Difference between revisions of "Team:Glasgow/Notebook"

 
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  <div class="timeline-start">Week1</div>
 
 
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             <!--------JUNE------->
 
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             <p>We went to Dundee University to attend the Scotland IGEM team's meeting. We had so much fun in the meeting, sharing different ideas with each team.</p>
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             <h1><strong>James’ muffins</strong></h1><p>Talked to Professors Marshall Stark and John Christie about our project. </p>
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        <div class="content-right-container">
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            <img src="https://static.igem.org/mediawiki/2015/4/42/2015-Glasgow-timeline2.jpg" style="max-width:350px;">
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              <img src="https://static.igem.org/mediawiki/2015/5/5b/2015-Glasgow-timeline3.jpg" style="max-width:350px">
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        <div class="content-right-container">
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          <div class="content-right">
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          <h1><strong>Repressors</strong></h1><p>Ordered P<sub><i>PhlF</i></sub> and P<sub><i>SrpR</i></sub> with BioBrick prefix and suffix as oligos. Ordered; PhlF and SrpR as G-blocks, with ribosome binding site B0032; Terminator B0010+ (a longer version of B0010, that we will characterise and submit as a new BioBrick) with BioBrick prefix and suffix.  Got E5501 and I13500 – GFP (E0040) with ribosome binding sites B0032 and B0034 respectively – in pSB1C3 in DH5α from glycerol stocks from last year’s iGEM team. </p>
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      <div class="timeline-article">
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        <div class="content-left-container">
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          <div class="content-left">
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            <h1><strong>UVA sensor</strong></h1><p>Designed primers to PCR <i>uirS</i>, <i>uirR</i>, and P<sub><i>lsiR</i></sub> from <i>Synechocystis</i> PCC6803 genomic DNA with ribosome binding site B0032 and BioBrick prefix/suffix. Tested UVB sensitivity of strains TOP10 and DH5α (<i>recA<sup>-</sup></i> mutants - very poor DNA repair), DS941 (<i>recF<sup>-</sup></i> - limited DNA repair), and MG1655.Z1 (wild type for DNA repair).</p>
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        <div class="content-right-container">
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            <img src="https://static.igem.org/mediawiki/2015/b/b6/2015-Glasgow-timeline4.jpg" style="max-width:350px;">
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        <div class="content-right-container">
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          <h1><strong>Bioluminescence</strong></h1><p>Retrieved plasmid DNA of pBAD.LuxCDABEG (Cambridge Luxbrick: K325909) and pBAD.LuxCDABEG.LumP (K769020) from 2015 iGEM distribution plates and transformed into TOP10 and DH5α strains. Designed primers to PCR LuxA, B, C, D, E, and G individually with ribosome binding site B0032 and BioBrick prefix/suffix from K325909. </p>
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  <div class="timeline-start">Week2</div>
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          <h1><strong>Vilija’s carrot cake</strong></h1><p>So good, honestly.</p>
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        <div class="content-right-container">
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          <img src="https://static.igem.org/mediawiki/2015/6/6b/2015-Glasgow-timeline6.jpg" style="max-width:350px;">
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      <div class="timeline-article">
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        <div class="content-left-container">
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          <img src="https://static.igem.org/mediawiki/2015/1/1a/2015-Glasgow-timeline11.jpg" style="max-width:350px;">
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        <div class="content-right-container">
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          <div class="content-right">
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          <h1><strong>Bioluminescence</strong></h1><p>Tested K325909 and K769020 in TOP10 to see how long until there is expression of bioluminescence after addition of arabinose inducer, how bright the bioluminescence is, and how LumP affects it in K769020.</p>
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        <h1><strong>Repressors</strong></h1><p>Ligated both P<i><sub>PhlF</sub></i> and P<i><sub>SrpR</sub></i> with E5501 and I13500 in pSB1C3, respectively, and transformed into TOP10 and DH5α. Retrieved C0040 (TetR), R0040 (P<i><sub>TetR</sub></i>) and backbone pSB3K3 (with insert J04450) off distribution plates – to use C0040 as a control for our repressors, and to use pSB3K3 as a second plasmid to have the promoters and repressors on separate plasmids. Ordered G-Block of R0011.B0032 (P<i><sub>LacI</sub></i> + RBS) for C0040 – as the plasmid on the plate has to RBS.</p>
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        <div class="content-right-container">
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            <img src="https://static.igem.org/mediawiki/2015/2/20/2015-Glasgow-timeline12.jpg" style="max-width:350px;">
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          <span class="date"></span>
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      <div class="timeline-article">
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        <div class="content-left-container">
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          <img src="https://static.igem.org/mediawiki/2015/d/dd/2015-Glasgow-timeline13.jpg" style="max-width:350px;">
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        <div class="content-right-container">
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          <div class="content-right">
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          <h1><strong>UVA sensor</strong></h1><p>Found genomic DNA from <i>Synechocystis</i> PCC6803 to use for PCR. Ordered P<sub><i>LsiR</i></sub> as a G-Block with BioBrick prefix and suffix.</p>
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<div id="Week3"></div>
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</br>
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</br>
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</br>
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  <div class="timeline-start">Week3</div>
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          <div class="content-left">
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          <h1><strong>Mhairi’s flapjacks</strong></h1><p>Talked to Amber Jones from the Glasgow School of Art about our project – changed to nightlight toys.</p>
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          <img src="https://static.igem.org/mediawiki/2015/e/ee/2015-Glasgow-timeline7.jpg" style="max-width:350px;">
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          <img src="https://static.igem.org/mediawiki/2015/1/18/2015-Glasgow-Team.jpg" style="max-width:350px">
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        <div class="content-right-container">
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          <div class="content-right">
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          <h1><strong>Bioluminescence</strong></h1><p>Started PCR of Lux genes from K325909 (twice), only LuxB worked.</p>
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          <div class="content-left">
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        <h1><strong>Repressors</strong></h1><p>Ligated R0040 with I13500 and E5501 separately in pSB1C3, R0011.B0032 oligo with C0040 in pSB1C3, and B0032.PhlF.B0010+ and B0032.SrpR.B0010+ G-Blocks separately in pSB1C3. Attempted transformations into TOP10 and DH5α with both electrocompetent cells, and chemically competent cells, but there was no growth, except on the control plates. Repeated this week’s ligations.</p>
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            <img src="https://static.igem.org/mediawiki/2015/6/65/2015-Glasgow-max.jpg" style="max-width:350px">
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          <img src="https://static.igem.org/mediawiki/2015/4/46/2015-Glasgow-timeline14.jpg" style="max-width:350px">
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        <div class="content-right-container">
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          <h1><strong>UVA sensor</strong></h1><p>Found <i>Synechocystis</i> PCC6803 genomic DNA in Sean's freezer. Started PCR of <i>uirR</I> and <i>uirS</I> (twice), didn’t work. Ligated P<sub><I>LsiR</i></sub> G-Block into pSB1C3.</p>
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<div id="Week4"></div>
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</br>
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  <div class="timeline-start">Week4</div>
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          <div class="content-left">
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          <h1><strong>Cara’s lemon drizzle cake</strong></h1><p>Pretty cool,^0^</p>
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        <div class="content-right-container">
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          <img src="https://static.igem.org/mediawiki/2015/8/80/2015-Glasgow-timeline9.jpg" style="max-width:350px;">
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          <img src="https://static.igem.org/mediawiki/2015/c/c2/2015-Glasgow-Mha.jpg" style="max-width:350px">
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        </div>
 +
        <div class="content-right-container">
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          <div class="content-right">
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          <h1><strong>Bioluminescence</strong></h1><p>PCR from K325909 worked for LuxA, B, C, D, E and G worked. Ligated into pSB1C3 and transformed into TOP10. No growth.</p>
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        <h1><strong>Repressors</strong></h1><p>Transformed redone ligations of R0040.I13500, R0040.E5501, R0011.B0032.C0040, B0032.PhlF.B0010+, and B0032.SrpR.B0010+ all in pSB1C3 into TOP10.</p>
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            <img src="https://static.igem.org/mediawiki/2015/6/68/2015-Glasgow-timeline15.jpg" style="max-width:350px;">
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          <h1><strong>UVA sensor</strong></h1><p>PCR of <i>uirS</i> and <i>uirR</i> from Synechocystis PCC6803 genomic DNA worked, ligated <i>uirR</i> into pSB1C3 and transformed into TOP10. PCR of <i>uirS</i> was ligated into TOPO TA cloning plasmid and transformed into TOP10. Designed primers for site-directed mutagenesis of <i>uirS</i> to remove incompatible restriction sites.</p>
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<div id="Week5"></div>
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  <div class="timeline-start">Week5</div>
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          <h1><strong>Adele’s millionaire shortcake</strong></h1><p>Feel like I'm millionaire, LOL.</p>
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 +
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 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
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 +
          <img src="https://static.igem.org/mediawiki/2015/a/a3/2015-Glasgow-timeline16.jpg" style="max-width:350px;">
 +
          </div>
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        </div>
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        <div class="content-right-container">
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          <div class="content-right">
 +
          <h1><strong>Bioluminescence</strong></h1><p>Redid PCR of LuxA, B, C, D, E and G from K325909 and gel purified the PCR product before ligating into pSB1C3 and transforming into TOP10. Designed primers for ribosome binding site library.</p>
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
        </div>
 +
      </div>
 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
        <h1><strong>Repressors</strong></h1><p>Ligated R0011N with B0032.PhlF.B0010+ and B0032.SrpR.B0010+, separately, and transformed into TOP10.</p>
 +
          </div>
 +
        </div>
 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
            <img src="https://static.igem.org/mediawiki/2015/6/67/2015-Glasgow-timeline18.jpg" style="max-width:350px;">
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
        </div>
 +
      </div>
 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
          <img src="https://static.igem.org/mediawiki/2015/a/ad/2015-Glasgow-timeline19.jpg" style="max-width:350px;">
 +
          </div>
 +
        </div>
 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
          <h1><strong>UVA sensor</strong></h1><p>Purified oligos for site-directed mutagenesis. Ligated P<sub><i>LsiR</i></sub> in pSB1C3 with E5501 and I13500, and transformed into TOP10.</p>
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
        </div>
 +
      </div>
 +
<div id="Week6"></div>
 +
</br>
 +
</br>
 +
</br>
 +
<div class="timeline-start">Week6</div>
 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
          <h1><strong>.</strong></h1><p>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
          <img src="https://static.igem.org/mediawiki/2015/6/6b/2015-Glasgow-timeline6.jpg" style="max-width:350px;">
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
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 +
        </div>
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      <div class="timeline-article">
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        <div class="content-left-container">
 +
          <div class="content-left">
 +
          <p></p>
 +
          </div>
 +
        </div>
 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
          <h1><strong>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
        </div>
 +
      </div>
 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
        <h1><strong>Repressors</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
−
            <img src="https://static.igem.org/mediawiki/2015/e/eb/2015-Glasgow-timeline1.jpg" style="max-width:350px;">
 
 
             <p></p>
 
             <p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">9th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
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              <p>The 4 strains of E. coli used are: MGI655ZI (which has tetR, and lacIq, DS941 (recF-), TOP10 (recA-), DH5α (recA-). We designed Phlf and SrpR promoters as BioBricks to be ordered from IDT as if their prefix and suffix were cut with EcoRI and SpeI.</p>
+
          <p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
 +
          <h1><strong>UVA sensor</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">15th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
 +
<div id="Week7"></div>
 +
</br>
 +
</br>
 +
</br>
 +
<div class="timeline-start">Week7</div>
 
       <div class="timeline-article">
 
       <div class="timeline-article">
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
          <h1><strong>.</strong></h1><p>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
          <img src="https://static.igem.org/mediawiki/2015/6/6b/2015-Glasgow-timeline6.jpg" style="max-width:350px;">
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
        </div>
 +
      </div>
 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
          <p></p>
 +
          </div>
 +
        </div>
 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
          <h1><strong>Bioluminescence</strong></h1><p>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
        </div>
 +
      </div>
 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
        <h1><strong>Repressors</strong></h1><p>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="content-right-container">
 +
          <div class="content-right">
 
             <p></p>
 
             <p></p>
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
        </div>
 +
      </div>
 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
          <p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
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−
            <p>           We grew bacteria from a single colony from an agar plate in liquid culture to prepare for the dilution series. We designed the Phlf and SrpR respressor sequences with a prefix, B0032 ribosome binding site, TACTAG scar sequence, the repressor itself, TACTAGAG scar sequence, B0010 extended terminator with the help of Steve, and suffix.</p>
+
          <h1><strong>UVA sensor</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">16th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
 +
<div id="Week8"></div>
 +
</br>
 +
</br>
 +
</br>
 +
<div class="timeline-start" >Week8</div>
 
       <div class="timeline-article">
 
       <div class="timeline-article">
 
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           <div class="content-left">
 +
          <h1><strong>Ye’s cakes</strong></h1><p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
 +
          <img src="https://static.igem.org/mediawiki/2015/c/ca/2015-Glasgow-timeline20.jpg" style="max-width:350px;">
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">9th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
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         </div>
 
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 +
          <img src="https://static.igem.org/mediawiki/2015/9/92/2015-Glasgow-timeline21.jpg" style="max-width:350px;">
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
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           <div class="content-right">
 +
          <h1><strong>Bioluminescence</strong></h1><ul>
 +
<li> Ligating promoter (pBAD) upstream of LuxAB – test by spraying with decanal</li>
 +
<li> Transformed LuxCD into DH5a and DS941.Z1 – ligate in R0011N (will be repressed in those strains); ligate Lux E downstream</li>
 +
<li> checking if intermediates of tetradecanal synthesis is toxic to cells (hence a repressed promoter)</li>
 +
<li> Checking sequenced of Lux genes with mutations (different from genomic Vibrio sequence) – if both colonies are the same, may be mutations in luxoperon (K325909) – need to design primers</li>
 +
<li> Ribosome binding site library</li>
 +
<ul>
 +
<li> PCR products of LuxA-G individually, transforming into</li>
 +
<li> LuxA ligated into pBAD-pSB1C3</li>
 +
<li> LuxC ligated into R0011N-pSB1C3</li>
 +
<li> LuxB,D,E, and G ligated into pSB1C3 with no promoter</li>
 +
<li> Ligate pBAD-(library)-LuxA with (library)-LuxB and spray with decanal</li>
 +
</ul>
 +
</ul>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">9th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
−
<!---------JULY------->
+
       <div class="timeline-article">
−
       <div class="timeline-article" id="July">
+
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
        <h1><strong>Repressors</strong></h1>
 +
<ul>
 +
<li> Have promoters+GFP in pSB3K3 in DS941.Z1</li>
 +
<li> Have R0011N+repressors in pSB1C3 in DS941.Z1 – make competent cells, and transform in promoters+GFP in pSB3K3</li>
 +
<li> Growth curve and fluorescence to test promoters</li>
 +
<li>Bi-stable switch: have PPhlF+SrpR, PSrpR+PhlF in pSB1C3 – need to make and check miniprep, then transform promoters+GFP in pSB3K3</li>
 +
<li>Make one promoter with GFP, one with RFP on same plasmid (one needs terminator)</li>
 +
<li>Terminators: ligate “steve” (B0010+), B0010, and B0015 into pSB1A10</li>
 +
 +
</ul>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
 +
            <img src="https://static.igem.org/mediawiki/2015/b/b8/2015-Glasgow-timeline22.jpg" style="max-width:350px;">
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">9th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
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         </div>
 
         </div>
 
       </div>
 
       </div>
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 +
          <img src="https://static.igem.org/mediawiki/2015/9/91/2015-Glasgow-timeline23.jpg" style="max-width:350px">
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
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           <div class="content-right">
 +
          <h1><strong>UVA sensor</strong></h1>
 +
<ul>
 +
<li>3rd PCR mutagenesis of UirS – so hopefully fixed, needs to be sequenced</li>
 +
<li>Chromophore (PCB) BioBricks as miniprep and checked by digest (has a constitutive promoter)</li>
 +
<li>Confirmed P<sub><i>LsiR</i></sub> is off when not activated (tested by GFP with growth curve)</li>
 +
</ul>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">9th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
 +
<div id="Week9"></div>
 +
</br>
 +
</br>
 +
</br>
 +
<div class="timeline-start">Week9</div>
 
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         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
          <h1><strong>.</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
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 +
          <img src="">
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">9th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
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         </div>
 
         </div>
 
       </div>
 
       </div>
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          <p></p>
 
           </div>
 
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         <div class="content-right-container">
 
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 +
          <h1><strong>.</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">9th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
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         </div>
 
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 +
        <h1><strong>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
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 +
            <p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">9th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
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         </div>
 
         </div>
 
       </div>
 
       </div>
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         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
          <p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
 +
          <h1><strong>.</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
−
        <!---------AUGUST------>
+
         <div class="meta-date">
−
         <div class="meta-date" id="August">
+
           <span class="date"></span>
−
           <span class="date">9th</span>
+
           <span class="month"></span>
−
           <span class="month">Jun</span>
+
 
         </div>
 
         </div>
 
       </div>
 
       </div>
 +
<div id="Week10"></div>
 +
</br>
 +
</br>
 +
</br>
 +
<div class="timeline-start">Week10</div>
 
       <div class="timeline-article">
 
       <div class="timeline-article">
 
         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
          <h1><strong>.</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
 +
          <img src="">
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">9th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
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         <div class="content-left-container">
 
         <div class="content-left-container">
 
           <div class="content-left">
 
           <div class="content-left">
 +
          <p></p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="content-right-container">
 
         <div class="content-right-container">
 
           <div class="content-right">
 
           <div class="content-right">
 +
          <h1><strong>.</strong></h1><p>.</p>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
         <div class="meta-date">
 
         <div class="meta-date">
−
           <span class="date">9th</span>
+
           <span class="date"></span>
−
           <span class="month">Jun</span>
+
           <span class="month"></span>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
        <h1><strong>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
            <p></p>
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
        </div>
 +
      </div>
 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
          <p></p>
 +
          </div>
 +
        </div>
 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
          <h1><strong>.</strong></h1><p>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
        </div>
 +
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 +
<div id="Week11"></div>
 +
</br>
 +
</br>
 +
</br>
 +
<div class="timeline-start">Week11</div>
 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
          <h1><strong>.</strong></h1><p>.</p>
 +
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 +
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 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
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 +
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 +
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 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
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 +
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 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
          <p></p>
 +
          </div>
 +
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 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
          <h1><strong>.</strong></h1><p>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
        </div>
 +
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 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
        <h1><strong>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
            <p></p>
 +
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 +
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 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
        </div>
 +
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 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
          <p></p>
 +
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 +
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 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
          <h1><strong>.</strong></h1><p>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
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 +
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 +
<div id="Week12"></div>
 +
</br>
 +
</br>
 +
</br>
 +
<div class="timeline-start">Week12</div>
 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
          <h1><strong>.</strong></h1><p>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
          <img src="">
 +
          </div>
 +
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 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
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      <div class="timeline-article">
 +
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          <p></p>
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 +
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 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
          <h1><strong>.</strong></h1><p>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
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 +
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 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
 +
          <div class="content-left">
 +
        <h1><strong>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
            <p></p>
 +
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 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month"></span>
 +
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 +
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 +
      <div class="timeline-article">
 +
        <div class="content-left-container">
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          <div class="content-left">
 +
          <p></p>
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 +
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 +
        <div class="content-right-container">
 +
          <div class="content-right">
 +
          <h1><strong>.</strong></h1><p>.</p>
 +
          </div>
 +
        </div>
 +
        <div class="meta-date">
 +
          <span class="date"></span>
 +
          <span class="month readMore"></span>
 +
        </div>
 +
      </div>
 +
 
        
 
        
 
     </div>
 
     </div>
 
   </section>
 
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+
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body {
 
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Line 448: Line 1,102:
 
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   @media screen and (max-width: 810px) {
−
     #June #July #August #September{
+
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         margin-left: 5px;
 
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.timeline-article .meta-date .month {
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 +
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.timeline-article .meta-date .month {
 +
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 +
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 +
}
 
</style>
 
</style>
−
</head>
+
</head>  
−
<body data-spy="scroll" data-target="#myScrollspy" data-offset="20">
+
<body data-spy="scroll" data-target="#myScrollspy" data-offset="20">
−
   
+
     <div class="row" style="margin-top: 30px;">
−
     <div class="row" style="margin-top: 80px;">
+
 
         <div class="container">
 
         <div class="container">
−
     <nav class="col-sm-3" id="myScrollspy">
+
     <div id="myScrollspy">
 
       <ul class="nav nav-pills nav-stacked">
 
       <ul class="nav nav-pills nav-stacked">
−
         <li class="active"><a href="#June">June</a></li>
+
         <li class="active"><a href="#Week1">Week1</a></li>
−
         <li><a href="#July">July</a></li>
+
         <li><a href="#Week2">Week2</a></li>
−
         <li><a href="#August">August</a></li>
+
         <li><a href="#Week3">Week3</a></li>
−
         <li><a href="#September">September</a></li>
+
         <li><a href="#Week4">Week4</a></li>
 +
        <li><a href="#Week5">Week5</a></li>
 +
        <li><a href="#Week6">Week6</a></li>
 +
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 +
        <li><a href="#Week8">Week8</a></li>
 +
        <li><a href="#Week9">Week9</a></li>
 +
        <li><a href="#Week10">Week10</a></li>
 
       </ul>
 
       </ul>
−
     </nav>
+
     </div>
 
   </div>
 
   </div>
 
</div>
 
</div>
 +
</div>
 +
<script type="text/javascript">
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{{Glasgowfooter}}

Latest revision as of 18:34, 15 November 2015

Responsive Vertical Timeline



Week1

James’ muffins

Talked to Professors Marshall Stark and John Christie about our project.

Repressors

Ordered PPhlF and PSrpR with BioBrick prefix and suffix as oligos. Ordered; PhlF and SrpR as G-blocks, with ribosome binding site B0032; Terminator B0010+ (a longer version of B0010, that we will characterise and submit as a new BioBrick) with BioBrick prefix and suffix. Got E5501 and I13500 – GFP (E0040) with ribosome binding sites B0032 and B0034 respectively – in pSB1C3 in DH5α from glycerol stocks from last year’s iGEM team.

UVA sensor

Designed primers to PCR uirS, uirR, and PlsiR from Synechocystis PCC6803 genomic DNA with ribosome binding site B0032 and BioBrick prefix/suffix. Tested UVB sensitivity of strains TOP10 and DH5α (recA- mutants - very poor DNA repair), DS941 (recF- - limited DNA repair), and MG1655.Z1 (wild type for DNA repair).

Bioluminescence

Retrieved plasmid DNA of pBAD.LuxCDABEG (Cambridge Luxbrick: K325909) and pBAD.LuxCDABEG.LumP (K769020) from 2015 iGEM distribution plates and transformed into TOP10 and DH5α strains. Designed primers to PCR LuxA, B, C, D, E, and G individually with ribosome binding site B0032 and BioBrick prefix/suffix from K325909.




Week2

Vilija’s carrot cake

So good, honestly.

Bioluminescence

Tested K325909 and K769020 in TOP10 to see how long until there is expression of bioluminescence after addition of arabinose inducer, how bright the bioluminescence is, and how LumP affects it in K769020.

Repressors

Ligated both PPhlF and PSrpR with E5501 and I13500 in pSB1C3, respectively, and transformed into TOP10 and DH5α. Retrieved C0040 (TetR), R0040 (PTetR) and backbone pSB3K3 (with insert J04450) off distribution plates – to use C0040 as a control for our repressors, and to use pSB3K3 as a second plasmid to have the promoters and repressors on separate plasmids. Ordered G-Block of R0011.B0032 (PLacI + RBS) for C0040 – as the plasmid on the plate has to RBS.

UVA sensor

Found genomic DNA from Synechocystis PCC6803 to use for PCR. Ordered PLsiR as a G-Block with BioBrick prefix and suffix.




Week3

Mhairi’s flapjacks

Talked to Amber Jones from the Glasgow School of Art about our project – changed to nightlight toys.

Bioluminescence

Started PCR of Lux genes from K325909 (twice), only LuxB worked.

Repressors

Ligated R0040 with I13500 and E5501 separately in pSB1C3, R0011.B0032 oligo with C0040 in pSB1C3, and B0032.PhlF.B0010+ and B0032.SrpR.B0010+ G-Blocks separately in pSB1C3. Attempted transformations into TOP10 and DH5α with both electrocompetent cells, and chemically competent cells, but there was no growth, except on the control plates. Repeated this week’s ligations.

UVA sensor

Found Synechocystis PCC6803 genomic DNA in Sean's freezer. Started PCR of uirR and uirS (twice), didn’t work. Ligated PLsiR G-Block into pSB1C3.




Week4

Cara’s lemon drizzle cake

Pretty cool,^0^

Bioluminescence

PCR from K325909 worked for LuxA, B, C, D, E and G worked. Ligated into pSB1C3 and transformed into TOP10. No growth.

Repressors

Transformed redone ligations of R0040.I13500, R0040.E5501, R0011.B0032.C0040, B0032.PhlF.B0010+, and B0032.SrpR.B0010+ all in pSB1C3 into TOP10.

UVA sensor

PCR of uirS and uirR from Synechocystis PCC6803 genomic DNA worked, ligated uirR into pSB1C3 and transformed into TOP10. PCR of uirS was ligated into TOPO TA cloning plasmid and transformed into TOP10. Designed primers for site-directed mutagenesis of uirS to remove incompatible restriction sites.




Week5

Adele’s millionaire shortcake

Feel like I'm millionaire, LOL.

Bioluminescence

Redid PCR of LuxA, B, C, D, E and G from K325909 and gel purified the PCR product before ligating into pSB1C3 and transforming into TOP10. Designed primers for ribosome binding site library.

Repressors

Ligated R0011N with B0032.PhlF.B0010+ and B0032.SrpR.B0010+, separately, and transformed into TOP10.

UVA sensor

Purified oligos for site-directed mutagenesis. Ligated PLsiR in pSB1C3 with E5501 and I13500, and transformed into TOP10.




Week6

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Repressors

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UVA sensor

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Week7

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Bioluminescence

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Repressors

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UVA sensor

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Week8

Ye’s cakes

Bioluminescence

  • Ligating promoter (pBAD) upstream of LuxAB – test by spraying with decanal
  • Transformed LuxCD into DH5a and DS941.Z1 – ligate in R0011N (will be repressed in those strains); ligate Lux E downstream
  • checking if intermediates of tetradecanal synthesis is toxic to cells (hence a repressed promoter)
  • Checking sequenced of Lux genes with mutations (different from genomic Vibrio sequence) – if both colonies are the same, may be mutations in luxoperon (K325909) – need to design primers
  • Ribosome binding site library
    • PCR products of LuxA-G individually, transforming into
    • LuxA ligated into pBAD-pSB1C3
    • LuxC ligated into R0011N-pSB1C3
    • LuxB,D,E, and G ligated into pSB1C3 with no promoter
    • Ligate pBAD-(library)-LuxA with (library)-LuxB and spray with decanal

Repressors

  • Have promoters+GFP in pSB3K3 in DS941.Z1
  • Have R0011N+repressors in pSB1C3 in DS941.Z1 – make competent cells, and transform in promoters+GFP in pSB3K3
  • Growth curve and fluorescence to test promoters
  • Bi-stable switch: have PPhlF+SrpR, PSrpR+PhlF in pSB1C3 – need to make and check miniprep, then transform promoters+GFP in pSB3K3
  • Make one promoter with GFP, one with RFP on same plasmid (one needs terminator)
  • Terminators: ligate “steve” (B0010+), B0010, and B0015 into pSB1A10

UVA sensor

  • 3rd PCR mutagenesis of UirS – so hopefully fixed, needs to be sequenced
  • Chromophore (PCB) BioBricks as miniprep and checked by digest (has a constitutive promoter)
  • Confirmed PLsiR is off when not activated (tested by GFP with growth curve)



Week9

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Week10

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Week11

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Week12

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Location

Bower Building, Wilkins Teaching Laboratory
University of Glasgow
University Avenue
G12 8QQ

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