Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun

From 2014.igem.org

(Difference between revisions)
Line 49: Line 49:
             </div>
             </div>
             <div class="content">
             <div class="content">
-
               <ul>
+
                
 +
        </div>
 +
      </div>
 +
    </div>
 +
    </div>
 +
 
 +
 
 +
<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
 +
  <div id="text">
 +
<div class="tab" id="Week2">
 +
            <div class="show">
 +
                <a href="#Week2">Week 2 &nbsp;&nbsp;&nbsp; 06/09 - 06/15</a>
 +
            </div>
 +
            <div class="hide">
 +
                <h6><a  style="font-size:24px" href="#"Week 2 &nbsp;&nbsp;&nbsp; 06/09 - 06/15</a></h6>
 +
            </div>
 +
            <div class="content">
 +
 
 +
        </div>
 +
      </div>
 +
    </div>
 +
    </div>
 +
 
 +
 
 +
<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
 +
  <div id="text">
 +
<div class="tab" id="Week3">
 +
            <div class="show">
 +
                <a href="#Week3">Week 3 &nbsp;&nbsp;&nbsp; 06/16 - 06/22</a>
 +
            </div>
 +
            <div class="hide">
 +
                <h6><a  style="font-size:24px" href="#">Week 3 &nbsp;&nbsp;&nbsp; 06/16 - 06/22</a></h6>
 +
            </div>
 +
            <div class="content">
 +
        </div>
 +
      </div>
 +
    </div>
 +
    </div>
 +
 
 +
 
 +
<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
 +
  <div id="text">
 +
<div class="tab" id="Week4">
 +
            <div class="show">
 +
                <a href="#Week4">Week 4 &nbsp;&nbsp;&nbsp; 06/23 - 06/29</a>
 +
            </div>
 +
            <div class="hide">
 +
                <h6><a  style="font-size:24px" href="#">Week 4 &nbsp;&nbsp;&nbsp; 06/23 - 06/29</a></h6>
 +
            </div>
 +
            <div class="content">
 +
            <ul>
          <li><b><i>kivD</i></b></li>
          <li><b><i>kivD</i></b></li>
          <ul>
          <ul>
Line 107: Line 157:
     </div>
     </div>
     </div>
     </div>
-
 
-
 
-
<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
 
-
  <div id="text">
 
-
<div class="tab" id="Week2">
 
-
            <div class="show">
 
-
                <a href="#Week2">Week 2 &nbsp;&nbsp;&nbsp; 06/09 - 06/15</a>
 
-
            </div>
 
-
            <div class="hide">
 
-
                <h6><a  style="font-size:24px" href="#"Week 2 &nbsp;&nbsp;&nbsp; 06/09 - 06/15</a></h6>
 
-
            </div>
 
-
            <div class="content">
 
-
        </div>
 
-
      </div>
 
-
    </div>
 
-
    </div>
 
-
 
-
 
-
<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
 
-
  <div id="text">
 
-
<div class="tab" id="Week3">
 
-
            <div class="show">
 
-
                <a href="#Week3">Week 3 &nbsp;&nbsp;&nbsp; 06/16 - 06/22</a>
 
-
            </div>
 
-
            <div class="hide">
 
-
                <h6><a  style="font-size:24px" href="#">Week 3 &nbsp;&nbsp;&nbsp; 06/16 - 06/22</a></h6>
 
-
            </div>
 
-
            <div class="content">
 
-
        </div>
 
-
      </div>
 
-
    </div>
 
-
    </div>
 
-
 
-
 
-
<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
 
-
  <div id="text">
 
-
<div class="tab" id="Week4">
 
-
            <div class="show">
 
-
                <a href="#Week4">Week 4 &nbsp;&nbsp;&nbsp; 06/23 - 06/29</a>
 
-
            </div>
 
-
            <div class="hide">
 
-
                <h6><a  style="font-size:24px" href="#">Week 4 &nbsp;&nbsp;&nbsp; 06/23 - 06/29</a></h6>
 
-
            </div>
 
-
            <div class="content">
 
-
        </div>
 
-
      </div>
 
-
    </div>
 
-
    </div>
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
<ul>
 
-
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation of electrocompotetent <i> E.coli </i> KRX cells:</a></li>
 
-
<ul>
 
-
<li>K538742: <i>pSB1C3-kivD</i> (parts distribution from 2013, plate 1, 24L)</li>
 
-
<li>K539621: <i>pSB1C3-ilvC</i> (parts distribution from 2013, plate 1, 24F)</li>
 
-
<li>K539626: <i>pSB1C3-ilvD</i> (parts distribution from 2013, plate 1, 22J)</li>
 
-
<li>K539627: <i>pSB1C3-alsS</i> (parts distribution from 2013, plate 1, 24H)</li>
 
-
</ul>
 
-
<li> Resulting colonies were selected to start liquid cultures: </li>
 
-
<ul>
 
-
<li>5 ml of LB in 20 ml reaction tube</li>
 
-
<li>incubation over night at 37°C and 250 rpm</li>
 
-
</ul>
 
-
<li>Plasmid isolation by ThermoScientific MiniPrep</li>
 
-
<li>Sanger sequencing using <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR</a> as sequencing primers</li>
 
-
<ul>
 
-
<li>Identities of all parts were confirmed, but not whole sequence of the parts was covered</li>
 
-
</ul>
 
-
<li>Glycerin stocks were created for all four strains:</li>
 
-
<ul>
 
-
<li><i>E. coli</i> KRX <i>pSB1C3-alsS</i></li>
 
-
<li><i>E. coli</i> KRX <i>pSB1C3-ilvC</i></li>
 
-
<li><i>E. coli</i> KRX <i>pSB1C3-ilvD</i></li>
 
-
<li><i>E. coli</i> KRX <i>pSB1C3-kivD</i></li>
 
-
</ul>
 
-
</ul>
 
-
 
-
 
-
 
-
 
-
 
Line 205: Line 167:
             </div>
             </div>
             <div class="hide">
             <div class="hide">
-
                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 5 06/30 - 07/06</p></a>
+
                 <h6><a  style="font-size:24px" href="#">Week 5 06/30 - 07/06</a></h6>
             </div>
             </div>
             <div class="content">
             <div class="content">
 +
            <ul>
 +
      <li><b><i>kivD</i></b></li>
 +
      <ul>
 +
  <li>This week we tried to combine kivD with the other using the Gibson Assembly.</li>
 +
                  <ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>)</li>
 +
            <ul>
 +
        <li>Elongation time: 90s</li>
 +
        <li>Bands (not) as expected (... bp)</li>
 +
            </ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>kivD</i>, <i>alsS</i>, <i>ilvC</i> and <i>ilvD</i> on pSB1C3</li>
 +
                  </ul>
 +
      </ul>
 +
              <li><b><i>alsS</i></b></li>
 +
      <ul>
 +
  <li>This week we tried to combine alsS with the other using the Gibson Assembly.</li>
 +
                  <ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_pSB1C3" target="_blank">fw_alsS_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS" target="_blank">rv_ilvC_alsS</a>)</li>
 +
            <ul>
 +
        <li>Elongation time: 90s</li>
 +
        <li>Bands (not) as expected (... bp)</li>
 +
            </ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>kivD</i>, <i>alsS</i>, <i>ilvC</i> and <i>ilvD</i> on pSB1C3</li>
 +
                  </ul>
 +
      </ul>
 +
              <li><b><i>ilvC</i></b></li>
 +
      <ul>
 +
  <li>This week we tried to combine ilvC with the other using the Gibson Assembly.</li>
 +
                  <ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvD_ilvC" target="_blank">rv_ilvD_ilvC</a>)</li>
 +
            <ul>
 +
        <li>Elongation time: 90s</li>
 +
        <li>Bands (not) as expected (... bp)</li>
 +
            </ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>kivD</i>, <i>alsS</i>, <i>ilvC</i> and <i>ilvD</i> on pSB1C3</li>
 +
                  </ul>
 +
      </ul>
 +
              <li><b><i>ilvD</i></b></li>
 +
      <ul>
 +
  <li>This week we tried to combine <i>ilvD</i> with the other using the Gibson Assembly.</li>
 +
                  <ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvC_ilvD" target="_blank">fw_ilvC_ilvD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li>
 +
            <ul>
 +
        <li>Elongation time: 90s</li>
 +
        <li>Bands (not) as expected (... bp)</li>
 +
            </ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>kivD</i>, <i>alsS</i>, <i>ilvC</i> and <i>ilvD</i> on pSB1C3</li>
 +
                  </ul>
 +
      </ul>
 +
              <li><b>Backbone</b></li>
 +
      <ul>
 +
  <li>This week we amplified the backbone to use it for the Gibson Assembly.</li>
 +
                  <ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_kivD_pSB1C3" target="_blank">fw_kivD_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_alsS_pSB1C3" target="_blank">rv_alsS_pSB1C3</a>)</li>
 +
            <ul>
 +
        <li>Elongation time: 90s</li>
 +
        <li>Bands (not) as expected (... bp)</li>
 +
            </ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>kivD</i>, <i>alsS</i>, <i>ilvC</i> and <i>ilvD</i> on pSB1C3</li>
 +
                  </ul>
 +
      </ul>
 +
            <li>PCR conditions need to be optimized due to low product quality and missing products</li>
 +
            </ul>
         </div>
         </div>
       </div>
       </div>
     </div>  
     </div>  
     </div>
     </div>
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
<ul>
 
-
<li><b>pSB1C3-alsS-ilvC-ilvD-kivD</b></li>
 
-
<ul>
 
-
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of all four coding sequences for a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> </li>
 
-
<ul>
 
-
<li>Elongation time: 90 seconds</li>
 
-
<li>Annealing temperature: 55°C</li>
 
-
<li>Combinations of primer and template combinations are listed in the table below. Sequences for Gibson Assembly are introduced by the primers. Additionally the forward primers contain a RBS (BBa_B0034).</li>
 
-
 
-
<table>
 
-
<tr>
 
-
<th>forward primer</th>
 
-
<th>reverse primer</th>
 
-
<th>template</th>
 
-
</tr>
 
-
<tr>
 
-
<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_pSB1C3_alsS" target="_blank">fw_pSB1C3_alsS</a></td>
 
-
<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvC_alsS" target="_blank">rv_ilvC_alsS</a></td>
 
-
<td><i>pSB1C3-alsS</i></td>
 
-
</tr>
 
-
<tr>
 
-
<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a></td>
 
-
<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvD_ilvC" target="_blank">rv_ilvD_ilvC</a></td>
 
-
<td><i>pSB1C3-ilvC</i></td>
 
-
</tr>
 
-
<tr>
 
-
<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvC_ilvD" target="_blank">fw_ilvC_ilvD</a></td>
 
-
<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a></td>
 
-
<td><i>pSB1C3-ilvD</i></td>
 
-
</tr>
 
-
<tr>
 
-
<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvD_kivD" target="_blank">fw_ilvD_kivD</a></td>
 
-
<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a></td>
 
-
<td><i>pSB1C3-kivD</i></td>
 
-
</tr>
 
-
<tr>
 
-
<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_kivD_pSB1C3" target="_blank">fw_kivD_pSB1C3</a></td>
 
-
<td><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_alsS_pSB1C3" target="_blank">rv_alsS_pSB1C3</a></td>
 
-
<td><i>pSB1C3-CFP</i></td>
 
-
</tr>
 
-
</table>
 
-
 
-
<li>Products were analyzed by agarose gel electrophoresis</li>
 
-
<ul>
 
-
<li> PCR conditions need to be optimized due to low product quality and missing products</li>
 
-
</ul>
 
-
</ul>
 
-
</ul>
 
-
</ul>
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
</html>
</html>

Revision as of 10:32, 27 August 2014


June

Week 4     06/23 - 06/29
Week 4     06/23 - 06/29
  • kivD
    • This week we aim to transform the construct from the parts distribution.
      • Transformation with electrocompotetent cells
        • K538742: pSB1C3-kivD (parts distribution from 2013, plate 1, 24L)
      • Plasmid isolation of kivD
      • Sequencing confirmed identities of all parts, but not whole sequence of the parts was covered. (VF and VR)
      • Glycerin stock created: E. coli KRX pSB1C3-kivD
  • alsS
    • This week we aim to transform the construct from the parts distribution.
      • Transformation with electrocompotetent cells
        • K539627: pSB1C3-alsS (parts distribution from 2013, plate 1, 24H)
      • Plasmid isolation of alsS
      • Sequencing confirmed identities of all parts, but not whole sequence of the parts was covered. (VF and VR)
      • Glycerin stock created: E. coli KRX pSB1C3-alsS
  • ilvC
    • This week we aim to transform the construct from the parts distribution.
      • Transformation with electrocompotetent cells
        • K539621: pSB1C3-ilvC (parts distribution from 2013, plate 1, 24F)
      • Plasmid isolation of ilC
      • Sequencing confirmed identities of all parts, but not whole sequence of the parts was covered. (VF and VR)
      • Glycerin stock created: E. coli KRX pSB1C3-ilvC
  • ilvD
    • This week we aim to transform the construct from the parts distribution.
      • Transformation with electrocompotetent cells
        • K539626: pSB1C3-ilvD (parts distribution from 2013, plate 1, 22J)
      • Plasmid isolation of ilvD
      • Sequencing confirmed identities of all parts, but not whole sequence of the parts was covered. (VF and VR)
      • Glycerin stock created: E. coli KRX pSB1C3-ilvD
Week 5     06/30 - 07/06
Week 5 06/30 - 07/06

Retrieved from "http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun"