Team/CINVESTAV-IPN-UNAM MX/Systems.htm

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Systems assembly
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<h1><em>Systems assembly!</em></h1>
 +
  <p><strong>12 July</strong></p>
 +
  <ul>
 +
    <li>ZR Miniprep Kit from all&nbsp; the genes
 +
      <ul>
 +
          <li>AppA</li>
 +
        <li>PpsR</li>
 +
        <li>PrrA</li>
 +
        <li>PrrB</li>
 +
        <li>PrrA Promoter</li>
 +
        <li>PpsR Promoter</li>
 +
      </ul>
 +
    </li>
 +
    <li>Transformation of E. coli TOP10</li>
 +
  </ul>
 +
  <p><strong>13 July</strong></p>
 +
  <ul>
 +
    <li>Colony PCR</li>
 +
    <ul>
 +
      <li>Taq Buffer&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 5&mu;L</li>
 +
      <li>MgCl2&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 5&mu;L</li>
 +
      <li>dnTP&rsquo;s&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1&mu;L</li>
 +
      <li>Forward&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1&mu;L</li>
 +
      <li>Reverse&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1&mu;L</li>
 +
      <li>Taq polymerase&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 0.5&mu;L</li>
 +
      <li>H<sub>2</sub>O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 36.5&mu;L</li>
 +
      <ul>
 +
        <li>TOTAL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 50&mu;L</li>
 +
      </ul>
 +
    </ul>
 +
  </ul>
 +
  <p>NEGATIVE</p>
 +
  <p><strong>14 July</strong></p>
 +
  <ul>
 +
    <li>Culture from 4 colonies per gene.  6hrs</li>
 +
    <li>ZR Miniprep Kit</li>
 +
    <li>Agarose gel electrophoresis 1%</li>
 +
    <li>PCR from plasmid</li>
 +
    <ul>
 +
      <li>Taq Buffer&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2&mu;L</li>
 +
      <li>MgCl2&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2&mu;L</li>
 +
      <li>dnTP&rsquo;s&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 0.5&mu;L</li>
 +
      <li>Forward&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;  1&mu;L</li>
 +
      <li>Reverse&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1&mu;L</li>
 +
      <li>Taq polymerase&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1&mu;L</li>
 +
      <li>H<sub>2</sub>O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 11.5&mu;L</li>
 +
      <ul>
 +
        <li>TOTAL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 20&mu;L</li>
 +
      </ul>
 +
    </ul>
 +
  </ul>
 +
  <p>POSITIVE</p>
 +
  <ul>
 +
    <li>Band purification</li>
 +
    <li>Plasmid digestion&nbsp; only&nbsp;  for genes (1.5h)</li>
 +
    <ul>
 +
      <li>Xba1 and Pst1</li>
 +
    </ul>
 +
    <li>Plasmid digestion&nbsp; only&nbsp;  for promoters (1.5h)</li>
 +
    <ul>
 +
      <li>EcoRI and SpeI</li>
 +
      <ul>
 +
        <li>Agarose gel electrophoresis 1%</li>
 +
        <li>Genes ( +)</li>
 +
        <li>Promoters&nbsp;&nbsp; (-)</li>
 +
      </ul>
 +
    </ul>
 +
    <li>Ligations</li>
 +
    <ul>
 +
      <li>RBS+GENES</li>
 +
      <ul>
 +
        <li>20&mu;L  Reaction (see protocols)</li>
 +
      </ul>
 +
    </ul>
 +
    <li>&nbsp;AGAIN!!!!&nbsp;  Plasmid digestion, only&nbsp; for  promoters (1.5h)</li>
 +
    <ul>
 +
      <li>EcoRI and SpeI</li>
 +
      <ul>
 +
        <li>Promoters&nbsp;&nbsp; (-)</li>
 +
      </ul>
 +
      <li>Agarose gel electrophoresis 4%</li>
 +
    </ul>
 +
  </ul>
 +
  <ul>
 +
    <ul>
 +
      <li>This was 4 more times!!!!!!!!!!!!!</li>
 +
    </ul>
 +
  </ul>
 +
  <p><strong>15 July</strong></p>
 +
  <ul>
 +
    <li>Transformation by heat-shock</li>
 +
  </ul>
 +
  <p><strong>16 July</strong></p>
 +
  <ul>
 +
    <li>Ligation of promoters in psB1A3</li>
 +
    <li>Cultures of RBS+GENES (6hr)</li>
 +
    <ul>
 +
      <li>ZR Miniprep kit</li>
 +
    </ul>
 +
  </ul>
 +
  <p><strong>17 July</strong></p>
 +
  <ul>
 +
    <li>Digestion of RBS+GENS (1.5h, see  protocols)</li>
 +
    <ul>
 +
      <li>RBS+PrrA &nbsp;&nbsp;&nbsp; S+P</li>
 +
      <li>RBS+PrrB&nbsp;&nbsp;&nbsp;&nbsp; X+P</li>
 +
      <li>RBS+PrrC&nbsp;&nbsp;&nbsp;&nbsp; E+S</li>
 +
      <li>DT&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; E+X</li>
 +
      <li>RBS+AppA&nbsp;&nbsp; X+P</li>
 +
      <li>Prom J23104&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; S+P</li>
 +
      <li>RBS+PpsR&nbsp;&nbsp;&nbsp; E+S</li>
 +
    </ul>
 +
    <li>Agarose gel electrophoresis 1%</li>
 +
    <ul>
 +
      <li>POSITIVE (+)</li>
 +
    </ul>
 +
  </ul>
 +
  <ul>
 +
    <li>Band Purification </li>
 +
    <li>Ligations A</li>
 +
    <ul>
 +
      <li><strong>1A</strong>&nbsp; &nbsp;&nbsp;&nbsp; RBS+PrrA  +RBS+PrrB&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </li>
 +
      <li><strong>2A</strong>&nbsp;&nbsp; &nbsp;&nbsp; RBS+PrrC+DT&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </li>
 +
      <li><strong>3A</strong>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;  Prom J23104+RBS+AppA </li>
 +
      <li><strong>5A</strong> &nbsp;&nbsp;&nbsp;&nbsp; RBS+PpsR+  DT</li>
 +
    </ul>
 +
    <li>Overnight cultures&nbsp; of promoters+psB1A3</li>
 +
  </ul>
 +
  <p><strong>18 July</strong></p>
 +
  <ul>
 +
    <li>Transformation of ligations A</li>
 +
    <li>ZR Miniprep Kit</li>
 +
    <li>Digestiion</li>
 +
    <ul>
 +
      <li>POSITIVE</li>
 +
    </ul>
 +
    <li>Band purification</li>
 +
    <li>Ligation D</li>
 +
    <ul>
 +
      <li>PrrA Promoter+GFP+DT</li>
 +
      <li>PpsR Promoter+GFP+DT</li>
 +
    </ul>
 +
  </ul>
 +
  <p><strong>19 July</strong></p>
 +
  <ul>
 +
    <li>Transformation of ligations D</li>
 +
    <li>Cultures of ligations A (6h)</li>
 +
    <ul>
 +
      <li>ZR Miniprep Kit</li>
 +
      <li>Digestion&nbsp; (1.5h)</li>
 +
      <ul>
 +
        <li><strong>1A</strong>&nbsp; &nbsp;&nbsp;&nbsp; S+P&nbsp;&nbsp;&nbsp;&nbsp; </li>
 +
        <li><strong>2A</strong>&nbsp;&nbsp; &nbsp;&nbsp; X+P&nbsp;&nbsp;&nbsp; </li>
 +
        <li><strong>3A</strong>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; S+P</li>
 +
        <li><strong>5A</strong> &nbsp;&nbsp;&nbsp;&nbsp; X+P</li>
 +
      </ul>
 +
      <li>Agarose gel electrophoresis 1%</li>
 +
      <li>POSITIVE (+)</li>
 +
      <li>Band purification</li>
 +
    </ul>
 +
    <li>Ligations C</li>
 +
    <ul>
 +
      <li><strong>1C</strong> &nbsp;&nbsp;&nbsp;&nbsp; RBS+PrrA +RBS+PrrB +RBS+PrrC+DT</li>
 +
      <li><strong>2C </strong>&nbsp;&nbsp;&nbsp;&nbsp; Prom  J23104+RBS+AppA+RBS+PpsR+ DT</li>
 +
    </ul>
 +
  </ul>
 +
  <p><strong>20 July</strong></p>
 +
  <ul>
 +
    <li>Overnight cultures of ligations D  (6h) (small colonies)</li>
 +
    <li>Transformation of ligations C</li>
 +
  </ul>
 +
  <p><strong>21 July</strong></p>
 +
  <ul>
 +
    <li>Cultures of Ligation C</li>
 +
    <ul>
 +
      <li>Miniprep Kit</li>
 +
      <li>Digestion E and E+P</li>
 +
      <li>Agarose gel electrophoresis 1%</li>
 +
      <li>POSITIVE (+)</li>
 +
    </ul>
 +
    <li>ZR Miniprep Kit of ligations D</li>
 +
    <ul>
 +
      <li>Digestion E and X+P</li>
 +
      <li>Agarose gel electrophoresis 1%</li>
 +
      <li>POSITIVE (+)</li>
 +
      <li>Band purification!</li>
 +
      <ul>
 +
        <li>NEGATIVE</li>
 +
      </ul>
 +
      <li>Overnight culture</li>
 +
    </ul>
 +
  </ul>
 +
  <p><strong>22 July</strong></p>
 +
  <ul>
 +
    <li>ZR Miniprep Kit ligations D</li>
 +
    <ul>
 +
      <li>Digestion E and E+P</li>
 +
      <ul>
 +
        <li>Ppsr promoter+GFP+DT&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; X+P</li>
 +
      </ul>
 +
      <li>Agarose gel electrophoresis 1%</li>
 +
      <li>POSITIVE (+)</li>
 +
      <li>High concentration! </li>
 +
      <li>Band purification</li>
 +
      <ul>
 +
        <li>POSITIVE +</li>
 +
      </ul>
 +
    </ul>
 +
    <li>Miniprep Kit ligation C</li>
 +
    <ul>
 +
      <li>Digestion</li>
 +
      <ul>
 +
        <li>PromJ23104 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; S+P</li>
 +
        <li>RBS+PrrA +RBS+PrrB +RBS+PrrC+DT&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; X+P</li>
 +
        <li>Prom J23104+RBS+AppA+RBS+PpsR+ DT&nbsp;&nbsp; S+P</li>
 +
      </ul>
 +
      <li>Agarose gel electrophoresis 1%</li>
 +
      <li>High concentration! J </li>
 +
      <li>Band purification</li>
 +
      <ul>
 +
        <li>POSITIVE +</li>
 +
      </ul>
 +
      <li>Ligations E</li>
 +
      <ul>
 +
        <li><strong>1E </strong>&nbsp;&nbsp;&nbsp;&nbsp; PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT</li>
 +
        <li><strong>2E</strong>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Prom J23104+RBS+AppA+RBS+PpsR+ DT+ Ppsr  promoter+GFP+DT </li>
 +
      </ul>
 +
    </ul>
 +
  </ul>
 +
  <p><strong>23 July</strong></p>
 +
  <ul>
 +
    <li>Transformation of ligations E</li>
 +
    <li>WE are also worked in the assembly of  the butanol pathway + regulatory systems</li>
 +
  </ul>
 +
  <p><strong>24 July</strong></p>
 +
  <ul>
 +
    <li><strong>1E  NOTHING!!!!!!!!!!</strong> </li>
 +
    <ul>
 +
      <li>Again ligation!!</li>
 +
      <li>Miniprep Kit ligation C</li>
 +
      <li>Digestion</li>
 +
      <ul>
 +
        <li>PromJ23104 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; S+P</li>
 +
        <li>RBS+PrrA +RBS+PrrB +RBS+PrrC+DT&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; X+P</li>
 +
      </ul>
 +
      <li>Agarose gel electrophoresis 1%</li>
 +
      <li>High concentration! J </li>
 +
      <li>Band purification</li>
 +
      <ul>
 +
        <li>POSITIVE +</li>
 +
      </ul>
 +
    </ul>
 +
    <li><strong>2E;  3 COLONIES!!... ;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Prom J23104+RBS+AppA+RBS+PpsR+  DT+ Ppsr promoter+GFP+DT</strong> </li>
 +
    <ul>
 +
      <li>6h culture!</li>
 +
      <li>ZR Miniprep Kit</li>
 +
      <li>Digestion (1.5h) &nbsp;&nbsp; S+P and X+P</li>
 +
      <li>Agarose gel electrophoresis 1%</li>
 +
      <ul>
 +
        <li>POSITIVE!!!!!!!!!!!!!!!!!!!</li>
 +
      </ul>
 +
      <li>Band purification </li>
 +
      <ul>
 +
        <li>FIRST SYSTEM READY</li>
 +
      </ul>
 +
    </ul>
 +
  </ul>
 +
  <p><strong>25 July</strong></p>
 +
  <ul>
 +
    <li>Transformation of ligation 1E</li>
 +
  </ul>
 +
  <p><strong>26 July</strong></p>
 +
  <ul>
 +
    <li><strong>1E; &nbsp;2 COLONIES!!! ; PromJ23104 +RBS+PrrA +RBS+PrrB  +RBS+PrrC+DT</strong>
 +
        <ul>
 +
          <li>6h culture!</li>
 +
          <li>ZR Miniprep Kit</li>
 +
          <li>Digestion (1.5h)</li>
 +
          <li>S+P and X+P</li>
 +
          <li>Agarose gel electrophoresis 1%
 +
            <ul>
 +
                <li>POSITIVE!!!!!!!!!!!!!!!!!!!</li>
 +
              <li>Band Purification</li>
 +
              <li>JUST ONE MORE LIGATION</li>
 +
              <li>Overnight digestion</li>
 +
            </ul>
 +
          </li>
 +
        </ul>
 +
    </li>
 +
  </ul>
 +
  <div align="center"><img src="http://2012.igem.org/wiki/images/1/1d/System04.jpg" alt="fe" width="480" height="385"></div>
 +
  <div align="center"><img src="http://2012.igem.org/wiki/images/8/80/System03.jpg" alt="fe" width="480" height="519"></div>
 +
  <p><strong>27 July</strong></p>
 +
  <ul>
 +
    <li>ZR Miniprep Kit ligations 1D</li>
 +
    <ul>
 +
      <li>Digestion X+P</li>
 +
      <ul>
 +
        <li>PrrA promoter+GFP+DT&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; X+P</li>
 +
      </ul>
 +
      <li>Agarose gel electrophoresis 1%</li>
 +
      <li>POSITIVE (+)</li>
 +
      <li>High concentration! </li>
 +
      <li>Band purification</li>
 +
      <li>POSITIVE</li>
 +
    </ul>
 +
    <li><strong>1E PromJ23104 +RBS+PrrA +RBS+PrrB  +RBS+PrrC+DT</strong></li>
 +
    <ul>
 +
      <li>ZR Miniprep Kit</li>
 +
      <li>Digestion (1.5h)</li>
 +
      <ul>
 +
        <li>S+P and X+P</li>
 +
      </ul>
 +
      <li>Agarose gel electrophoresis 1%</li>
 +
      <ul>
 +
        <li>POSITIVE!!!!!!!!!!!!!!!!!!!</li>
 +
        <li>Band Purification</li>
 +
        <li>POSITIVE</li>
 +
      </ul>
 +
    </ul>
 +
    <li>Ligation 2F: </li>
 +
    <ul>
 +
      <li>PromJ23104 +RBS+PrrA +RBS+PrrB  +RBS+PrrC+DT+ PrrA promoter+GFP+DT&nbsp;&nbsp;&nbsp;&nbsp; </li>
 +
    </ul>
 +
  </ul>
 +
  <p><strong>28 July</strong></p>
 +
  <ul>
 +
    <li>Transformation ligation 2F</li>
 +
  </ul>
 +
  <p><strong>29 July</strong></p>
 +
  <ul>
 +
    <li><strong>NOTHING,  AGAIN!&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </strong> </li>
 +
    <li>ZR Miniprep Kit ligations 1D</li>
 +
    <ul>
 +
      <li>Digestion X+P</li>
 +
      <ul>
 +
        <li>PrrA promoter+GFP+DT&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; X+P</li>
 +
      </ul>
 +
      <li>Agarose gel electrophoresis 1%</li>
 +
      <li>POSITIVE (+)</li>
 +
      <li>High concentration! </li>
 +
      <li>Band purification</li>
 +
      <li>POSITIVE</li>
 +
    </ul>
 +
    <li><strong>1E PromJ23104 +RBS+PrrA +RBS+PrrB  +RBS+PrrC+DT</strong></li>
 +
    <ul>
 +
      <li>ZR Miniprep Kit</li>
 +
      <li>Digestion (1.5h)</li>
 +
      <ul>
 +
        <li>S+P and X+P</li>
 +
      </ul>
 +
      <li>Agarose gel electrophoresis 1%</li>
 +
      <ul>
 +
        <li>POSITIVE!!!!!!!!!!!!!!!!!!!</li>
 +
        <li>Band Purification</li>
 +
        <li>POSITIVE</li>
 +
      </ul>
 +
    </ul>
 +
    <li>Ligation 2F</li>
 +
  </ul>
 +
  <p><strong>30 July</strong></p>
 +
  <ul>
 +
    <li><strong>Transformation</strong></li>
 +
  </ul>
 +
  <p><strong>1 August</strong></p>
 +
  <ul>
 +
    <li><strong>2F,  4 COLONIES&nbsp;&nbsp;&nbsp;&nbsp; </strong>
 +
        <ul>
 +
          <li>6h<strong> cultures</strong></li>
 +
          <li>ZR Miniprep Kit</li>
 +
          <li>Digestion (1.5h)
 +
            <ul>
 +
                <li>E and E+P</li>
 +
            </ul>
 +
          </li>
 +
          <li>Agarose gel electrophoresis 1%
 +
            <ul>
 +
                <li>POSITIVE!!!!!!!!!!!!!!!!!!!</li>
 +
            </ul>
 +
          </li>
 +
        </ul>
 +
    </li>
 +
    <ul>
 +
      <ul>
 +
        <li>Band Purification</li>
 +
        <li>POSITIVE</li>
 +
        <li><strong>SECOND  SYSTEM READY</strong></li>
 +
      </ul>
 +
    </ul>
 +
  </ul>
 +
  <div align="center"><img src="http://2012.igem.org/wiki/images/1/16/System05.jpg" alt="fe" width="480" height="378"></div>
 +
  <div align="center"><img src="http://2012.igem.org/wiki/images/7/79/System02.jpg" alt="fe" width="480" height="697"></div>
 +
  <ul>
 +
    <li><strong>Party! all the systems are ready&hellip;</strong></li>
 +
  </ul>
 +
  <p align="center"><strong>Biobricks Final assembly to psB1C3</strong></p>
 +
  <ul>
 +
  <ul>
 +
    <li>From 3 of  August to 15 of August, we did the following things.
 +
      <ul>
 +
          <li>Overnight and 6h cultures</li>
 +
        <li>ZR Miniprep Kit</li>
 +
        <li>Digestion (1.5h)
 +
          <ul>
 +
                <li>For ligate</li>
 +
            <li>For confirmation</li>
 +
          </ul>
 +
        </li>
 +
        <li>Agarose gel electrophoresis 1%</li>
 +
        <li>Band purification</li>
 +
        <li>Ligation</li>
 +
        <li>Transformation; and again!</li>
 +
        <li><strong>TOTAL  &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 11 parts  in psB1C3</strong></li>
 +
      </ul>
 +
    </li>
 +
  </ul>
 +
  <div align="center"><img src="http://2012.igem.org/wiki/images/4/4e/System01.jpg" alt="fe" width="480" height="358"></div>
 +
 
 +
</div>
 +
 
 +
<div id="sidebar">
 +
  <div id="updates" class="orangebox">
 +
 +
    <h2>Notebook</h2>
 +
<ul>
 +
<li><a href="February.htm" target="_parent">February</a></li>
 +
<li><a href="March.htm">March</a></li>
 +
<li><a href="April.htm">April</a></li>
 +
<li><a href="May.htm">May</a></li>
 +
<li><a href="June.htm">June</a></li>
 +
<li><a href="July.htm">July</a></li>
 +
<li><a href="August.htm">August</a></li>
 +
<li><a href="September.htm">September</a></li>
 +
<li><a href="http://2012.igem.org/wiki/images/2/25/Systems_assembly.pdf">Systems assembly</a></li>
 +
</ul>
 +
</div>
 +
  </div>  
 +
  <div style="clear: both;">&nbsp;</div>
 +
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Latest revision as of 06:13, 26 October 2012

Rho

Systems assembly!

12 July

  • ZR Miniprep Kit from all  the genes
    • AppA
    • PpsR
    • PrrA
    • PrrB
    • PrrA Promoter
    • PpsR Promoter
  • Transformation of E. coli TOP10

13 July

  • Colony PCR
    • Taq Buffer                        5μL
    • MgCl2                             5μL
    • dnTP’s                             1μL
    • Forward                           1μL
    • Reverse                            1μL
    • Taq polymerase                0.5μL
    • H2O                                 36.5μL
      • TOTAL                 50μL

NEGATIVE

14 July

  • Culture from 4 colonies per gene. 6hrs
  • ZR Miniprep Kit
  • Agarose gel electrophoresis 1%
  • PCR from plasmid
    • Taq Buffer                       2μL
    • MgCl2                             2μL
    • dnTP’s                             0.5μL
    • Forward                          1μL
    • Reverse                            1μL
    • Taq polymerase                1μL
    • H2O                                 11.5μL
      • TOTAL                  20μL

POSITIVE

  • Band purification
  • Plasmid digestion  only  for genes (1.5h)
    • Xba1 and Pst1
  • Plasmid digestion  only  for promoters (1.5h)
    • EcoRI and SpeI
      • Agarose gel electrophoresis 1%
      • Genes ( +)
      • Promoters   (-)
  • Ligations
    • RBS+GENES
      • 20μL Reaction (see protocols)
  •  AGAIN!!!!  Plasmid digestion, only  for promoters (1.5h)
    • EcoRI and SpeI
      • Promoters   (-)
    • Agarose gel electrophoresis 4%
    • This was 4 more times!!!!!!!!!!!!!

15 July

  • Transformation by heat-shock

16 July

  • Ligation of promoters in psB1A3
  • Cultures of RBS+GENES (6hr)
    • ZR Miniprep kit

17 July

  • Digestion of RBS+GENS (1.5h, see protocols)
    • RBS+PrrA     S+P
    • RBS+PrrB     X+P
    • RBS+PrrC     E+S
    • DT                  E+X
    • RBS+AppA   X+P
    • Prom J23104         S+P
    • RBS+PpsR    E+S
  • Agarose gel electrophoresis 1%
    • POSITIVE (+)
  • Band Purification
  • Ligations A
    • 1A      RBS+PrrA +RBS+PrrB     
    • 2A      RBS+PrrC+DT       
    • 3A      Prom J23104+RBS+AppA
    • 5A      RBS+PpsR+ DT
  • Overnight cultures  of promoters+psB1A3

18 July

  • Transformation of ligations A
  • ZR Miniprep Kit
  • Digestiion
    • POSITIVE
  • Band purification
  • Ligation D
    • PrrA Promoter+GFP+DT
    • PpsR Promoter+GFP+DT

19 July

  • Transformation of ligations D
  • Cultures of ligations A (6h)
    • ZR Miniprep Kit
    • Digestion  (1.5h)
      • 1A      S+P    
      • 2A      X+P   
      • 3A      S+P
      • 5A      X+P
    • Agarose gel electrophoresis 1%
    • POSITIVE (+)
    • Band purification
  • Ligations C
    • 1C      RBS+PrrA +RBS+PrrB +RBS+PrrC+DT
    • 2C      Prom J23104+RBS+AppA+RBS+PpsR+ DT

20 July

  • Overnight cultures of ligations D (6h) (small colonies)
  • Transformation of ligations C

21 July

  • Cultures of Ligation C
    • Miniprep Kit
    • Digestion E and E+P
    • Agarose gel electrophoresis 1%
    • POSITIVE (+)
  • ZR Miniprep Kit of ligations D
    • Digestion E and X+P
    • Agarose gel electrophoresis 1%
    • POSITIVE (+)
    • Band purification!
      • NEGATIVE
    • Overnight culture

22 July

  • ZR Miniprep Kit ligations D
    • Digestion E and E+P
      • Ppsr promoter+GFP+DT            X+P
    • Agarose gel electrophoresis 1%
    • POSITIVE (+)
    • High concentration!
    • Band purification
      • POSITIVE +
  • Miniprep Kit ligation C
    • Digestion
      • PromJ23104                                                      S+P
      • RBS+PrrA +RBS+PrrB +RBS+PrrC+DT         X+P
      • Prom J23104+RBS+AppA+RBS+PpsR+ DT   S+P
    • Agarose gel electrophoresis 1%
    • High concentration! J
    • Band purification
      • POSITIVE +
    • Ligations E
      • 1E      PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT
      • 2E      Prom J23104+RBS+AppA+RBS+PpsR+ DT+ Ppsr promoter+GFP+DT

23 July

  • Transformation of ligations E
  • WE are also worked in the assembly of the butanol pathway + regulatory systems

24 July

  • 1E NOTHING!!!!!!!!!!
    • Again ligation!!
    • Miniprep Kit ligation C
    • Digestion
      • PromJ23104                                                         S+P
      • RBS+PrrA +RBS+PrrB +RBS+PrrC+DT            X+P
    • Agarose gel electrophoresis 1%
    • High concentration! J
    • Band purification
      • POSITIVE +
  • 2E; 3 COLONIES!!... ;       Prom J23104+RBS+AppA+RBS+PpsR+ DT+ Ppsr promoter+GFP+DT
    • 6h culture!
    • ZR Miniprep Kit
    • Digestion (1.5h)    S+P and X+P
    • Agarose gel electrophoresis 1%
      • POSITIVE!!!!!!!!!!!!!!!!!!!
    • Band purification
      • FIRST SYSTEM READY

25 July

  • Transformation of ligation 1E

26 July

  • 1E;  2 COLONIES!!! ; PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT
    • 6h culture!
    • ZR Miniprep Kit
    • Digestion (1.5h)
    • S+P and X+P
    • Agarose gel electrophoresis 1%
      • POSITIVE!!!!!!!!!!!!!!!!!!!
      • Band Purification
      • JUST ONE MORE LIGATION
      • Overnight digestion
fe
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27 July

  • ZR Miniprep Kit ligations 1D
    • Digestion X+P
      • PrrA promoter+GFP+DT            X+P
    • Agarose gel electrophoresis 1%
    • POSITIVE (+)
    • High concentration!
    • Band purification
    • POSITIVE
  • 1E PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT
    • ZR Miniprep Kit
    • Digestion (1.5h)
      • S+P and X+P
    • Agarose gel electrophoresis 1%
      • POSITIVE!!!!!!!!!!!!!!!!!!!
      • Band Purification
      • POSITIVE
  • Ligation 2F:
    • PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT+ PrrA promoter+GFP+DT    

28 July

  • Transformation ligation 2F

29 July

  • NOTHING, AGAIN!           
  • ZR Miniprep Kit ligations 1D
    • Digestion X+P
      • PrrA promoter+GFP+DT            X+P
    • Agarose gel electrophoresis 1%
    • POSITIVE (+)
    • High concentration!
    • Band purification
    • POSITIVE
  • 1E PromJ23104 +RBS+PrrA +RBS+PrrB +RBS+PrrC+DT
    • ZR Miniprep Kit
    • Digestion (1.5h)
      • S+P and X+P
    • Agarose gel electrophoresis 1%
      • POSITIVE!!!!!!!!!!!!!!!!!!!
      • Band Purification
      • POSITIVE
  • Ligation 2F

30 July

  • Transformation

1 August

  • 2F, 4 COLONIES    
    • 6h cultures
    • ZR Miniprep Kit
    • Digestion (1.5h)
      • E and E+P
    • Agarose gel electrophoresis 1%
      • POSITIVE!!!!!!!!!!!!!!!!!!!
      • Band Purification
      • POSITIVE
      • SECOND SYSTEM READY
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  • Party! all the systems are ready…

Biobricks Final assembly to psB1C3

    • From 3 of August to 15 of August, we did the following things.
      • Overnight and 6h cultures
      • ZR Miniprep Kit
      • Digestion (1.5h)
        • For ligate
        • For confirmation
      • Agarose gel electrophoresis 1%
      • Band purification
      • Ligation
      • Transformation; and again!
      • TOTAL                                  11 parts in psB1C3
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Rhodofactory 2012

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