Team:Aix-Marseille/Notebook

From 2014.igem.org

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           </div>
           </div>
         </div>
         </div>
 +
 +
          <!-- ======= WEEK 6 ======= -->
 +
<div class="panel panel-info notes-panel last-note">
 +
  <span class="note-tag" id="week6"></span>
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title">Week 6 : 08/04/2014 &#10145; 08/10/2014 <div class="pull-right"></div></h3>
 +
  </div>
 +
  <div class="panel-body">
 +
 
 +
    <ul class="nav nav-tabs" role="tablist">
 +
      <li class="active"><a href="#0728" role="tab" data-toggle="tab" class="date-notebook">07/28/14</a></li>
 +
      <li><a href="#0729" role="tab" data-toggle="tab" class="date-notebook">07/29/14</a></li>
 +
      <li><a href="#0730" role="tab" data-toggle="tab" class="date-notebook">07/30/14</a></li>
 +
      <li><a href="#0731" role="tab" data-toggle="tab" class="date-notebook">07/31/14</a></li>
 +
      <li><a href="#0801" role="tab" data-toggle="tab" class="date-notebook">08/01/14</a></li>
 +
      <li><a href="#0803" role="tab" data-toggle="tab" class="date-notebook">08/03/14</a></li>
 +
    </ul>
 +
 +
    <div class="tab-content">
 +
      <div class="tab-pane fade in active" id="0804">
 +
        <ul class="list-notebook">
 +
<li>
 +
Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :
 +
<ul>
 +
<li>SLIC RelA without STOP PCR product 7/8 Clone 28 and 29 &rArr; PiGEM_02.16 and PiGEM_02.17</li>
 +
<li>SLIC RelA with STOP PCR product 7/8 Clone 31, 32 and 33 &rArr; PiGEM_02.18, PiGEM_02.19 and PiGEM_02.20</li>
 +
</ul>
 +
<div class="media notes-media">
 +
<img class="media-object img-rounded pull-left" src="AMU_Team-Week_6-080414-1.png" style="width:350px">
 +
<div class="media-body">
 +
All plasmids seemed to be correctly purified. No DNA contamination was noticeable.
 +
</div>
 +
</div>
 +
</li>
 +
          <li>
 +
            <p>Several DNA fragments were amplified thanks to iGEM PCR protocol (Q5 polymerase).</p>
 +
           
 +
            <table class="table table-hover table-bordered notes-table">
 +
              <thead>
 +
                <tr>
 +
                  <th>#</th>
 +
                  <th>DNA Template</th>
 +
                  <th>Primers Numbers</th>
 +
                  <th>Primers Names</th>
 +
                  <th>Weight of amplified DNA</th>
 +
                </tr>
 +
              </thead>
 +
              <tbody>
 +
                <tr>
 +
                  <td>14</td>
 +
                  <td>Genomic DNA of W3110</td>
 +
                  <td>33 - 34</td>
 +
                  <td>CusR_iProm-up<br>CusR_iProm-down</td>
 +
                  <td>117 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>15</td>
 +
                  <td>PiGEM_01.04</td>
 +
                  <td>37 - 38</td>
 +
                  <td>BBa_E1010-up<br>BBa_E1010_NoStop-down</td>
 +
                  <td>708 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>16</td>
 +
                  <td>PiGEM_02.03</td>
 +
                  <td>39 - 40</td>
 +
                  <td>BBa_E0040-up<br>BBa_E0040_NoStop-down</td>
 +
                  <td>714 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>17</td>
 +
                  <td>PiGEM_01.09</td>
 +
                  <td>45 - 46</td>
 +
              <td>(SH3)4-up<br>(SH3)4-down</td>
 +
                  <td>932 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>18</td>
 +
                  <td>PiGEM_02.08</td>
 +
                  <td>47 - 48</td>
 +
                  <td>cusR_L_LZA-up<br>cusR_L_LZA_down</td>
 +
                  <td>857 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>19</td>
 +
                  <td>PiGEM_02.07</td>
 +
                  <td>49 - 50</td>
 +
                  <td>SH3pep_L_LZA-up<br>SH3pep_L_LZA-down</td>
 +
                  <td>192 bp</td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
           
 +
            <p>The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.</p>
 +
            <div class="media notes-media">
 +
              <img class="media-object img-rounded pull-left" src="AMU_Team-Week_6-080414-2.png" style="width:350px">
 +
              <img class="media-object img-rounded pull-right" src="AMU_Team-Week_6-080414-3.png" style="width:350px; margin-right:21px">
 +
            </div>
 +
            <p>Fragments 19, 15, 16 and 18 seemed to be correctly amplified. They were going to be cloned in iGEM plasmids. Unfortunately, fragments 14 and 17 were not amplified. Another PCR had to be made.</p>
 +
          </li>
 +
          <li>
 +
            <p>Several control PCR were realized to verify whether SLIC, bricks 200 and bricks 202 worked. Taq polymerase was used.</p>
 +
           
 +
            <table class="table table-hover table-bordered notes-table">
 +
              <thead>
 +
                <tr>
 +
                  <th>#</th>
 +
                  <th>DNA Template</th>
 +
                  <th>Primers Numbers</th>
 +
                  <th>Primers Names</th>
 +
                  <th>Weight of amplified DNA</th>
 +
                </tr>
 +
              </thead>
 +
              <tbody>
 +
                <tr>
 +
                  <td>20</td>
 +
                  <td>Clone 1 of SLIC SerA</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>1032 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>21</td>
 +
                  <td>Clone 2 of SLIC SerA</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>1032 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>22</td>
 +
                  <td>Clone 28 of SLIC SerA</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>2260 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>23</td>
 +
                  <td>Clone 29 of SLIC SerA</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>2260 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>24</td>
 +
                  <td>Clone 31 of SLIC SerA</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>2260 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>25</td>
 +
                  <td>Clone 32 of SLIC SerA</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>2260 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>26</td>
 +
                  <td>Clone 33 of SLIC SerA</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>2260 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>27</td>
 +
                  <td>Clone 34 of SLIC CheA</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>2432 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>28</td>
 +
                  <td>Clone 35 of SLIC CheA</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>2432 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>29</td>
 +
                  <td>Clone 36 of SLIC CheA</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>2432 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>30</td>
 +
                  <td>Clone 37 of SLIC CheA</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>2432 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>31</td>
 +
                  <td>Clone 38 of SLIC CheA</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>2432 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>32</td>
 +
                  <td>Clone 39 of SLIC CheA</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>2432 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>33</td>
 +
                  <td>Brick 202 (1)</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>730 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>34</td>
 +
                  <td>Brick 202 (2)</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>769 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>35</td>
 +
                  <td>Brick 200 (3)</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>769 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>36</td>
 +
                  <td>Brick 200 (4)</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>769 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>37</td>
 +
                  <td>Brick 200 (5)</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>769 bp</td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
           
 +
            <p>The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.</p>
 +
            <div class="media notes-media">
 +
              <img class="media-object img-rounded pull-left" src="AMU_Team-Week_6-080414-4.png" style="width:350px">
 +
              <img class="media-object img-rounded pull-right" src="AMU_Team-Week_6-080414-5.png" style="width:350px; margin-right:21px">
 +
            </div>
 +
            <p>No fragment seemed to be correctly amplified. SLIC were going to be done again with another protocol. Bricks 200 and 202 were also going to be remade.</p>
 +
          </li>
 +
          <li>
 +
          <p>PiGEM_01.03 (pSB1C3, BBa_B0033) was digested with XbaI and SpeI to be used for stability label cloning.</p>
 +
          <p>The efficiency of the digestion was tested thanks to agarose gel electrophoresis. Results are presented below.</p>
 +
          <div class="media notes-media">
 +
<img class="media-object img-rounded pull-left" src="AMU_Team-Week_6-080414-6.png" style="width:350px">
 +
<div class="media-body">
 +
PSB1C3 of PiGEM_01.03  seemed to be correctly digested.
 +
</div>
 +
</div>
 +
          </li>
 +
        </ul>
 +
      </div>
 +
 +
      <div class="tab-pane fade in active" id="0805">
 +
        <ul class="list-notebook">
 +
          <li>
 +
            <p>PiGEM_01.03 (pSB1C3, BBa_B0033) was digested with EcoRI and SpeI to be used for PCR products 15 and 16 cloning. PCR products 15 and 16 (respectively RFP without STOP codon and GFP without STOP codon) were digested by EcoRI and SpeI. Then, PCR products 15 and 16 were leagued in pSB1C3 digested. DH5 alpha supercompetent were transformed  with these ligation products and spread on Cm dish.</p>
 +
          </li>
 +
          <li>
 +
            <p>PiGEM_02.27 (pSB1A2, BBa_J04450) was digested with EcoRI and PstI to be used for PCR products 18 and 19 cloning. PCR products 18 and 19 (respectively cusR-L-LZa and SH3pep-L-LZa) were digested by EcoRI and PstI. Then, PCR products 18 and 19 were leagued in pSB1A2 digested. DH5 alpha supercompetent were transformed  with these ligation products and spread on Amp dish.</p>
 +
          </li>
 +
          <li>
 +
            <p>Because PCR 17 did not work, (SH3)4  was amplified again thanks to iGEM PCR protocol (Q5 polymerase).</p>
 +
           
 +
            <table class="table table-hover table-bordered notes-table">
 +
              <thead>
 +
                <tr>
 +
                  <th>#</th>
 +
                  <th>DNA Template</th>
 +
                  <th>Primers Numbers</th>
 +
                  <th>Primers Names</th>
 +
                  <th>Weight of amplified DNA</th>
 +
                </tr>
 +
              </thead>
 +
              <tbody>
 +
                <tr>
 +
                  <td>38</td>
 +
                  <td>PiGEM_02.07</td>
 +
                  <td>49 - 50</td>
 +
                  <td>SH3pep_L_LZA-up<br>SH3pep_L_LZA-down</td>
 +
                  <td>192 bp</td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
           
 +
            <p>The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.</p>
 +
            <div class="media notes-media">
 +
<img class="media-object img-rounded pull-left" src="AMU_Team-Week_6-080514-1.png" style="width:350px">
 +
<div class="media-body">
 +
(SH3)<sub>4</sub> amplification did not work again. A specific treatment of this cloning was needed.
 +
</div>
 +
</div>
 +
          <li>
 +
            <p>PiGEM_01.03 (pSB1C3, BBa_B0033) had be digested with XbaI and SpeI the day before to be used for stability label cloning. A gel extract was made this day to collect only the backbone. The backbone obtained was digested by PstI because XbaI and SpeI are compatible restriction sites. Stability labels were assembled by annealing.</p>
 +
           
 +
            <table class="table table-hover table-bordered notes-table">
 +
              <thead>
 +
                <tr>
 +
                  <th>Primers numbers</th>
 +
                  <th>Primers names</th>
 +
                  <th>Brick created</th>
 +
                </tr>
 +
              </thead>
 +
              <tbody>
 +
                <tr>
 +
                  <td>27 - 28</td>
 +
                  <td>BBa_K1051206-up<br>BBa_K1051206-down</td>
 +
                  <td>BBa_K1051206</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>29 -30</td>
 +
                  <td>BBa_K1051207-up<br>BBa_K1051207-down</td>
 +
                  <td>BBa_K1051207</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>31 -32</td>
 +
                  <td>BBa_K1051208-up<br>BBa_K1051208-down</td>
 +
                  <td>BBa_K1051208</td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
           
 +
            <p>Each annealing oligonucleotides were leagued in pSB1C3 digested XSP. DH5 alpha supercompetent were transformed  with these ligation products and spread on Cm dish.</p>
 +
          </li>
 +
          <li>
 +
            <p>Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :</p>
 +
            <p>
 +
              SLIC CheA PCR product 5/6 Clone 34, 35, 36, 37, 38 and 39 &rArr; PiGEM_02.21, PiGEM_02.22, PiGEM_02.23, PiGEM_02.24, PiGEM_02.25, PiGEM_02.26
 +
            </p>
 +
          </li>
 +
          <li>
 +
          <p>Several control PCR were realized to verify one more time whether SLIC worked. Besides, several control PCR were realized to verify whether bricks 200 and bricks 202 worked. Taq polymerase was used. </p>
 +
           
 +
            <table class="table table-hover table-bordered notes-table">
 +
              <thead>
 +
                <tr>
 +
                  <th>#</th>
 +
                  <th>DNA Template</th>
 +
                  <th>Primers Numbers</th>
 +
                  <th>Primers Names</th>
 +
                  <th>Weight of amplified DNA</th>
 +
                </tr>
 +
              </thead>
 +
              <tbody>
 +
                <tr>
 +
                  <td>39</td>
 +
                  <td>PiGEM_02.10</td>
 +
                  <td>5 - 6</td>
 +
                  <td>serA_PrefRFC10RBS_up<br>serA_tronq_SufRFC23_down</td>
 +
                  <td>1032 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>40</td>
 +
                  <td>PiGEM_02.11</td>
 +
                  <td>5 - 6</td>
 +
                  <td>serA_PrefRFC10RBS_up<br>serA_tronq_SufRFC23_down</td>
 +
                  <td>1032 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>41</td>
 +
                  <td>PiGEM_02.16</td>
 +
                  <td>17 - 18</td>
 +
                  <td>relA_RFC10-RBS_up<br>relA_sans_stop_SufRFC23_down</td>
 +
                  <td>2259 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>42</td>
 +
                  <td>PiGEM_02.17</td>
 +
                  <td>17 - 18</td>
 +
                  <td>relA_RFC10-RBS_up<br>relA_sans_stop_SufRFC23_down</td>
 +
                  <td>2259 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>43</td>
 +
                  <td>PiGEM_02.18</td>
 +
                  <td>17 - 19</td>
 +
                  <td>relA_RFC10-RBS_up<br>relA_2_stop_SufRFC23_down</td>
 +
                  <td>2259 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>44</td>
 +
                  <td>PiGEM_02.19</td>
 +
                  <td>17 - 19</td>
 +
                  <td>relA_RFC10-RBS_up<br>relA_2_stop_SufRFC23_down</td>
 +
                  <td>2259 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>45</td>
 +
                  <td>PiGEM_02.20</td>
 +
                  <td>9 - 10</td>
 +
                  <td>cheA_PrefRFC23_up<br>cheA_SufRFC23_down</td>
 +
                  <td>2432 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>46</td>
 +
                  <td>PiGEM_02.21</td>
 +
                  <td>9 - 10</td>
 +
                  <td>cheA_PrefRFC23_up<br>cheA_SufRFC23_down</td>
 +
                  <td>2432 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>47</td>
 +
                  <td>PiGEM_02.22</td>
 +
                  <td>9 - 10</td>
 +
                  <td>cheA_PrefRFC23_up<br>cheA_SufRFC23_down</td>
 +
                  <td>2432 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>48</td>
 +
                  <td>PiGEM_02.23</td>
 +
                  <td>9 - 10</td>
 +
                  <td>cheA_PrefRFC23_up<br>cheA_SufRFC23_down</td>
 +
                  <td>2432 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>49</td>
 +
                  <td>PiGEM_02.24</td>
 +
                  <td>9 - 10</td>
 +
                  <td>cheA_PrefRFC23_up<br>cheA_SufRFC23_down</td>
 +
                  <td>2432 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>50</td>
 +
                  <td>PiGEM_02.25</td>
 +
                  <td>9 - 10</td>
 +
                  <td>cheA_PrefRFC23_up<br>cheA_SufRFC23_down</td>
 +
                  <td>2432 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>51</td>
 +
                  <td>PiGEM_02.26</td>
 +
                  <td>9 - 10</td>
 +
                  <td>cheA_PrefRFC23_up<br>cheA_SufRFC23_down</td>
 +
                  <td>2432 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>52</td>
 +
                  <td>PiGEM_01.04</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>706 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>53</td>
 +
                  <td>PiGEM_02.10</td>
 +
                  <td>43 - 44</td>
 +
                  <td>Prefix_up<br>Suffix_down</td>
 +
                  <td>1032 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>54</td>
 +
                  <td>Brick 202 (1)</td>
 +
                  <td>40 - 51</td>
 +
                  <td>BBa_E0040_NoStop-down<br>BBa_B0034-up</td>
 +
                  <td>730 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>55</td>
 +
                  <td>Brick 202 (2)</td>
 +
                  <td>40 - 51</td>
 +
                  <td>BBa_E0040_NoStop-down<br>BBa_B0034-up</td>
 +
                  <td>730 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>56</td>
 +
                  <td>Brick 202 (3)</td>
 +
                  <td>40 - 51</td>
 +
                  <td>BBa_E0040_NoStop-down<br>BBa_B0034-up</td>
 +
                  <td>730 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>57</td>
 +
                  <td>Brick 202 (4)</td>
 +
                  <td>40 - 51</td>
 +
                  <td>BBa_E0040_NoStop-down<br>BBa_B0034-up</td>
 +
                  <td>730 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>58</td>
 +
                  <td>Brick 200 (3)</td>
 +
                  <td>38 - 51</td>
 +
                  <td>BBa_E0040_NoStop-down<br>BBa_B0034-up</td>
 +
                  <td>711 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>59</td>
 +
                  <td>Brick 200 (4)</td>
 +
                  <td>38 - 51</td>
 +
                  <td>BBa_E0040_NoStop-down<br>BBa_B0034-up</td>
 +
                  <td>730 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>60</td>
 +
                  <td>Brick 200 (6)</td>
 +
                  <td>38 - 51</td>
 +
                  <td>BBa_E0040_NoStop-down<br>BBa_B0034-up</td>
 +
                  <td>711 bp</td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
          </li>
 +
          <li>
 +
          <p>Because PCR 14 did not work, cusR promoter was amplified again thanks to iGEM PCR protocol (Q5 polymerase).</p>
 +
           
 +
            <table class="table table-hover table-bordered notes-table">
 +
              <thead>
 +
                <tr>
 +
                  <th>#</th>
 +
                  <th>DNA Template</th>
 +
                  <th>Primers Numbers</th>
 +
                  <th>Primers Names</th>
 +
                  <th>Weight of amplified DNA</th>
 +
                </tr>
 +
              </thead>
 +
              <tbody>
 +
                <tr>
 +
                  <td>62</td>
 +
                  <td>Genomic DNA of W3110</td>
 +
                  <td>33 - 34</td>
 +
                  <td>CusR_iProm-up<br>CusR_iProm-down</td>
 +
                  <td>117 bp</td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
          </li>
 +
        </ul>
 +
      </div>
 +
 +
      <div class="tab-pane fade in active" id="0806">
 +
        <ul class="list-notebook">
 +
          <li>
 +
            <p>The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.</p>
 +
            <div class="media notes-media">
 +
              <img class="media-object img-rounded pull-left" src="AMU_Team-Week_6-080614-1.png" style="width:350px">
 +
              <img class="media-object img-rounded pull-right" src="AMU_Team-Week_6-080614-2.png" style="width:350px; margin-right:21px">
 +
            </div>
 +
            <div class="media notes-media">
 +
              <img class="media-object img-rounded pull-left" src="AMU_Team-Week_6-080614-3.png" style="width:350px">
 +
              <img class="media-object img-rounded pull-right" src="AMU_Team-Week_6-080614-4.png" style="width:350px; margin-right:21px">
 +
            </div>
 +
            <p>SLIC had to be made again. Bricks 200 were correctly amplified but bricks 202 were not. CusR promoter was not amplified.</p>
 +
          </li>
 +
          <li>
 +
          <p>Because PCR 62 did not work, cusR promoter was amplified again thanks to iGEM PCR protocol (Q5 polymerase).</p>
 +
           
 +
            <table class="table table-hover table-bordered notes-table">
 +
              <thead>
 +
                <tr>
 +
                  <th>#</th>
 +
                  <th>DNA Template</th>
 +
                  <th>Primers Numbers</th>
 +
                  <th>Primers Names</th>
 +
                  <th>Weight of amplified DNA</th>
 +
                </tr>
 +
              </thead>
 +
              <tbody>
 +
                <tr>
 +
                  <td>63</td>
 +
                  <td>Genomic DNA of W3110</td>
 +
                  <td>33 - 34</td>
 +
                  <td>CusR_jProm-up<br>CusR_jProm-down</td>
 +
                  <td>117 bp</td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
          </li>
 +
          <li>
 +
            <p>Several control PCR were realized to verify whether bricks 200 worked. Taq polymerase was used. </p>
 +
           
 +
            <table class="table table-hover table-bordered notes-table">
 +
              <thead>
 +
                <tr>
 +
                  <th>#</th>
 +
                  <th>DNA Template</th>
 +
                  <th>Primers Numbers</th>
 +
                  <th>Primers Names</th>
 +
                  <th>Weight of amplified DNA</th>
 +
                </tr>
 +
              </thead>
 +
              <tbody>
 +
                <tr>
 +
                  <td>64</td>
 +
                  <td>Brick 202 (5)</td>
 +
                  <td>40 - 51</td>
 +
                  <td>BBa_E0040_NoStop-down<br>BBa_B0034-up</td>
 +
                  <td>730 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>65</td>
 +
                  <td>Brick 202 (6)</td>
 +
                  <td>40 - 51</td>
 +
                  <td>BBa_E0040_NoStop-down<br>BBa_B0034-up</td>
 +
                  <td>730 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>646</td>
 +
                  <td>Brick 202 (7)</td>
 +
                  <td>40 - 51</td>
 +
                  <td>BBa_E0040_NoStop-down<br>BBa_B0034-up</td>
 +
                  <td>730 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>67</td>
 +
                  <td>Brick 202 (8)</td>
 +
                  <td>40 - 51</td>
 +
                  <td>BBa_E0040_NoStop-down<br>BBa_B0034-up</td>
 +
                  <td>730 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>68</td>
 +
                  <td>Brick 202 (9)</td>
 +
                  <td>40 - 51</td>
 +
                  <td>BBa_E0040_NoStop-down<br>BBa_B0034-up</td>
 +
                  <td>730 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>69</td>
 +
                  <td>Brick 202 (10)</td>
 +
                  <td>40 - 51</td>
 +
                  <td>BBa_E0040_NoStop-down<br>BBa_B0034-up</td>
 +
                  <td>730 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>70</td>
 +
                  <td>Brick 202 (11)</td>
 +
                  <td>40 - 51</td>
 +
                  <td>BBa_E0040_NoStop-down<br>BBa_B0034-up</td>
 +
                  <td>730 bp</td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
          </li>
 +
          <li>
 +
            <p>A gel extract of PCR product 38 (SH3)<sub>4</sub> was made.</p>
 +
          </li>
 +
        </ul>
 +
      </div>
 +
 +
      <div class="tab-pane fade in active" id="0807">
 +
        <ul class="list-notebook">
 +
          <li>
 +
            <p>Several control PCR were realized to verify whether RFP without STOP codon and GFP without STOP codon worked. Taq polymerase was used.</p>
 +
           
 +
            <table class="table table-hover table-bordered notes-table">
 +
              <thead>
 +
                <tr>
 +
                  <th>#</th>
 +
                  <th>DNA Template</th>
 +
                  <th>Primers Numbers</th>
 +
                  <th>Primers Names</th>
 +
                  <th>Weight of amplified DNA</th>
 +
                </tr>
 +
              </thead>
 +
              <tbody>
 +
                <tr>
 +
                  <td>72</td>
 +
                  <td>RFP without STOP codon<br>Clone 40</td>
 +
                  <td>37 - 38</td>
 +
                  <td>BBa_E1010-up<br>BBa_E1010_NoStop-down</td>
 +
                  <td>708 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>73</td>
 +
                  <td>RFP without STOP codon<br>Clone 41</td>
 +
                  <td>37 - 38</td>
 +
                  <td>BBa_E1010-up<br>BBa_E1010_NoStop-down</td>
 +
                  <td>708 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>74</td>
 +
                  <td>RFP without STOP codon<br>Clone 42</td>
 +
                  <td>37 - 38</td>
 +
                  <td>BBa_E1010-up<br>BBa_E1010_NoStop-down</td>
 +
                  <td>708 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>75</td>
 +
                  <td>RFP without STOP codon<br>Clone 43</td>
 +
                  <td>37 - 38</td>
 +
                  <td>BBa_E1010-up<br>BBa_E1010_NoStop-down</td>
 +
                  <td>708 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>76</td>
 +
                  <td>RFP without STOP codon<br>Clone 44</td>
 +
                  <td>37 - 38</td>
 +
                  <td>BBa_E1010-up<br>BBa_E1010_NoStop-down</td>
 +
                  <td>708 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>77</td>
 +
                  <td>GFP without STOP codon<br>Clone 45</td>
 +
                  <td>39 - 40</td>
 +
                  <td>BBa_E0040-up<br>BBa_E0040_NoStop-down</td>
 +
                  <td>714 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>78</td>
 +
                  <td>GFP without STOP codon<br>Clone 46</td>
 +
                  <td>39 - 40</td>
 +
                  <td>BBa_E0040-up<br>BBa_E0040_NoStop-down</td>
 +
                  <td>714 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>79</td>
 +
                  <td>GFP without STOP codon<br>Clone 47</td>
 +
                  <td>39 - 40</td>
 +
                  <td>BBa_E0040-up<br>BBa_E0040_NoStop-down</td>
 +
                  <td>714 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>80</td>
 +
                  <td>GFP without STOP codon<br>Clone 48</td>
 +
                  <td>39 - 40</td>
 +
                  <td>BBa_E0040-up<br>BBa_E0040_NoStop-down</td>
 +
                  <td>714 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>81</td>
 +
                  <td>GFP without STOP codon<br>Clone 49</td>
 +
                  <td>39 - 40</td>
 +
                  <td>BBa_E0040-up<br>BBa_E0040_NoStop-down</td>
 +
                  <td>714 bp</td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
          </li>
 +
          <li>
 +
            <p>The efficiency of the purification was tested thanks to agarose gel electrophoresis. Results are presented following.</p>
 +
            <div class="media notes-media">
 +
              <img class="media-object img-rounded pull-left" src="AMU_Team-Week6-080714-1.png" style="width:350px">
 +
              <div class="media-body">
 +
                <p>PCR product 63 was correctly amplified. PiGEM_02.27 (pSB1A2, BBa_J04450) was digested with EcoRI and Pst. PCR product 63 (cusR promoter) was digested by EcoRI and PstI. Then, PCR product 63  was leagued in pSB1A2 digested. DH5 alpha supercompetent were transformed  with this ligation product and spread on Amp dish.</p>
 +
              </div>
 +
            </div>
 +
          </li>
 +
          <li>
 +
            <p>To confirm the cloning of stability label and SLIC, each plasmid containing constructions were digested with specific restriction enzymes.</p>
 +
            <table class="table table-hover table-bordered notes-table">
 +
            <thead>
 +
<tr>
 +
<th rowspan="2">Plasmid Numbers</th>
 +
<th rowspan="2">Plasmid Names</th>
 +
<th colspan="4">Digestion</th>
 +
<th rowspan="2">Weight of expected bands if constructions are correct</th>
 +
</tr>
 +
<tr>
 +
<th>EcoRI</th>
 +
<th>PstI</th>
 +
<th>SpeI</th>
 +
<th>XbaI</th>
 +
</tr>
 +
</thead>
 +
              <tbody>
 +
                <tr>
 +
                  <td>PiGEM_01.10</td>
 +
                  <td>BBa_K1051207<br>Clone St1</td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td>X</td>
 +
                  <td>X</td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>2082 pb</li>
 +
                  <li>80 pb</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>PiGEM_01.11</td>
 +
                  <td>BBa_K1051207<br>Clone St2</td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td>X</td>
 +
                  <td>X</td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>2082 pb</li>
 +
                  <li>80 pb</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>PiGEM_01.12</td>
 +
                  <td>BBa_K1051208<br>Clone St3</td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td>X</td>
 +
                  <td>X</td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>2082 pb</li>
 +
                  <li>80 pb</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>PiGEM_02.10</td>
 +
                  <td>SLIC SerA produit PCR 2/3<br>Clone 1</td>
 +
                  <td>X</td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>2352 pb</li>
 +
                  <li>847 pb</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>PiGEM_02.11</td>
 +
                  <td>SLIC SerA produit PCR 2/3<br>Clone 2</td>
 +
                  <td>X</td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>2352 pb</li>
 +
                  <li>847 pb</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>PiGEM_02.12</td>
 +
                  <td>SLIC Mesh1 sans STOP produit PCR 10/11/12<br>Clone 4</td>
 +
                  <td></td>
 +
                  <td>X</td>
 +
                  <td>X</td>
 +
                  <td></td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>2422 pb</li>
 +
                  <li>180 pb</li>
 +
                  <li>84</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>PiGEM_02.13</td>
 +
                  <td>SLIC Mesh1 sans STOP produit PCR 10/11/12<br>Clone 6</td>
 +
                  <td></td>
 +
                  <td>X</td>
 +
                  <td>X</td>
 +
                  <td></td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>2422 pb</li>
 +
                  <li>180 pb</li>
 +
                  <li>84</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>PiGEM_02.14</td>
 +
                  <td>SLIC Mesh1 sans STOP produit PCR 10/11/13<br>Clone 7</td>
 +
                  <td></td>
 +
                  <td>X</td>
 +
                  <td>X</td>
 +
                  <td></td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>2422 pb</li>
 +
                  <li>180 pb</li>
 +
                  <li>84</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>PiGEM_02.15</td>
 +
                  <td>SLIC Mesh1 sans STOP produit PCR 10/11/13<br>Clone 8</td>
 +
                  <td></td>
 +
                  <td>X</td>
 +
                  <td>X</td>
 +
                  <td></td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>2422 pb</li>
 +
                  <li>180 pb</li>
 +
                  <li>84</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>PiGEM_02.16</td>
 +
                  <td>SLIC RelA sans STOP produit PCR 7/8<br>Clone 28</td>
 +
                  <td></td>
 +
                  <td>X</td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>3530 pb</li>
 +
                  <li>872 pb</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>PiGEM_02.17</td>
 +
                  <td>SLIC RelA sans STOP produit PCR 7/8<br>Clone 29</td>
 +
                  <td></td>
 +
                  <td>X</td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>3530 pb</li>
 +
                  <li>872 pb</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>PiGEM_02.18</td>
 +
                  <td>SLIC RelA sans STOP produit PCR 7/8<br>Clone 31</td>
 +
                  <td></td>
 +
                  <td>X</td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>3530 pb</li>
 +
                  <li>872 pb</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>PiGEM_02.19</td>
 +
                  <td>SLIC RelA sans STOP produit PCR 7/8<br>Clone 32</td>
 +
                  <td></td>
 +
                  <td>X</td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>3530 pb</li>
 +
                  <li>872 pb</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>PiGEM_02.24</td>
 +
                  <td>SLIC CheA produit PCR 5/6<br>Clone 37</td>
 +
                  <td>X</td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>3457 pb</li>
 +
                  <li>675 pb</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>PiGEM_02.25</td>
 +
                  <td>SLIC CheA produit PCR 5/6<br>Clone 38</td>
 +
                  <td>X</td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>3457 pb</li>
 +
                  <li>675 pb</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
                <tr>
 +
                  <td>PiGEM_02.26</td>
 +
                  <td>SLIC CheA produit PCR 5/6<br>Clone 39</td>
 +
                  <td>X</td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td></td>
 +
                  <td>
 +
                  <ul>
 +
                  <li>3457 pb</li>
 +
                  <li>675 pb</li>
 +
                  </ul>
 +
                  </td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
           
 +
            <p>The efficiency of the digestion was tested thanks to agarose gel electrophoresis. Results are presented bellow.</p>
 +
            <div class="media notes-media">
 +
              <img class="media-object img-rounded pull-left" src="AMU_Team-Week_6-080714-2.png" style="width:350px">
 +
              <img class="media-object img-rounded pull-right" src="AMU_Team-Week_6-080714-3.png" style="width:350px; margin-right:21px">
 +
            </div>
 +
           
 +
            <p>For stability label, linearized plasmid was the only fragment visible. For SLIC, no fragment matched with the weight of expected bands. All SLIC construction had to be made again.</p>
 +
          </li>
 +
          <li>
 +
          <p>PiGEM_02.27 (pSB1A2, BBa_J04450) was digested with XbaI, SpeI and PstI to be used for stability label cloning. Stability labels were assembled again by annealing.</p>
 +
         
 +
          <table class="table table-hover table-bordered notes-table">
 +
              <thead>
 +
                <tr>
 +
                  <th>Primers numbers</th>
 +
                  <th>Primers names</th>
 +
                  <th>Brick created</th>
 +
                </tr>
 +
              </thead>
 +
              <tbody>
 +
                <tr>
 +
                  <td>27 - 28</td>
 +
                  <td>BBa_K1051206-up<br>BBa_K1051206-down</td>
 +
                  <td>BBa_K1051206</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>29 -30</td>
 +
                  <td>BBa_K1051207-up<br>BBa_K1051207-down</td>
 +
                  <td>BBa_K1051207</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>31 -32</td>
 +
                  <td>BBa_K1051208-up<br>BBa_K1051208-down</td>
 +
                  <td>BBa_K1051208</td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
           
 +
            <p>Each annealing oligonucleotides were leagued in pSB1A2 digested XSP. DH5 alpha supercompetent were transformed  with these ligation products and spread on Amp dish.</p>
 +
          </li>
 +
          <li>
 +
          <p>PiGEM_02.27 (pSB1A2, BBa_J04450) was digested with XbaI and SpeI. CusR box and Flag were assembled by annealing.</p>
 +
         
 +
          <table class="table table-hover table-bordered notes-table">
 +
              <thead>
 +
                <tr>
 +
                  <th>Primers numbers</th>
 +
                  <th>Primers names</th>
 +
                  <th>Brick created</th>
 +
                </tr>
 +
              </thead>
 +
              <tbody>
 +
                <tr>
 +
                  <td>35 - 36</td>
 +
                  <td>CusR_Box-up<br>CusR_Box-down</td>
 +
                  <td>CusR Box</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>41 - 42</td>
 +
                  <td>Flag-up<br>Flag-down</td>
 +
                  <td>Flag</td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
           
 +
            <p>Each annealing oligonucleotides were leagued in pSB1A2 digested XS. DH5 alpha supercompetent were transformed  with these ligation products and spread on Amp dish.</p>
 +
          </li>
 +
        </ul>
 +
      </div>
 +
 +
      <div class="tab-pane fade in active" id="0808">
 +
        <ul class="list-notebook">
 +
          <li>
 +
            <p>SLIC assembly of CheA, SerA, RelA with and without STOP codon and Mesh 1 with and without STOP codon were restarted. DNA fragments were amplified thanks to iGEM PCR protocol (Q5 polymerase). </p>
 +
           
 +
            <table class="table table-hover table-bordered notes-table">
 +
              <thead>
 +
                <tr>
 +
                  <th>#</th>
 +
                  <th>DNA Template</th>
 +
                  <th>Primers Numbers</th>
 +
                  <th>Primers Names</th>
 +
                  <th>Weight of amplified DNA</th>
 +
                </tr>
 +
              </thead>
 +
              <tbody>
 +
                <tr>
 +
                  <td>82</td>
 +
                  <td>Genomic DNA of W3110</td>
 +
                  <td>6 - 7</td>
 +
                  <td>serA_mut_ A849G_up<br>serA_tronq_SufRFC23_down</td>
 +
                  <td>171 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>83</td>
 +
                  <td>Genomic DNA of W3110</td>
 +
                  <td>5 - 8</td>
 +
                  <td>serA_PrefRFC10RBS_up<br>serA_mut_A849_down</td>
 +
                  <td>861 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>84</td>
 +
                  <td>PiGEM_02.05</td>
 +
                  <td>15 - 17</td>
 +
                  <td>relA_mut_C1366G_up<br>relA_RFC10-RBS_up</td>
 +
                  <td>1377 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>85</td>
 +
                  <td>PiGEM_02.05</td>
 +
                  <td>16 - 18</td>
 +
                  <td>relA_mut_C1366_down<br>relA_sans_stop_SufRFC23_down</td>
 +
                  <td>882 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>86</td>
 +
                  <td>PiGEM_02.05</td>
 +
                  <td>16 - 19</td>
 +
                  <td>relA_mut_C1366_down<br>relA_2_stop_SufRFC23_down</td>
 +
                  <td>885 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>87</td>
 +
                  <td>PiGEM_02.06</td>
 +
                  <td>20 - 24</td>
 +
                  <td>Mesh_1_mut_A255T_up<br>Mesh_1_RFC10-RBS_up</td>
 +
                  <td>267 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>88</td>
 +
                  <td>PiGEM_02.06</td>
 +
                  <td>21 - 22</td>
 +
                  <td>Mesh_1_mut_A255T_down<br>Mesh_1_mut_A343G_up</td>
 +
                  <td>120 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>89</td>
 +
                  <td>PiGEM_02.06</td>
 +
                  <td>23 - 25</td>
 +
                  <td>Mesh_1_mut_A343G_down<br>Mesh1_sans_stop_SufRFC23_down</td>
 +
                  <td>192 bp</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>90</td>
 +
                  <td>PiGEM_02.06</td>
 +
                  <td>23 - 26</td>
 +
                  <td>Mesh_1_mut_A343G_down<br>Mesh_1_2_stop_SufRFC23_down</td>
 +
                  <td>192 bp</td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
           
 +
            <p>The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.</p>
 +
            <div class="media notes-media">
 +
              <img class="media-object img-rounded pull-left" src="AMU_Team-Week_6-080814-1.png" style="width:350px">
 +
              <img class="media-object img-rounded pull-right" src="AMU_Team-Week_6-080814-2.png" style="width:350px; margin-right:21px">
 +
            </div>
 +
            <p>All DNA fragments were correctly amplified. Thus, SLIC protocol was realized to assemble each fragment of CheA, SerA, RelA and Mesh1. Plasmid pSB1A2 digested XbaI and SpeI was used.</p>
 +
          </li>
 +
          <li>
 +
          <p>Several control PCR had be realized the day before to verify whether RFP without STOP codon and GFP without STOP codon worked. The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented following.</p>
 +
          <div class="media notes-media">
 +
              <img class="media-object img-rounded pull-left" src="AMU_Team-Week6-080814-3.png" style="width:350px">
 +
              <div class="media-body">
 +
                <p>Only clones 41, 42 43 and 44 of RPF without STOP codon and clones 47 and 48 of GFP without STOP codon seemed to be correct.</p>
 +
              </div>
 +
            </div>
 +
          </li>
 +
        </ul>
 +
      </div>
 +
     
 +
      <div class="tab-pane fade in active" id="0809">
 +
        <ul class="list-notebook">
 +
          <li>
 +
          <p>Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :</p>
 +
          <ul>
 +
<li>PCR product 15 RFP without STOP Clone 41 &rArr; PiGEM_01.14</li>
 +
<li>PCR product 15 RFP without STOP Clone 43 &rArr; PiGEM_01.15</li>
 +
<li>PCR product 16 GFP without STOP Clone 47 &rArr; PiGEM_01.16</li>
 +
<li>PCR product 16 GFP without STOP Clone 48 &rArr; PiGEM_01.17</li>
 +
</ul>
 +
          </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
 +
  </div>
 +
  <div class="panel-footer">
 +
    Protocols used : ...
 +
  </div>
 +
</div
       </div><!-- /NOTES -->
       </div><!-- /NOTES -->

Revision as of 13:04, 22 September 2014

Notebook



Week 1 : 06/30/2014 ➡ 07/06/2014
Preparation of strains

  • W3110 Escherichia coli strain and DH5alpha bacteria were put in culture on Petri dish.
  • Both strains were incubated in Erlenmeyer flasks at 37 ° C.
  • Preparation to electrocompetence : W3110 strain was washed in a 10% glycerol solution to remove extracellular salts that are lethal in this processing. W3110 competent bacteria were then frozen and stored.
  • Preparation to chemocompetence : working at cold temperature, bacteria were washed and resuspended with CaCl2. Again, bacteria were washed and resuspended with CaCl2 and glycerol. Cells were frozen at -80°C.
  • Electrocompetence : W3110 bacteria were transformed with pKOBEG plasmid by electroporation and put in culture at 30°C on petri dish.
  • Colonies were observed in W3110 control cultures. Transformed bacteria (W3110pKOBEG) were put in culture at 30°C.
  • Chemocompetence : A heat shock at 37°C was made with DH5α strain and the bacteria were then transformed with pBba_JO4450 plasmid and put in culture on petri dish.
  • W3110pKOBEG bacteria were washed and arabinose was added to provide the expression of the recombinase encoded by pKOBEG plasmid. They were then incubated at 42 °C in order to destroy the thermosensitive pKOBEG plasmid.
  • Preparation of electrocompetence : W3110 bacteria expressing the recombinase were prepared to electrocompetence and frozen at -80 ° C.
    No colony was observed in the petri dish where DH5α transformed bacteria were previously put in culture.
  • Chemocompetence : bacteria were prepared using a formerly thawed aliquot. A heat shock at 42°C were made on competent DH5α containing pBba-J04450. They were finally put in culture at 37°C.
  • DH5α control : Colonies were observed in the “negative control” culture. These colonies did not express RFP (no red fluorescent colour could be observed).
The LB we used in our experiments were contaminated, leading to the growth of undesirable colonies. The transformation of W3110 using pKOBEG plasmid was succesfull. The recombinase was expressed and the pKOBEG plasmid was destroyed using a thermosensitive origin of replication.
Protocols used : Chemocompetent and Electrocompetent E.coli cells

Week 2 : 07/07/2014 ➡ 07/13/2014

  • Supercompetent DH5alpha preculture was made.
  • Electroporation of W3110 strain with J04450 plasmid (20ng) and the telltale without plasmid was made.
  • Preparation of supercompetent cells
The plasmid concentration contained in the iGEM kit was enough to transform bacteria by electroporation.
Protocols used : Supercompetent and Electrocompetent E.coli cells

Week 3 : 07/14/2014 ➡ 07/20/2014

  • DH5α supercompetent bacteria were transformed using 3 ng of J04450 plasmid or 60 pg of J04450 plasmid. Both cultures showed colonies meaning transformations were successful.
  • DH5α supercompetent bacteria were transformed with respectively 1,5 ng of the following plasmids :
    • K864600 plasmid
    • B0032 plasmid
    • B0033 plasmid
    • E1010 plasmid
    • B0034 plasmid
    • B0010 plasmid
    • B0030 plasmid
    • E0040 plasmid
    • pT18 RelA
    • pBAD Mesh1
  • DH5α bacteria containing respectively the following plasmids were put in liquid culture in order to perform miniprep the day after.
    • K864600 plasmid
    • B0032 plasmid
    • B0033 plasmid
    • E1010 plasmid
    • B0034 plasmid
    • B0010 plasmid
    • B0030 plasmid
    • E0040 plasmid
  • DH5α supercompetent bacteria were transformed with respectively :
    • pSB1C3 K608006 Cm resistant plasmid
    • pSB1C3 K823017 Cm resistant plasmid
    • pSB1C3 J04500 Cm resistant plasmid
    • pWW2179 (SH3)4 Cm resistant plasmid
    • pWW2021 CusR-L-LZa Kan resistant plasmid
    • pWW2181 Amp resistant plasmid

    • Note :

    Petri dishes containing respectively bacteria transformed with pT18 RelA plasmid and bacteria transformed with pBAD Mesh1 plasmid showed so many colonies that it was not possible to transfer isolated colonies into liquid culture. So, new cultures of both transformed bacteria were made again using a technique leading to isolated colonies.

  • As a negative control, a culture of empty bacteria and Ampicillin was made.
  • After the Miniprep was performed on bacteria containing pSB1AC3-J04450 plasmid, an electrophoresis was realised. It shows one high molecular weight band. The plasmid pSB1AC3-J04450 was successfully purified. It is stocked at -20°C and the strain is conserved in glycerol at -80°C.
  • After the Miniprep was performed on bacteria containing K864600 plasmid to E0040 plasmid, the following electrophoresis* was obtained :
    • Well 1: B0010
    • Well 2: B0030
    • Well 3: B0032
    • Well 4: B0033
    • Well 5: B0034
    • Well 6: E0040
    • Well 7: E1010
    • Well 8: K864600
    * Be aware that Well 1 was filled with 5μL whereas the others were filled with only 3μL.
  • Petri dish containing the bacteria transformed the day before with p5B1C3 K608006 Cm resistant plasmid to DH5α
  • DH5α supercompetent bacteria previously transformed ( on 07/17/2014) with pSB1C3 K608006 Cm resistant plasmid to p5B1C3 K608006 Cm resistant plasmid were put in liquid culture in order to perform miniprep the day after
Protocols used : ...

Week 4 : 07/21/2014 ➡ 07/27/2014

  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :
    • pSB1C3 K608006
    • pSB1C3 K823017
    • pSB1C3 J04500
    • pWW2179 (SH3)4 Cm resistant
    • pWW2021 CusR-L-LZa Kan resistant
    • pWW2181 Amp resistant
    • pT18 RelA Amp resistant
    • pBAD Mesh1 Amp resistant
    The efficiency of the purification was tested thanks to agarose gel electrophoresis. Results are presented below.
    Each well contains one or two fragments of DNA (corresponding to the different plasmid compaction degrees), thus all plasmids were successfully purified. They were stocked at -20°C and strains were put in glycerol at -80°C.
  • Bacteria transformed with the pSB1A2 BBa_B0010 plasmid were strangely red. That's why the plasmid was twice digested with EcoR1 and with XBa1 and Pst1. Digestion products were tested thanks to agarose gel electrophoresis. Results are presented below.
    The digestion with EcoR1 made the plasmid linear. The digestion with Xba1 and Pst1 was supposed to produce two bands, corresponding to the backbone (2070pb) and to the BBa_B0010 part (80pb). No low molecular weight band appeared, the only visible band has a too low molecular weight to be the expected backbone. Something is wrong. We have to clarify this. Further experiments should be made to clarify these surprising results.
  • The pSB1A2 BBa_B0010 plasmid was anew twice digested with EcoR1 and with XBa1 and Pst1. Digestion products are tested thanks to agarose gel electrophoresis. Results are presented below.
    Again, these results did not make sense. It looks as if restriction enzymes did not cut the plasmid. This part is set-aside for the moment.
  • pSB1C3 BBa_E1010, pSB1A2 BBa_E0040 and pSB1A2 BBa_B0034 plasmids were digested and ligated according to the BioBrick Assembly Kit of BioLabs. The destination plasmid DNA used is the backbone pSB1K3.m1 linearized plasmid. DH5α supercompetent bacteria were transformed with ligation products and spread over Kan dish
  • DH5α supercompetent bacteria were transformed with BBA_J23100 (17D Plate 4) and spread over Amp dish.
  • DH5α supercompetent bacteria previously transformed (on 23/17/2014) with BBA_J23100 Amp resistant plasmid and with Brick 200 and 202 Kan resistant plasmid were put in liquid culture in order to perform miniprep the day after
  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :
    • Brick 200 (1)
    • Brick 200 (2)
    • Brick 202 (1)
    • Brick 202 (2)
    • J23100
    The efficiency of the purification is tested thanks to agarose gel electrophoresis. Results are presented below.
    Each well contains DNA fragments, thus all plasmids were successfully purified.
  • Bricks 200 (1), 200 (2), 202 (1) and 202 (2) were digested by EcoR1 and Spe1. Digestion products are tested thanks to agarose gel electrophoresis. Results are presented below.
    Brick 200 (1), 200 (2) and 202 (1) did not present any insert. Only Brick 200 (2) seemed to present an insert.
    To control it was the correct insert, a second electrophoresis was made with the RFP and GFP digestion products. Results are presented below.
    Results were not conclusive. A control PCR might be made in order to determine the presence or the absence of BBa_B0034 RBS.
Protocols used : ...

Week 5 : 07/28/2014 ➡ 08/03/2014

  • Several DNA fragments were amplified thanks to iGEM PCR protocol (Q5 polymerase).

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    2 Genomic DNA of W3110 6 - 7 serA_mut_ A849G_up
    serA_tronq_SufRFC23_down    		 171 bp
    3 Genomic DNA of W3110 5 - 8 serA_PrefRFC10RBS_up
    sera_mut_A849_down    		 861 bp
    6 Genomic DNA of W3110 10 - 13 cheA_SufRFC23_down
    cheA_mut_T1290C_up    		 726 bp
    8 PiGEM_02.05 16 - 18 relA_mut_C1366_down
    relA_sans_stop_SufRFC23_down    		 882 bp
    9 PiGEM_02.05 16 - 19 relA_mut_C1366_down
    relA_2_stop_SufRFC23_down    		 885 bp
    10 PiGEM_02.06 20 - 24 Mesh_1_mut_A255T_up
    Mesh_1_RFC10-RBS_up    		 267 bp
    11 PiGEM_02.06 21 - 22 Mesh_1_mut_A255T_down
    Mesh_1_mut_A343G_up    		 120 bp
    12 PiGEM_02.06 23 - 25 Mesh_1_mut_A343G_down
    Mesh1_sans_stop_SufRFC23_down    		 192 bp
    13 PiGEM_02.06 23 - 26 Mesh_1_mut_A343G_down
    Mesh_1_2_stop_SufRFC23_down    		 192 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    All DNA fragments seemed to be correctly amplified.

  • PCR products were purified thanks to Macherey Nagel PCR Clean up Kit.

  • The DNA concentration of PCR products was tested thanks to the Nanodrop. Results are presented bellow.

    # Concentration of DNA (ng/µL)
    2 52.28
    3 44.86
    6 49.93
    8 73.38
    9 76.77
    10 57.00
    11 39.87
    12 50.88
    13 62.42
  • PCR products number 6, 8 and 9 were stocked at -20°C whereas PCR products number 2, 3, 10, 11, 12 and 13 were used to realize SLIC protocol. This protocol permits to assemble all parts of SerA and Mesh1 genes with their directed mutation. The destination plasmid DNA used is the backbone pSB1A3 linearized plasmid. DH5α supercompetent bacteria were transformed with SLIC products and spread over Amp dish.
  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :
    • Brick 200 (3)
    • Brick 200 (4)
    • Brick 200 (5)
    • Brick 200 (6)
  • The efficiency of the purification was tested thanks to agarose gel electrophoresis. Results are presented following.
    Brick 200 (6) well did not present any DNA fragment. Other wells contain one or two fragments of DNA, so Brick 200 (3), 200 (4) and 200 (5) were successfully purified.

  • Several control PCR were realized to verify if SLIC worked. Taq polymerase was used. Three clones of each SLIC were tested. They were also pricked up on Amp dish.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    S1 Clone 1 of SLIC SerA 5 - 6 serA_PrefRFC10RBS_up
    serA_tronq_SufRFC23_down    		 1032 bp
    S2 Clone 2 of SLIC SerA 5 - 6 serA_PrefRFC10RBS_up
    serA_tronq_SufRFC23_down    		 1032 bp
    S3 Clone 3 of SLIC SerA 5 - 6 serA_PrefRFC10RBS_up
    serA_tronq_SufRFC23_down    		 1032 bp
    S4 Clone 4 of SLIC Mesh1 without STOP 24 - 25 Mesh_1_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 579 bp
    S5 Clone 5 of SLIC Mesh1 without STOP 24 - 25 Mesh_1_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 579 bp
    S6 Clone 6 of SLIC Mesh1 without STOP 24 - 25 Mesh_1_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 579 bp
    S7 Clone 7 of SLIC Mesh1 with STOP 24 - 26 Mesh_1_RFC10-RBS_up
    Mesh_1_2_stop_SufRFC23_down    		 582 bp
    S8 Clone 8 of SLIC Mesh1 with STOP 24 - 26 Mesh_1_RFC10-RBS_up
    Mesh_1_2_stop_SufRFC23_down    		 582 bp
    S9 Clone 9 of SLIC Mesh1 with STOP 24 - 26 Mesh_1_RFC10-RBS_up
    Mesh_1_2_stop_SufRFC23_down    		 582 bp

  • Several DNA fragments were amplified thanks to iGEM PCR protocol (Q5 polymerase).

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    1 pKD4 plasmid 1 - 2 sdaCsdaB_mut_pKD4_up
    sdaCsdaB_ mut_pKD4_down    		 1032 bp
    4 pKD4 plasmid 3 - 4 cheA_mut_pKD4_up
    cheA_mut_pKD4_down    		 1032 bp
    5 Genomic DNA of W3110 9 - 14 cheA_PrefRFC23_up
    cheA_mut_T1290C_down    		 1032 bp
    7 Genomic DNA of W3110 15 - 17 relA_mut_C1366G_up
    relA_RFC10-RBS_up    		 579 bp
    PCR product 1 and 4 were correctly amplified. Thus, they were purified thanks to Macherey Nagel PCR Clean up Kit. PCR product 5 and 7 seemed to be compound with too many different DNA fragments. That's why, a new amplification by PCR was made overnight (iGEM PCR protocol Q5 polymerase).
  • Realization of "Day 2" of lambda red pKOBEG protocol. W3110 with active recombinase was electroporated by PCR product 1 (sdaCsdaB) and 4 (cheA). Transformed bacteria were spread over a Kan dish.

  • The efficiency of the purification of PCR product number S1 to S9 was tested thanks to agarose gel electrophoresis. At the same time, the efficiency of the amplification of the beginning sequences of CheA and RelA (PCR product 5 and 7) was tested thanks to agarose gel electrophoresis. Results are presented bellow.

    PCR products 5 and 7 were good for SLIC. Clone 1 and 2 (serA), clone 4 and 6 (Mesh1 without STOP) and clone 7 and 8 (Mesh1 with STOP) were put in culture in LB with Amp.
  • The DNA concentration of PCR products 5 and 7 was tested thanks to the Nanodrop. Results are presented bellow.
    # Concentration of DNA (ng/µL)
    5 29.78
    7 85.51
  • PCR products number 5, 6, 7, 8 and 9 were used to realize SLIC protocol. This protocol permits to assemble all parts of CheA (5 and 6) and RelA (7 and 8 without STOP, 7 and 9 with STOP) genes with their directed mutation. The destination plasmid DNA used is the backbone pSB1A3 linearized plasmid. DH5α supercompetent bacteria were transformed with SLIC products and spread over Amp dish.
  • SLIC assembly of RelA was a success but SLIC assembly of CheA failed. That's why, a new SLIC assembly of CheA with PCR products 5 and 6 was made. The destination plasmid DNA used is the backbone pSB1A3 linearized plasmid. DH5α supercompetent bacteria were transformed with SLIC products and spread over Amp dish.
  • Realization of "Day 2" of lambda red pKOBEG protocol a second time. PCR product 1 and 4 were purified a second time. The efficiency of the purification was tested thanks to agarose gel electrophoresis. Results are presented bellow. W3110 with active recombinase was electroporated by PCR product 1 (sdaCsdaB) and 4 (cheA). Transformed bacteria were spread over a Kan dish.

  • Several control PCR were realized to verify whether RelA SLIC worked. Taq polymerase was used. Three clones of each SLIC were tested. They were also pricked up on Amp dish.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    S22 Clone 22 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    S23 Clone 22 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    S24 Clone 22 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    S25 Clone 25 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2262 bp
    S26 Clone 26 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2262 bp
    S27 Clone 27 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2262 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented bellow.

    PCR products 1 and 4 were correctly amplified. Thus, they were purified thanks to Macherey Nagel PCR Clean up Kit. Transplanting of S22 to S27 failed, those clones were gave up.
  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :
    • Clone 1 and 2 (SerA) ⇒ PiGEM_02.10 and PiGEM_02.11
    • Clone 4 and 6 (Mesh1 without STOP) ⇒ PiGEM_02.12 and PiGEM_02.13
    • Clone 7 and 8 (Mesh1 with STOP) ⇒ PiGEM_02.14 and PiGEM_02.15
  • SLIC assembly of CheA failed. That's why, a new SLIC assembly of CheA with PCR products 5 and 6 was made. The destination plasmid DNA used is the backbone pSB1A3 linearized plasmid. DH5α supercompetent bacteria were transformed with SLIC products and spread over Amp dish.

  • Several control PCR were realized to verify whether RelA SLIC worked. Taq polymerase was used. Three clones of each SLIC were tested. They were also pricked up on Amp dish.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    S28 Clone 28 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    S29 Clone 28 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    S30 Clone 28 of SLIC RelA without Stop 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    S31 Clone 31 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2262 bp
    S32 Clone 31 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2262 bp
    S33 Clone 31 of SLIC RelA with STOP 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2262 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented bellow.

    PCR product S28, S29, S31, S32 and S33 were good for SLIC. Clones 28 and 29 (RelA without STOP) and clones 31, 32 and 33 (RelA with STOP) were put in culture in LB with Amp.
  • Realization of "Day 2" of lambda red pKOBEG protocol for the third time. PCR products 1 and 4 were purified a third time. pKOBEG electrocompetent were prepared a third time. W3110 with active recombinase was electroporated with PCR products 1 (sdaCsdaB) and 4 (cheA). Transformed bacteria were spread over a Kan dish.
  • The efficiency of the purification of PiGEM_02.10, PiGEM_02.11, PiGEM_02.12, PiGEM_02.13, PiGEM_02.14 and PiGEM_02.15 was tested thanks to agarose gel electrophoresis. Results are presented bellow.
    These plasmids seemed to be in low quantity. The DNA concentration of plasmids was tested thanks to the Nanodrop. Results are presented following
    # Concentration of DNA (ng/µL)
    PiGEM_02.10 30.6
    PiGEM_02.11 37.8
    PiGEM_02.12 12.2
    PiGEM_02.13 15.11
    PiGEM_02.14 14.11
    PiGEM_02.15 19.22
  • The W3110 with active recombinase was electroporated with PCR products 1 (sdaCsdaB) and 4 (cheA). Transformed bacteria were spread over a Kan dish.
  • Bacteria transformed with PCR products 1 (sdaCsdaB) and 4 (cheA) put in culture on Kan dish on the 08/01/14 showed colonies.
  • 5 different colonies of SdaCsdaB and CheA mutants were spread over Kan dish and Cm30 dish in order to verify whether the pKOBEG plasmid was eliminated (by the previously heat shock) or not as we wanted to, and whether the transformed bacteria contained the kanamycin resistance. The bacteria we want must show colonies on Kan dish and no colonies on Cm 30 dish at the same time.
Protocols used : ...

Week 6 : 08/04/2014 ➡ 08/10/2014

  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :
    • SLIC RelA without STOP PCR product 7/8 Clone 28 and 29 ⇒ PiGEM_02.16 and PiGEM_02.17
    • SLIC RelA with STOP PCR product 7/8 Clone 31, 32 and 33 ⇒ PiGEM_02.18, PiGEM_02.19 and PiGEM_02.20
    All plasmids seemed to be correctly purified. No DNA contamination was noticeable.

  • Several DNA fragments were amplified thanks to iGEM PCR protocol (Q5 polymerase).

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    14 Genomic DNA of W3110 33 - 34 CusR_iProm-up
    CusR_iProm-down    		 117 bp
    15 PiGEM_01.04 37 - 38 BBa_E1010-up
    BBa_E1010_NoStop-down    		 708 bp
    16 PiGEM_02.03 39 - 40 BBa_E0040-up
    BBa_E0040_NoStop-down    		 714 bp
    17 PiGEM_01.09 45 - 46 (SH3)4-up
    (SH3)4-down    		 932 bp
    18 PiGEM_02.08 47 - 48 cusR_L_LZA-up
    cusR_L_LZA_down    		 857 bp
    19 PiGEM_02.07 49 - 50 SH3pep_L_LZA-up
    SH3pep_L_LZA-down    		 192 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    Fragments 19, 15, 16 and 18 seemed to be correctly amplified. They were going to be cloned in iGEM plasmids. Unfortunately, fragments 14 and 17 were not amplified. Another PCR had to be made.

  • Several control PCR were realized to verify whether SLIC, bricks 200 and bricks 202 worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    20 Clone 1 of SLIC SerA 43 - 44 Prefix_up
    Suffix_down    		 1032 bp
    21 Clone 2 of SLIC SerA 43 - 44 Prefix_up
    Suffix_down    		 1032 bp
    22 Clone 28 of SLIC SerA 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    23 Clone 29 of SLIC SerA 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    24 Clone 31 of SLIC SerA 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    25 Clone 32 of SLIC SerA 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    26 Clone 33 of SLIC SerA 43 - 44 Prefix_up
    Suffix_down    		 2260 bp
    27 Clone 34 of SLIC CheA 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    28 Clone 35 of SLIC CheA 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    29 Clone 36 of SLIC CheA 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    30 Clone 37 of SLIC CheA 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    31 Clone 38 of SLIC CheA 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    32 Clone 39 of SLIC CheA 43 - 44 Prefix_up
    Suffix_down    		 2432 bp
    33 Brick 202 (1) 43 - 44 Prefix_up
    Suffix_down    		 730 bp
    34 Brick 202 (2) 43 - 44 Prefix_up
    Suffix_down    		 769 bp
    35 Brick 200 (3) 43 - 44 Prefix_up
    Suffix_down    		 769 bp
    36 Brick 200 (4) 43 - 44 Prefix_up
    Suffix_down    		 769 bp
    37 Brick 200 (5) 43 - 44 Prefix_up
    Suffix_down    		 769 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    No fragment seemed to be correctly amplified. SLIC were going to be done again with another protocol. Bricks 200 and 202 were also going to be remade.

  • PiGEM_01.03 (pSB1C3, BBa_B0033) was digested with XbaI and SpeI to be used for stability label cloning.

    The efficiency of the digestion was tested thanks to agarose gel electrophoresis. Results are presented below.

    PSB1C3 of PiGEM_01.03 seemed to be correctly digested.

  • PiGEM_01.03 (pSB1C3, BBa_B0033) was digested with EcoRI and SpeI to be used for PCR products 15 and 16 cloning. PCR products 15 and 16 (respectively RFP without STOP codon and GFP without STOP codon) were digested by EcoRI and SpeI. Then, PCR products 15 and 16 were leagued in pSB1C3 digested. DH5 alpha supercompetent were transformed with these ligation products and spread on Cm dish.

  • PiGEM_02.27 (pSB1A2, BBa_J04450) was digested with EcoRI and PstI to be used for PCR products 18 and 19 cloning. PCR products 18 and 19 (respectively cusR-L-LZa and SH3pep-L-LZa) were digested by EcoRI and PstI. Then, PCR products 18 and 19 were leagued in pSB1A2 digested. DH5 alpha supercompetent were transformed with these ligation products and spread on Amp dish.

  • Because PCR 17 did not work, (SH3)4 was amplified again thanks to iGEM PCR protocol (Q5 polymerase).

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    38 PiGEM_02.07 49 - 50 SH3pep_L_LZA-up
    SH3pep_L_LZA-down    		 192 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    (SH3)4 amplification did not work again. A specific treatment of this cloning was needed.

  • PiGEM_01.03 (pSB1C3, BBa_B0033) had be digested with XbaI and SpeI the day before to be used for stability label cloning. A gel extract was made this day to collect only the backbone. The backbone obtained was digested by PstI because XbaI and SpeI are compatible restriction sites. Stability labels were assembled by annealing.

    Primers numbers Primers names Brick created
    27 - 28 BBa_K1051206-up
    BBa_K1051206-down    		 BBa_K1051206
    29 -30 BBa_K1051207-up
    BBa_K1051207-down    		 BBa_K1051207
    31 -32 BBa_K1051208-up
    BBa_K1051208-down    		 BBa_K1051208

    Each annealing oligonucleotides were leagued in pSB1C3 digested XSP. DH5 alpha supercompetent were transformed with these ligation products and spread on Cm dish.

  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :

    SLIC CheA PCR product 5/6 Clone 34, 35, 36, 37, 38 and 39 ⇒ PiGEM_02.21, PiGEM_02.22, PiGEM_02.23, PiGEM_02.24, PiGEM_02.25, PiGEM_02.26

  • Several control PCR were realized to verify one more time whether SLIC worked. Besides, several control PCR were realized to verify whether bricks 200 and bricks 202 worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    39 PiGEM_02.10 5 - 6 serA_PrefRFC10RBS_up
    serA_tronq_SufRFC23_down    		 1032 bp
    40 PiGEM_02.11 5 - 6 serA_PrefRFC10RBS_up
    serA_tronq_SufRFC23_down    		 1032 bp
    41 PiGEM_02.16 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    42 PiGEM_02.17 17 - 18 relA_RFC10-RBS_up
    relA_sans_stop_SufRFC23_down    		 2259 bp
    43 PiGEM_02.18 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2259 bp
    44 PiGEM_02.19 17 - 19 relA_RFC10-RBS_up
    relA_2_stop_SufRFC23_down    		 2259 bp
    45 PiGEM_02.20 9 - 10 cheA_PrefRFC23_up
    cheA_SufRFC23_down    		 2432 bp
    46 PiGEM_02.21 9 - 10 cheA_PrefRFC23_up
    cheA_SufRFC23_down    		 2432 bp
    47 PiGEM_02.22 9 - 10 cheA_PrefRFC23_up
    cheA_SufRFC23_down    		 2432 bp
    48 PiGEM_02.23 9 - 10 cheA_PrefRFC23_up
    cheA_SufRFC23_down    		 2432 bp
    49 PiGEM_02.24 9 - 10 cheA_PrefRFC23_up
    cheA_SufRFC23_down    		 2432 bp
    50 PiGEM_02.25 9 - 10 cheA_PrefRFC23_up
    cheA_SufRFC23_down    		 2432 bp
    51 PiGEM_02.26 9 - 10 cheA_PrefRFC23_up
    cheA_SufRFC23_down    		 2432 bp
    52 PiGEM_01.04 43 - 44 Prefix_up
    Suffix_down    		 706 bp
    53 PiGEM_02.10 43 - 44 Prefix_up
    Suffix_down    		 1032 bp
    54 Brick 202 (1) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    55 Brick 202 (2) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    56 Brick 202 (3) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    57 Brick 202 (4) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    58 Brick 200 (3) 38 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 711 bp
    59 Brick 200 (4) 38 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    60 Brick 200 (6) 38 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 711 bp

  • Because PCR 14 did not work, cusR promoter was amplified again thanks to iGEM PCR protocol (Q5 polymerase).

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    62 Genomic DNA of W3110 33 - 34 CusR_iProm-up
    CusR_iProm-down    		 117 bp

  • The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    SLIC had to be made again. Bricks 200 were correctly amplified but bricks 202 were not. CusR promoter was not amplified.

  • Because PCR 62 did not work, cusR promoter was amplified again thanks to iGEM PCR protocol (Q5 polymerase).

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    63 Genomic DNA of W3110 33 - 34 CusR_jProm-up
    CusR_jProm-down    		 117 bp

  • Several control PCR were realized to verify whether bricks 200 worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    64 Brick 202 (5) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    65 Brick 202 (6) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    646 Brick 202 (7) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    67 Brick 202 (8) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    68 Brick 202 (9) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    69 Brick 202 (10) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp
    70 Brick 202 (11) 40 - 51 BBa_E0040_NoStop-down
    BBa_B0034-up    		 730 bp

  • A gel extract of PCR product 38 (SH3)4 was made.

  • Several control PCR were realized to verify whether RFP without STOP codon and GFP without STOP codon worked. Taq polymerase was used.

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    72 RFP without STOP codon
    Clone 40    		 37 - 38 BBa_E1010-up
    BBa_E1010_NoStop-down    		 708 bp
    73 RFP without STOP codon
    Clone 41    		 37 - 38 BBa_E1010-up
    BBa_E1010_NoStop-down    		 708 bp
    74 RFP without STOP codon
    Clone 42    		 37 - 38 BBa_E1010-up
    BBa_E1010_NoStop-down    		 708 bp
    75 RFP without STOP codon
    Clone 43    		 37 - 38 BBa_E1010-up
    BBa_E1010_NoStop-down    		 708 bp
    76 RFP without STOP codon
    Clone 44    		 37 - 38 BBa_E1010-up
    BBa_E1010_NoStop-down    		 708 bp
    77 GFP without STOP codon
    Clone 45    		 39 - 40 BBa_E0040-up
    BBa_E0040_NoStop-down    		 714 bp
    78 GFP without STOP codon
    Clone 46    		 39 - 40 BBa_E0040-up
    BBa_E0040_NoStop-down    		 714 bp
    79 GFP without STOP codon
    Clone 47    		 39 - 40 BBa_E0040-up
    BBa_E0040_NoStop-down    		 714 bp
    80 GFP without STOP codon
    Clone 48    		 39 - 40 BBa_E0040-up
    BBa_E0040_NoStop-down    		 714 bp
    81 GFP without STOP codon
    Clone 49    		 39 - 40 BBa_E0040-up
    BBa_E0040_NoStop-down    		 714 bp

  • The efficiency of the purification was tested thanks to agarose gel electrophoresis. Results are presented following.

    PCR product 63 was correctly amplified. PiGEM_02.27 (pSB1A2, BBa_J04450) was digested with EcoRI and Pst. PCR product 63 (cusR promoter) was digested by EcoRI and PstI. Then, PCR product 63 was leagued in pSB1A2 digested. DH5 alpha supercompetent were transformed with this ligation product and spread on Amp dish.

  • To confirm the cloning of stability label and SLIC, each plasmid containing constructions were digested with specific restriction enzymes.

    Plasmid Numbers Plasmid Names Digestion Weight of expected bands if constructions are correct
    EcoRI PstI SpeI XbaI
    PiGEM_01.10 BBa_K1051207
    Clone St1    		 X X
    • 2082 pb
    • 80 pb
    PiGEM_01.11 BBa_K1051207
    Clone St2    		 X X
    • 2082 pb
    • 80 pb
    PiGEM_01.12 BBa_K1051208
    Clone St3    		 X X
    • 2082 pb
    • 80 pb
    PiGEM_02.10 SLIC SerA produit PCR 2/3
    Clone 1    		 X
    • 2352 pb
    • 847 pb
    PiGEM_02.11 SLIC SerA produit PCR 2/3
    Clone 2    		 X
    • 2352 pb
    • 847 pb
    PiGEM_02.12 SLIC Mesh1 sans STOP produit PCR 10/11/12
    Clone 4    		 X X
    • 2422 pb
    • 180 pb
    • 84
    PiGEM_02.13 SLIC Mesh1 sans STOP produit PCR 10/11/12
    Clone 6    		 X X
    • 2422 pb
    • 180 pb
    • 84
    PiGEM_02.14 SLIC Mesh1 sans STOP produit PCR 10/11/13
    Clone 7    		 X X
    • 2422 pb
    • 180 pb
    • 84
    PiGEM_02.15 SLIC Mesh1 sans STOP produit PCR 10/11/13
    Clone 8    		 X X
    • 2422 pb
    • 180 pb
    • 84
    PiGEM_02.16 SLIC RelA sans STOP produit PCR 7/8
    Clone 28    		 X
    • 3530 pb
    • 872 pb
    PiGEM_02.17 SLIC RelA sans STOP produit PCR 7/8
    Clone 29    		 X
    • 3530 pb
    • 872 pb
    PiGEM_02.18 SLIC RelA sans STOP produit PCR 7/8
    Clone 31    		 X
    • 3530 pb
    • 872 pb
    PiGEM_02.19 SLIC RelA sans STOP produit PCR 7/8
    Clone 32    		 X
    • 3530 pb
    • 872 pb
    PiGEM_02.24 SLIC CheA produit PCR 5/6
    Clone 37    		 X
    • 3457 pb
    • 675 pb
    PiGEM_02.25 SLIC CheA produit PCR 5/6
    Clone 38    		 X
    • 3457 pb
    • 675 pb
    PiGEM_02.26 SLIC CheA produit PCR 5/6
    Clone 39    		 X
    • 3457 pb
    • 675 pb

    The efficiency of the digestion was tested thanks to agarose gel electrophoresis. Results are presented bellow.

    For stability label, linearized plasmid was the only fragment visible. For SLIC, no fragment matched with the weight of expected bands. All SLIC construction had to be made again.

  • PiGEM_02.27 (pSB1A2, BBa_J04450) was digested with XbaI, SpeI and PstI to be used for stability label cloning. Stability labels were assembled again by annealing.

    Primers numbers Primers names Brick created
    27 - 28 BBa_K1051206-up
    BBa_K1051206-down    		 BBa_K1051206
    29 -30 BBa_K1051207-up
    BBa_K1051207-down    		 BBa_K1051207
    31 -32 BBa_K1051208-up
    BBa_K1051208-down    		 BBa_K1051208

    Each annealing oligonucleotides were leagued in pSB1A2 digested XSP. DH5 alpha supercompetent were transformed with these ligation products and spread on Amp dish.

  • PiGEM_02.27 (pSB1A2, BBa_J04450) was digested with XbaI and SpeI. CusR box and Flag were assembled by annealing.

    Primers numbers Primers names Brick created
    35 - 36 CusR_Box-up
    CusR_Box-down    		 CusR Box
    41 - 42 Flag-up
    Flag-down    		 Flag

    Each annealing oligonucleotides were leagued in pSB1A2 digested XS. DH5 alpha supercompetent were transformed with these ligation products and spread on Amp dish.

  • SLIC assembly of CheA, SerA, RelA with and without STOP codon and Mesh 1 with and without STOP codon were restarted. DNA fragments were amplified thanks to iGEM PCR protocol (Q5 polymerase).

    # DNA Template Primers Numbers Primers Names Weight of amplified DNA
    82 Genomic DNA of W3110 6 - 7 serA_mut_ A849G_up
    serA_tronq_SufRFC23_down    		 171 bp
    83 Genomic DNA of W3110 5 - 8 serA_PrefRFC10RBS_up
    serA_mut_A849_down    		 861 bp
    84 PiGEM_02.05 15 - 17 relA_mut_C1366G_up
    relA_RFC10-RBS_up    		 1377 bp
    85 PiGEM_02.05 16 - 18 relA_mut_C1366_down
    relA_sans_stop_SufRFC23_down    		 882 bp
    86 PiGEM_02.05 16 - 19 relA_mut_C1366_down
    relA_2_stop_SufRFC23_down    		 885 bp
    87 PiGEM_02.06 20 - 24 Mesh_1_mut_A255T_up
    Mesh_1_RFC10-RBS_up    		 267 bp
    88 PiGEM_02.06 21 - 22 Mesh_1_mut_A255T_down
    Mesh_1_mut_A343G_up    		 120 bp
    89 PiGEM_02.06 23 - 25 Mesh_1_mut_A343G_down
    Mesh1_sans_stop_SufRFC23_down    		 192 bp
    90 PiGEM_02.06 23 - 26 Mesh_1_mut_A343G_down
    Mesh_1_2_stop_SufRFC23_down    		 192 bp

    The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented below.

    All DNA fragments were correctly amplified. Thus, SLIC protocol was realized to assemble each fragment of CheA, SerA, RelA and Mesh1. Plasmid pSB1A2 digested XbaI and SpeI was used.

  • Several control PCR had be realized the day before to verify whether RFP without STOP codon and GFP without STOP codon worked. The efficiency of the amplification was tested thanks to agarose gel electrophoresis. Results are presented following.

    Only clones 41, 42 43 and 44 of RPF without STOP codon and clones 47 and 48 of GFP without STOP codon seemed to be correct.

  • Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :

    • PCR product 15 RFP without STOP Clone 41 ⇒ PiGEM_01.14
    • PCR product 15 RFP without STOP Clone 43 ⇒ PiGEM_01.15
    • PCR product 16 GFP without STOP Clone 47 ⇒ PiGEM_01.16
    • PCR product 16 GFP without STOP Clone 48 ⇒ PiGEM_01.17
Protocols used : ...

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