</strong>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">digest</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">gel</span></a></div>
+<br/>
+<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>
+<h4>Creation of azo-reductase BioBrick parts from plasmids</h4>
+<div class="byline"><i class="icon-user"></i> Adam Denyer &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="Monday, October 15, 2013,  8:21 PM">October 15, 2013</abbr>
+<strong>&nbsp;&nbsp;Protocols&nbsp;&nbsp;</strong>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">competent cells</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">transformation</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">miniprep</span></a></div>
+<br/>
+<p>senectus et netus et malesuada</p>
+<h4>Diagnostic digest of azo-reductase BioBrick parts</h4>
+<div class="byline"><i class="icon-user"></i> Adam Denyer &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="Monday, October 15, 2013,  8:21 PM">October 15, 2013</abbr>
+<strong>&nbsp;&nbsp;Protocols&nbsp;&nbsp;</strong>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">digest</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">gel</span></a>
+</div>
+<br/>
+<p>senectus et netus et malesuada</p>
+<h4>Extraction of useful BioBrick plasmids from iGEM 2014 Distribution Kit</h4>
+<div class="byline"><i class="icon-user"></i> Adam Denyer &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="Monday, October 15, 2013,  8:21 PM">October 15, 2013</abbr>
+&nbsp;&nbsp;<strong>Protocols&nbsp;&nbsp;</strong>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">competent cells</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">transformation</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">miniprep</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">digest</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">gel</span></a></div>
+<br/>
+<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 expresing 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>
+<h4>Assembling azo-reductase BioBrick Device(s)</h4>
+<div class="byline"><i class="icon-user"></i> Adam Denyer &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="Monday, October 15, 2013,  8:21 PM">October 15, 2013</abbr>
+<strong>&nbsp;&nbsp;Protocols&nbsp;&nbsp;</strong>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">competent cells</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">transformation</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">miniprep</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">digest</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">gel</span></a></div>
+<br/>
+<p>senectus et netus et malesuada</p>
+<h4>Characterisation of azo-reductase BioBrick devices</h4>
+<div class="byline"><i class="icon-user"></i> Adam Denyer &nbsp;&nbsp; <i class="icon-time"></i> <abbr class="published" title="Monday, October 15, 2013,  8:21 PM">October 15, 2013</abbr>
+<strong>Protocols&nbsp;&nbsp;</strong>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">competent cells</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">transformation</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">miniprep</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">digest</span></a>
+<a href="/Team:UCL/Science/Protocols"><span class="label label-warning">gel</span></a></div>
+<br/>
+<p>senectus et netus et malesuada</p>
+            </div>
+</div><!-- This is the css of the page. Dont change it unless you have consulted with Lewis or Adam about what your changing-->
+<style>
+/*=======PAGE HEADER=======*/
+.pageTitle {
+    height:200px;
+    width:100%;
+    background-color:darkgrey;
+    padding-top:50px;
+    display:inline-block;
+}
+.floater {
+float:left;
+}
+/*=======Body=======*/
+.textArena {
+    background-color:white;
+    padding: 5% 5% 5% 5%;
+    font-size:90%;
+}
+.widthCorrect {
+    width:100%;
+}
+.label-warning {
+    background: black;
+    width: 100%;
+    height: 100%;
+    padding: 5px 10px;
+}
+table, th, td {
+   border: 1px solid black;
+}
+</style>
+</html>
+{{:Team:UCL/Template:footerx}}
+oes, play with it but dont change the class names-->
 <div class="textTitle"><h3 class="widthCorrect">List of Experiments</h3></div>