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| − | {{NTNU Trondheim_Draft}} | + | {{NTNU_Trondheim_Draft}} |
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| − | <!-- -------------------Month Filters------------------- -->
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| − | <li><h6> Jump to: </h6></li>
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| − | <!--li class="nb-month">
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| − | <div style="top:67px;margin-left:-6px">Spring</div>
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| − | </li>
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| − | <li id="weekA" class="active" onclick="weekFilter(this)">
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| − | <a class="nb-week" href="#A">January</a>
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| − | </li>
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| − | <li id="weekB" onclick="weekFilter(this)">
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| − | <a class="nb-week" href="#B">February</a>
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| − | <li id="weekC" onclick="weekFilter(this)">
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| − | <a class="nb-week" href="#C">March</a>
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| − | <a class="nb-week" href="#E">May</a>
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| − | <div style="top:45px">June</div>
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| − | <a class="nb-week" href="#23">Week 23</a>
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| − | <a class="nb-week" href="#24">Week 24</a>
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| − | <a class="nb-week" href="#25">Week 25</a>
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| − | <a class="nb-week" href="#26">Week 26</a>
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| − | <div style="top:65px;margin-left:2px">July</div>
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| − | <a class="nb-week" href="#27">Week 27</a>
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| − | <a class="nb-week" href="#28">Week 28</a>
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| − | <a class="nb-week" href="#29">Week 29</a>
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| − | <a class="nb-week" href="#30">Week 30</a>
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| − | <a class="nb-week" href="#31">Week 31</a>
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| − | <div style="top:52px;margin-left:-9px">August</div>
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| − | <li id="week32" onclick="weekFilter(this)">
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| − | <a class="nb-week" href="#32">Week 32</a>
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| − | <a class="nb-week" href="#33">Week 33</a>
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| − | <a class="nb-week" href="#34">Week 34</a>
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| − | <a class="nb-week" href="#35">Week 35</a>
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| − | <div style="top:52px;margin-left:-22px">September</div>
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| − | <a class="nb-week" href="#36">Week 36</a>
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| − | <a class="nb-week" href="#37">Week 37</a>
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| − | <a class="nb-week" href="#38">Week 38</a>
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| − | <a class="nb-week" href="#39">Week 39</a>
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| − | </li>
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| − | </ul>
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| − | </div>
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| − | <div class="ten columns team-bios-container">
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| − | <div class="twelve columns">
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| | <div class="page-header"> | | <div class="page-header"> |
| − | <h2>Notebook</h2> | + | <h2>Software</h2> |
| − | </div> </div>
| + | </div> |
| − | </div>
| + | <ul class="nav nav-pills"> |
| − | <!-- -------------------Subteam Filters------------------- -->
| + | <li role="presentation" class="active"><a href="https://2015.igem.org/Team:NTNU_Trondheim/Software">Overview</a></li> |
| − | <div class="row last-ele" style="padding-bottom:0px">
| + | <li role="presentation"><a href="https://2015.igem.org/Team:NTNU_Trondheim/Software/iGEM Matchmaker">iGEM Matchmaker</a></li> |
| − | <h5 style="margin-left:15px;text-align:left">Filter by category:
| + | <li role="presentation"><a href="https://2015.igem.org/Team:NTNU_Trondheim/Software/Quantitative_Analysis_Tool">Quantitative Analysis Tool</a></li> |
| − | <div id="nb-all" class="nb-all" onclick="showAll()">show all categories</div>
| + | <li role="presentation"><a href="https://2015.igem.org/Team:NTNU_Trondheim/Software/Brixells_Tool">Brixells Modeling Tool</a></li> |
| − | </h5>
| + | </ul> |
| − | </div>
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| − | <div class="row last-ele nb-labels" style="padding-bottom:0px">
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| − | <div style="opacity:0.00;width:0px">"_"</div>
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| − | <div>Genetics</div>
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| − | <div>Encapsulation</div>
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| − | <div>Interlab Study</div>
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| − | <div>Modeling</div>
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| − | <div>Practices</div>
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| − | <div>Wiki</div>
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| − | <img id="dry" src="https://static.igem.org/mediawiki/2015/4/41/NTNU_Trondheim_calculations_on.png">
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| − | </div>
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| − | <div id="dry-only" class="nb-only" onclick="onlyFilter(this)">only</div>
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| − | <div class="filter-container" onclick="filter(this)" style="background-image:url(https://static.igem.org/mediawiki/2015/8/8c/Team_NTNU_Trondheim_nb-hprac-sg.png)">
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| − | <img id="hprac" src="https://static.igem.org/mediawiki/2015/9/9b/Team_NTNU_Trondheim_nb-hprac-sc.png">
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| − | <img id="wiki" src="https://static.igem.org/mediawiki/2015/9/98/Team_NTNU_Trondheim_nb-wiki-sc.png">
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| − | </div>
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| − | <!-- PLACEHOLDER --> <div class="two columns nb-filter" style="opacity:0.00;width:0px">
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| − | </div>
| + | |
| − | <!-- ===================Notebook entries=================== -->
| + | |
| − | <p>
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| − | </p>
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| | | | |
| − | <div class=" nb- week" id=" week23entry"> | + | <div class="col-md-3 pull-right"> |
| − | <div class=" twelve columns"> | + | <div class="panel panel-default"> |
| − | <h3>Week 23</h3>
| + | <div class=" panel-heading"> |
| − | <h6>(01/06 - 07/06)</h6>
| + | <h3 class="panel-title">Software repository</h3> |
| | + | </div> |
| | + | <div class="panel-body"> |
| | + | Our software is available on the iGEM <a href="https://github.com/igemsoftware/NTNU-Trondheim-2015">GitHub software repository!</a> |
| | + | </div> |
| | + | </div> |
| | + | </div> |
| | | | |
| − | <div class="entry nb-dry">
| + | <br> |
| − | <div>
| + | <p>Three computational tools have been developed in the course of the summer: |
| − | <h6>February 02</h6>
| + | <ol> |
| − |
| + | <li><a href="https://2015.igem.org/Team:NTNU_Trondheim/Software/Quantitative_Analysis_Tool">Quantitative image analysis tool</a> for high throughput processing of confocal microscopy images.</li> |
| − | <p>The iGEM Matchmaker (V2.0) is deployed for 2015.</p>
| + | <li><a href="http://almaaslab.nt.ntnu.no/igem_matchmaker/">iGEM Matchmaker</a>, updated from the one originally developed by the NTNU 2012 team with automatic team matching using keyword extraction.</li> |
| − | </div>
| + | <li><a href="https://2015.igem.org/Team:NTNU_Trondheim/Collaborations">Brixells Modeling Tool</a>, created to assist <a href="https://2015.igem.org/Team:Warwick">Team Warwick</a> to predict the probability of bonding of 3D structures built with E. Coli expressing Zinc fingers and DNA strands. |
| − | </div>
| + | </ol> |
| | + | </p> |
| | + | <br> |
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| − | </div>
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| − | </div>
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| − | <div class="nb-week" id="week24entry">
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| − | <div class="twelve columns">
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| − | <h3>Week 24</h3>
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| − | <h6>(08/06 - 14/06)</h6>
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| − | <div class="entry nb-wet">
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| − | <div>
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| − | <h6>June 11</h6>
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| − | <p>Discussed <i>Escherichia coli</i> glucose uptake and possible promoters in <i>E. coli</i>.</p>
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| − | </div>
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| − | <div class="nb-week" id="week25entry">
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| − | <div class="twelve columns">
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| − | <h3>Week 25</h3>
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| − | <h6>(15/06 - 21/06)</h6>
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| − | <div class="entry nb-wet">
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| − | <div>
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| − | <h6>June 15</h6>
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| − | <p>Discussed <i>Pseudomonas putida</i> as a candidate microorganism.<br>Discussion of a system for glucose uptake in <i>P. putida</i>.</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-hprac">
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| − | <div>
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| − | <h6>June 15</h6>
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| − | <p>Interviews.</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-wet">
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| − | <div>
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| − | <h6>June 17</h6>
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| − | <p>Design of promoter regions for <i>P. putida</i>.<br>Ordered promoter regions for <i>P. putida</i>.</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-meas">
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| − | <div>
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| − | <h6>June 18</h6>
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| − | <p>Discussion of InterLab Study.<br>Preparations for the InterLab Study</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-meas">
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| − | <div>
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| − | <h6>June 19</h6>
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| − | <p>Prepared and autoclaved LB medium 300 ml, LA medium 3 x 500 ml, Eppendorf tubes, distilled water and pipette tips.<br>Prepared antibiotic stock solutions (Ampicillin, Chloramphenicol and Kanamycin).</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-hprac">
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| − | <div>
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| − | <h6>June 20</h6>
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| − | <p>Created survey for students and employees at NTNU.<br>Interviews with diabetic patients.</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-meas">
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| − | <div>
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| − | <h6>June 21</h6>
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| − | <p>Inoculation of DH5α in 3 ml, incubation over night at 37 <sup>0</sup>C.</p>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="nb-week" id="week26entry">
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| − | <div class="twelve columns">
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| − | <h3>Week 26</h3>
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| − | <h6>(22/06 - 28/06)</h6>
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| − | <div class="entry nb-meas">
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| − | <div>
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| − | <h6>June 22</h6>
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| − | <p>Making <i>E. coli</i>electrocompetent (after 3h incubation, OD<SUB>600</SUB> = 0.44).<br>Electroporation of E. coli.</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-meas">
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| − | <div>
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| − | <h6>June 23</h6>
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| − | <p>Transformation.</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-anim">
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| − | <div>
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| − | <h6>June 24</h6>
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| − | <p>Introduction to alginate encapsulation + demo on how to operate the electrostatic capsule generator.</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-meas">
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| − | <div>
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| − | <h6>June 24</h6>
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| − | <p>Inoculation of a colony from plate in 3 ml LB.</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-anim">
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| − | <div>
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| − | <h6>June 25</h6>
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| − | <p>Playing around with the electrostatic bead generator, making capsules of different sizes by varying the different parameters of the instrument.</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-meas">
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| − | <div>
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| − | <h6>June 25</h6>
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| − | <p>Miniprep of transformed E. coli.<br>Nanodrop measurement of DNA concentration.</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-meas">
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| − | <div>
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| − | <h6>June 26</h6>
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| − | <p>Enzyme digestion.<br>Ligation.<br>Transformation.<br>Prepared LA plates.</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-meas">
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| − | <div>
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| − | <h6>June 27</h6>
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| − | <p>Put plates from incubator in the fridge (there were also colonies on the control plate, so we have to validate that the colonies we picked for inoculation really have the desired plasmid).</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-meas">
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| − | <div>
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| − | <h6>June 28</h6>
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| − | <p>Inoculation of 3 colonies from each sample from the 50 μl plate, and 3 colonies from the positive and negative control plates (from June 23rd).</p>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="nb-week" id="week27entry">
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| − | <div class="twelve columns">
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| − | <h3>Week 27</h3>
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| − | <h6>(29/06 - 05/07)</h6>
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| − | <div class="entry nb-anim">
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| − | <div>
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| − | <h6>June 29</h6>
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| − | <p>Moved the cell encapsulation equipment into our lab, so it was ready for the GM bacteria.</p>
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| − | </div>
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| − | </div>
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| − |
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| − | <div class="entry nb-meas">
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| − | <div>
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| − | <h6>July 01</h6>
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| − | <p>Miniprep.<br>Nanodrop measurement after miniprep (58.38 + 64.98 + 70.48 ~ 64.63).</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-anim">
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| − | <div>
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| − | <h6>July 01</h6>
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| − | <p>Preparation of alginate solution, gelling solution and washing solution.</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-anim">
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| − | <div>
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| − | <h6>July 02</h6>
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| − | <p>Inoculation of <i>Pseudomonas putida</i> pHH+GFP from -80 <sup>0</sup>C in 3 ml LB + Kan medium. Incubation at 30 <sup>0</sup>C.</p>
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| − | </div>
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| − | </div>
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| − | <div class="entry nb-anim">
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| − | <div>
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| − | <h6>July 03</h6>
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| − |
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| − | <p>5 % inoculation of overnight culture in fresh LB + Kan.<br>Incubation at 30 <sup>0</sup>C for two hours. Induction of <i>P. putida</i> pHH+GFP with m-Toluic acid.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-meas">
| |
| − | <div>
| |
| − | <h6>July 03</h6>
| |
| − |
| |
| − | <p>Introduction to confocal microscopy.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-dry">
| |
| − | <div>
| |
| − | <h6>July 05</h6>
| |
| − |
| |
| − | <p>Created the website design using Bootstrap, and set up the Notebook and Protocols pages.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="nb-week" id="week28entry">
| |
| − | <div class="twelve columns">
| |
| − | <h3>Week 28</h3>
| |
| − | <h6>(06/07 - 12/07)</h6>
| |
| − |
| |
| − | <div class="entry nb-meas">
| |
| − | <div>
| |
| − | <h6>July 06</h6>
| |
| − |
| |
| − | <p>PCR (1 μl template DNA = miniprepped from <i>P. putida</i> pHH+GFP, annealing temperature = 60 <sup>0</sup>C).<br>Prepared LB (200 ml with Kan, 100 ml with Chl).<br>GelRed to test the PCR. It did not work. <br>PCR repeated but with Rahmi’s DNA, did not work either.<br>PCR repeated again and gel shows the right band (but also an unknown second band).</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>July 06</h6>
| |
| − |
| |
| − | <p>Inoculation of <i>Pseudomonas putida</i> pHH+GFP from -80 <sup>0</sup>C in 3 ml LB + kan medium. Incubation at 30 <sup>0</sup>C.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-meas">
| |
| − | <div>
| |
| − | <h6>July 07</h6>
| |
| − |
| |
| − | <p>Repeated PCR because of the unknown second band. This time annealing temperature is 61 °C, annealing time 10 sec.<br>OD measurement on devices (to make a standard curve with the plate reader, but the plate reader did not work).<br>2h incubation of InterLab samples at 37 °C, 1h at room temperature, then measurement of samples C by spectrophotometry and plate reader (Since that failed, I would also delete that point). </p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>July 07</h6>
| |
| − |
| |
| − | <p>Preparation of LB + kan medium.<br>1 % inoculation of overnight culture in fresh LB + kan. Incubation at 30 <sup>0</sup>C for 6 hours. Measurement of the OD every 2h and observation of the bacteria number to the microscopy.<br>Results: 2 hours OD = 0.047, 4 hours OD = 0.3, 6 hours, OD = 0.5. Observation: 2 hours, microscopy shows the culture is not dense enough. 4 hours, microscopy shows the culture may be suitable for encapsulation. 6 hours, microscopy shows the culture may be a little too dense for encapsulation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-meas">
| |
| − | <div>
| |
| − | <h6>July 08</h6>
| |
| − |
| |
| − | <p>Inoculation of InterLab samples in 3 ml LB (+ Kan/Chl).<br>Prepared 400 ml PBS.<br>Plate reader measurement of InterLab samples (undiluted from ON culture).</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-meas">
| |
| − | <div>
| |
| − | <h6>July 09</h6>
| |
| − |
| |
| − | <p>1 % inoculation of samples Dev 1A – Dev 3B in 3 ml LB + Kan for flow cytometry.<br>1 % inoculation of samples Dev 3C – NCC in 3 ml LB (+ Kan/Chl) in the same way.<br>Purification of backbone PCR.<br>Nanodrop measurement of concentrations. 1: (9.0 + 9.3 + 9.6) ng/μl : 3 = 9.3 ng/μl. 2: (17.2 + 16.8 + 16.9) ng/μl : 3 = 17.0 ng/μl.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>July 09</h6>
| |
| − |
| |
| − | <p>Preparation of alginate solution, and put this in the 4 <sup>0</sup>C fridge for future use.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 10</h6>
| |
| − |
| |
| − | <p>Prepared TE buffer.<br>Added TE buffer to DNA.<br>HIFI (0.5 μl backbone DNA, 1 μl insert DNA Edd promoter), once with each of the purified backbones.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 11</h6>
| |
| − |
| |
| − | <p>Heat transformation of <i>E. coli</i> DH5α competent cells with Edd promoter (HIFI).<br>Electroporation of <i>P. putida</i> with cells with Edd promoter.<br>Plating <i>P. putida</i> for overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-meas">
| |
| − | <div>
| |
| − | <h6>July 11</h6>
| |
| − |
| |
| − | <p>Plate reader measurement of InterLab samples.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 12</h6>
| |
| − |
| |
| − | <p>Checked plates, they did not grow.<br>Inoculation of <i>P. putida</i>.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-meas">
| |
| − | <div>
| |
| − | <h6>July 12</h6>
| |
| − |
| |
| − | <p>Inoculation of InterLab samples in 3 ml LB (+Kan/+Chl).<br>Plate reader calibration.<br>Plate reader measurement of InterLab samples for 5 hours.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="nb-week" id="week29entry">
| |
| − | <div class="twelve columns">
| |
| − | <h3>Week 29</h3>
| |
| − | <h6>(13/07 - 19/07)</h6>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 13</h6>
| |
| − |
| |
| − | <p>HIFI (0.5 μl backbone DNA, 1 μl insert DNA KguE1 and KguE2 promoter parts), once with each of the purified backbones.<br>Heat transformation of <i>E. coli</i> DH5α competent cell with KguE promoter (HIFI).<br>HIFI (0.5 μl backbone DNA, 1 μl Insert DNA edd promoter), once with each of the purified backbones, again.<br>Heat transformation of <i>E. coli</i> DH5α competent cell with Edd promoter (HIFI).<br>Preparation of LA plates (+ Kan).<br>Plating <i>E. coli</i> DH5α and ET12567 competent cells for overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>July 13</h6>
| |
| − |
| |
| − | <p>Production of alginate capsules (first attempt). Parameters: 2 % alginate solution, needle inner diameter 130 micron, voltage 5000 V, 15 ml/h, distance needle to gelling bath 1 cm.<br>Microscopy confirmed many capsules, and the diameter of nine of them was measured and was ranging from 214 and 275 micron, with an average of 249 micron. We will attempt to produce smaller capsules with a narrower size distribution.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 14</h6>
| |
| − |
| |
| − | <p>Checked plates, they didn’t grow (only few colonies of edd promoter cells on some plates).<br>Inoculations of these colonies in LB for few hours and plating on LA for overnight.<br>Since we received very low amount of ordered DNA we decide to order primers to amplify it.<br>Design of primer sequence for KguE1, KguE2 and Kdd promoters.<br>Left yesterday’s plates for another day.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>July 14</h6>
| |
| − |
| |
| − | <p>Production of alginate capsules (second attempt). Parameters: 2 % alginate solution, needle inner diameter 130 micron, voltage 4000V, 15 ml/h, distance needle to gelling bath 1 cm.<br>Microscopy showed many capsules, the diameter of eight of them was measured and was ranging from 206 and 347 micron, with an average of 235 micron. We will attempt to produce smaller capsules with a narrower size distribution. <br>Production of alginate capsules (third attempt). Parameters: 2 % alginate solution, needle inner diameter 130 micron, voltage 4000V, 15 ml/h, distance needle to gelling bath 1 cm.<br>Only big capsules, around 400 micron in diameter. We will attempt to produce smaller capsules with a narrower size distribution. Find out if keeping all parameters constant really makes capsules with the same size properties, or if something else in the procedure affects size and shape.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 15</h6>
| |
| − |
| |
| − | <p>Yesterday’s plates grew, but very few like before.<br>2 days inoculated cells grew more.<br>No control plates did grow as expected.<br>ET12567 competent cells grew better than <i>E. coli</i> DH5α cells.<br>Inoculation of a single (doubt of taking only one) colony of edd1 and edd2 to LB + Kan medium.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-meas">
| |
| − | <div>
| |
| − | <h6>July 15</h6>
| |
| − |
| |
| − | <p>Changed the medium of the Devices.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-dry">
| |
| − | <div>
| |
| − | <h6>July 15</h6>
| |
| − |
| |
| − | <p>Written first version of the image analysis tool.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>July 15</h6>
| |
| − |
| |
| − | <p>Production of alginate capsules. Parameters: 2 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1 cm.<br>The capsules are bigger than expected with diameters such as 286, 283, 263 microns.<br>Inoculation of a <i>P. putida</i> pHH+GFP that was stored in the 4 <sup>0</sup>C fridge since last week, 1 % in LB + kan medium. Incubation at 30 <sup>0</sup>C for 3 hours and OD check. After 3 hours of incubation, OD = 0.308.<br>Encapsulation of <i>P. putida</i> pHH+GFP in alginate. Parameters: 2 % alginate solution, needle inner diameter 130 micron, voltage 4000V, 15 ml/h, distance needle to gelling bath 1 cm. Induction of the bacteria with m-Toluic acid.<br>Confocal microscopy, observation of encapsulated cells. Induction of GFP with m-Toluic acid did not work. The inducer should diffuse readily into the encapsulated cells through the alginate gel matrix, the lack of fluorescence is most likely due to using an old culture. Will attempt encapsulated cells from a fresh culture next time.<br>Inoculation of <i>P. putida</i> PHH+GFP from -80 <sup>0</sup>C in 3 ml LB + kan medium. Incubation at 30 <sup>0</sup>C.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 16</h6>
| |
| − |
| |
| − | <p>Overnight inoculated cultures grew.<br>OD measurement: 0.3 to 0.5.<br>Plated them again to obtain single colonies.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>July 16</h6>
| |
| − |
| |
| − | <p>1 % inoculation of overnight culture in fresh LB + kan. Incubation at 30 <sup>0</sup>C for 2h15. OD = 0.132.<br>Encapsulation of <i>P. putida</i> pHH+GFP in alginate. Parameters: 2 % alginate solution, needle inner diameter 130 micron, voltage 4000V, 15 ml/h, distance needle to gelling bath 1 cm. Induction of the bacteria with m-Toluic acid.<br>Confocal microscopy, observation of encapsulated cells. Induction of GFP with m-Toluic acid did work. Presence of capsule with the fluorescent bacteria. The size of the capsules was ranging from 238 to 286 microns with an average of 272 micron.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 17</h6>
| |
| − |
| |
| − | <p>Plates grew.<br>Single colony inoculation in LB + Kan.<br>Overnight incubation 37 <sup>0</sup>C.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>July 17</h6>
| |
| − |
| |
| − | <p>Confocal microscopy of the encapsulated bacteria of the previous batch that did not work. This time the bacteria were induced with 5 mM of m-toluic acid. Fluorescence was observed.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 18</h6>
| |
| − |
| |
| − | <p>Cells grew well in LB + Kan.<br>OD measurement: 0.2344.<br>2 % inoculation of cells. </p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 19</h6>
| |
| − |
| |
| − | <p>Gel results were not clear.<br>Cells grew well in LB + Kan.<br>OD measurement: 0.2344.<br>Miniprep of Edd1 and Edd2.<br>Gel run to see sizes.<br>1 % inoculation of cells with Edd.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>July 19</h6>
| |
| − |
| |
| − | <p>Inoculation of <i>P. putida</i> pHH+GFP from -80 <sup>0</sup>C in 3 ml LB + kan medium. Incubation at 30 <sup>0</sup>C.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="nb-week" id="week30entry">
| |
| − | <div class="twelve columns">
| |
| − | <h3>Week 30</h3>
| |
| − | <h6>(20/07 - 26/07)</h6>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 20</h6>
| |
| − |
| |
| − | <p>Gel run to see sizes – results were not clear.<br>Gel run again – bands clearly visible, but ladder was not clear.<br>OD measurement: Edd1 – 1.9858, Edd2 – 1.8782.<br>HIFI (1 μl Backbone DNA, 2 μl Insert DNA KguE1 and KguE2 promoter parts), once with each of the purified backbones.<br>Heat transformation of <i>E. coli</i> DH5α competent cells with KguE promoter (HIFI).<br>Plating cells with KguE promoter for overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>July 20</h6>
| |
| − |
| |
| − | <p>Preparation of gelling solution, washing solution + MQ water with NaCl for tomorrow’s alginate solution.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 21</h6>
| |
| − |
| |
| − | <p>Checked plates, they didn’t grow.<br>PCR for KguE promoter, because our new primers arrived.<br>Gel run.<br>Inoculation of wild type <i>P. putida</i> from -80 <sup>0</sup>C and overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>July 21</h6>
| |
| − |
| |
| − | <p>Preparation of alginate solution. Reduced the alginate concentration from 4.0% to 3.6% (1.8% in the finished encapsulation mixture).</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 22</h6>
| |
| − |
| |
| − | <p>Inoculation of wild type <i>P. putida</i> from overnight incubation.<br>Incubation for 2h.<br>OD measurement: 0.3 to 0.4.<br>Electrocompetent cells preparation.<br>Electroporation of <i>P. putida</i> with cells with Edd promoter.<br>Plating <i>P. putida</i> for overnight incubation.<br>Gel run to see sizes of Edd again – results were better this time, the band was between 3500 - 4000 bp.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 23</h6>
| |
| − |
| |
| − | <p>Checked the electroporation plates, they didn’t grow.<br>Electrocompetent cells preparation, again.<br>Electroporation of <i>P. putida</i> with cells with KguE promoter (Last time something was wrong with volt shock).<br>Plating <i>P. putida</i> for overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 24</h6>
| |
| − |
| |
| − | <p>Checked the electroporation plates, they didn’t grow.<br>PCR of KguE.<br>Gel run.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 25</h6>
| |
| − |
| |
| − | <p>Checked plates, they didn’t grow.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 26</h6>
| |
| − |
| |
| − | <p>Heat transformation of <i>P. putida</i> cells with Edd promoter.<br>Plating <i>P. putida</i> cells with Edd promoter for overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="nb-week" id="week31entry">
| |
| − | <div class="twelve columns">
| |
| − | <h3>Week 31</h3>
| |
| − | <h6>(27/07 - 02/08)</h6>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 27</h6>
| |
| − |
| |
| − | <p>Checked plates, they didn’t grow.<br>Inoculation of wild type <i>P. putida</i> from -80 <sup>0</sup>C and overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 28</h6>
| |
| − |
| |
| − | <p>Enzyme digest of KguE (EcoR1 and Pst1).<br>Backbone preparation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 29</h6>
| |
| − |
| |
| − | <p>PCR of KguE without primers.<br>Enzyme digestion.<br>Gel run.<br>Electrocompetent cells preparation.<br>Electroporation of <i>P. putida</i> with cells with Edd promoter.<br>Plating <i>P. putida</i> for overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 30</h6>
| |
| − |
| |
| − | <p>Checked electroporation plates, they grew.<br>Overnight single colony inoculation (<i>P. putida</i>).<br>Enzyme digestion of KguE (EcoR1 and Pst1) again with 4x concentration.<br>Preparation of new LA plates (+ Kan).<br>Gel run, results in no band.<br>Ligation of backbone and KguE insert.<br>Heat transformation of DH5α with KguE.<br>Plating cells with KguE promoter for overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>July 31</h6>
| |
| − |
| |
| − | <p>Checked plates, they didn’t grow.<br>Gel preparation (agarose).</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 01</h6>
| |
| − |
| |
| − | <p>Glucose preparation for medium.<br><i>P. putida</i> incubation with new glucose medium.<br>Plate reading of <i>P. putida</i> with and without glucose in medium.<br>Results showed no fluorescence (mCherry).<br>HIFI and heat transformation for KguE promoter with DH5α competent cells.<br>Plating cells with KguE promoter for overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 01</h6>
| |
| − |
| |
| − | <p>Prepared LA + kan plates for capsule leakage tests.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 02</h6>
| |
| − |
| |
| − | <p>Checked plates, they didn’t grow.<br>Inoculation of <i>P. putida</i> with Edd promoter in new medium.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 02</h6>
| |
| − |
| |
| − | <p>Inoculation of <i>P. putida</i> pHH+GFP in LB + kan.<br>Sterilisation of equipment and glassware for cell encapsulation by autoclavation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="nb-week" id="week32entry">
| |
| − | <div class="twelve columns">
| |
| − | <h3>Week 32</h3>
| |
| − | <h6>(03/08 - 09/08)</h6>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 03</h6>
| |
| − |
| |
| − | <p>PCR of KguE with different annealing temperature.<br>Enzyme digest of KguE.<br>Miniprep of DH5α Edd and KguE.<br>Gel run.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 03</h6>
| |
| − |
| |
| − | <p>5 % Inoculation of overnight culture in LB + kan.<br>Encapsulation of <i>P. putida</i> pHH+GFP in alginate. Parameters: 1.8 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1 cm. <br>Capsule leakage tests (encapsulated <i>P. putida</i> pHH+GFP stored in LB).</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 04</h6>
| |
| − |
| |
| − | <p>PCR of edd promoter.<br>Gel run.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 04</h6>
| |
| − |
| |
| − | <p>Checked plates from leakage tests, substantial leakage observed.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 05</h6>
| |
| − |
| |
| − | <p>Confocal microscopy after 1 day incubation of capsules.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="nb-week" id="week33entry">
| |
| − | <div class="twelve columns">
| |
| − | <h3>Week 33</h3>
| |
| − | <h6>(10/08 - 16/08)</h6>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 10</h6>
| |
| − |
| |
| − | <p>Inoculation of pHH+mCherry DH5α cells to new medium + Kan.<br>Miniprep.<br>Gel run.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 10</h6>
| |
| − |
| |
| − | <p>Prepared LA + kan plates.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 11</h6>
| |
| − |
| |
| − | <p>Inoculation of pHH+mCherry DH5α cells to new medium + Kan.<br>Ordered new primers.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 11</h6>
| |
| − |
| |
| − | <p>Inoculation of <i>P. putida</i> PHH+GFP in LB + kan.<br>Sterilisation of equipment and glassware for cell encapsulation by autoclavation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 12</h6>
| |
| − |
| |
| − | <p>Cell encapsulation in alginate + poly-L-lysine HBr coating. Parameters: 1.8 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1 cm. <br>Capsule leakage tests (encapsulated <i>P. putida</i> pHH+GFP with poly-L-lysine HBr coating stored in LB).</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 13</h6>
| |
| − |
| |
| − | <p>Looked at BioBrick protocol and preparations.<br>New primers arrived.<br>PCR of all promoters: Edd, KguE, Zwf and Gad.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 13</h6>
| |
| − |
| |
| − | <p>Checked plates from leakage tests, substantial leakage observed. No improvement with poly-L-lysine HBr coating.<br>Analysis of results.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 14</h6>
| |
| − |
| |
| − | <p>Measurements of PCR products and miniprep (backbone plasmid) concentrations on Nanodrop.<br>Digestion of backbone with Nde1 and Bste2 enzymes.<br>Digestion of PCR products (promoters).<br>PCR purification of all promoters (from PCR) and backbone.<br>Gel run to check sizes – results showed multiple bands (we needed only 2 bands).<br>Gel cut of highest 3 bands (ladder on gel was not so clear).<br>Gel cut DNA extraction.<br>Backbone plasmid separately digested with Nde1 and Bste2 enzymes.<br>Gel run again with all digested products and 2nd digestions.<br>Gel results showed that the Bste2 enzyme was cutting in many places.<br>Ligation of three gel cut DNA and one insert (Gad promoter).<br>Heat transformation of Dh5α competent cells with Gad promoter.<br>Plated cells on LA with Gad promoter for overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 14</h6>
| |
| − |
| |
| − | <p>Prepared LA + kan plates for capsule leakage tests.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 15</h6>
| |
| − |
| |
| − | <p>Checked plates, they didn’t grow.<br>Inoculation of new pHH+mCherry Dh5α cells to medium + Kan from -80 <sup>0</sup>C.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 16</h6>
| |
| − |
| |
| − | <p>Miniprep of overnight inoculation pHH+mCherry Dh5α cells (backbone plasmid).<br>Measurements of miniprep (backbone plasmid) concentrations on Nanodrop.<br>Digestion of backbone with Nde1 and Bste2 enzymes (backbone preparation).<br>Gel run with digested plasmid backbone to see size.<br>Gel results showed Bste2 was cutting in many places again but this time less.<br>Ligation of plasmid backbone and one insert (Zwf promoter).<br>Heat transformation of Dh5α competent cells with Zwf promoter.<br>Plating cells on LA with Zwf promoter for overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="nb-week" id="week34entry">
| |
| − | <div class="twelve columns">
| |
| − | <h3>Week 34</h3>
| |
| − | <h6>(17/08 - 23/08)</h6>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 17</h6>
| |
| − |
| |
| − | <p>Checked plates, they grew.<br>Single colony inoculation.<br>Digestion of all promoters (PCR products) with Nde1 and Bste2 enzymes (insert preparation).<br>PCR purification of digested products.<br>Ligation of backbone and other promoters.<br>Heat transformation of Dh5α competent cells with other promoter.<br>Plating cells on LA with other promoter for overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 17</h6>
| |
| − |
| |
| − | <p>Inoculation of <i>P. putida</i> pHH+GFP in LB + kan.<br>Sterilisation of equipment and glassware for cell encapsulation by autoclavation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 18</h6>
| |
| − |
| |
| − | <p>Checked plates, they grew (Edd, Kgu and Zwf).<br>Single colony inoculation (5 colonies from each plate).<br>Miniprep of overnight single inoculation (Zwf promoter plasmid).<br>Measurements of miniprep (Zwf promoter plasmid) concentrations on Nanodrop.<br>PCR it with our primers to check presence of promoter sequence.<br>Gel run to see PCR product (confirmation).<br>Gel results confirmed.<br>Electrocompetent cells preparation (Wild type <i>P. putida</i> with no plasmid).<br>Electroporation of WT <i>P. putida</i>.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 18</h6>
| |
| − |
| |
| − | <p>Cell encapsulation in Alginate + poly-L-lysine HCl coating. Parameters: 1.8 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1 cm. <br>Capsule leakage tests (encapsulated <i>P. putida</i> pHH+GFP + poly-L-lysine HCl coating stored in LB).</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 19</h6>
| |
| − |
| |
| − | <p>Checked plates: <i>P. putida</i> didn’t grow, electroporation didn’t work.<br>Miniprep of overnight single inoculation (Kgu and Edd).<br>Measurements of miniprep (Kgu and Edd) concentrations on Nanodrop.<br>PCR it with our primers to check presence of promoter sequence.<br>Gel run to see PCR product (confirmation).</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 19</h6>
| |
| − |
| |
| − | <p>Checked plates from capsule leakage tests, substantial leakage observed. No improvement with poly-L-lysine HCl coating.<br>Analysis of results.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 22</h6>
| |
| − |
| |
| − | <p>Blunt ligation of Gad overnight.<br>Lig1 - PshA1, Lig2 - Pml1 and Lig3 - HPA1.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 23</h6>
| |
| − |
| |
| − | <p>Heat transformation of Dh5α competent cells with overnight ligated plasmids.<br>Plating cells on LA for overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="nb-week" id="week35entry">
| |
| − | <div class="twelve columns">
| |
| − | <h3>Week 35</h3>
| |
| − | <h6>(24/08 - 30/08)</h6>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 24</h6>
| |
| − |
| |
| − | <p><i>P. putida</i> with Kgu and Zwf promoters are ready.<br>Checked plates, they grew (Gad promoter).<br>Single colony inoculation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 24</h6>
| |
| − |
| |
| − | <p>Sterilisation of equipment and glassware for cell encapsulation by autoclavation.<br>Inoculation of <i>P. putida</i> pHH+GFP in LB + kan.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 25</h6>
| |
| − |
| |
| − | <p>Miniprep of overnight single inoculation (Gad).<br>Measurements of miniprep (Gad) concentrations on Nanodrop.<br>PCR it with our primers to check presence of promoter sequence.<br>Gel run to see PCR product (confirmation). Result of gel: was not confirmed.<br>Glucose medium preparation.<br>Incubation of <i>P. putida</i> with Kgu and Zwf promoters with glucose medium.<br>Digestion of Edd and Gad PCR products with PshA1, Pml1 and HPA1.<br>Gel run to see digestion result.<br>Gel extraction.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 25</h6>
| |
| − |
| |
| − | <p>5 % inoculation of overnight culture in LB + kan + m-toluic acid (inducer).<br>Cell encapsulation in Alginate. Parameters: 1.8 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1 cm. <br>Capsule leakage tests (encapsulated <i>P. putida</i> pHH+GFP in PBS).</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 26</h6>
| |
| − |
| |
| − | <p>OD measurement of overnight incubated <i>P. putida</i>.<br>Plate reader (Spectrophotometry) of <i>P. putida</i> with Kgu and Zwf promoters.<br>Enzyme digestion.<br>Blunt ligation overnight.<br>Analysis of results.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-meas">
| |
| − | <div>
| |
| − | <h6>August 26</h6>
| |
| − |
| |
| − | <p>Overnight inoculation of samples 1A, 1B, 1C, 2A, 2B, 2C, 3A, 3B and 3C.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 26</h6>
| |
| − |
| |
| − | <p>Checked plates from leakage tests, much better results in PBS compared to LB.<br>Analysis of results.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 27</h6>
| |
| − |
| |
| − | <p>Heat transformation of DH5α competent cells.<br>Plating cells on LA for overnight incubation.<br>Inoculation of <i>P. putida</i> in glucose medium.<br>2 hour incubation.<br>OD measurement of overnight incubated <i>P. putida</i>.<br>Plate reader (Spectrophotometry) of <i>P. putida</i> with Kgu and Zwf promoters.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-meas">
| |
| − | <div>
| |
| − | <h6>August 27</h6>
| |
| − |
| |
| − | <p>Acquisition of images by confocal microscopy, devices 1A, 1B, 1C, 2A, 2B, 2C, 3A, 3B and 3C (each in technical triplicates).</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 28</h6>
| |
| − |
| |
| − | <p>Checked plates, they didn’t grow.<br>PCR amplification of all four promoters.<br>Inoculation of <i>P. putida</i> in different glucose media.<br>OD measurement of overnight incubated <i>P. putida</i>.<br>2 hour incubation.<br>Plate reader (Spectrophotometry) of <i>P. putida</i> with Kgu and Zwf promoters.<br>PCR of miniprep DH5α Kgu and Zwf plasmids with Prefix/Suffix primers.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 28</h6>
| |
| − |
| |
| − | <p>Prepared LA + kan + 2mM m-Toluic acid plates for leakage tests.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-meas">
| |
| − | <div>
| |
| − | <h6>August 28</h6>
| |
| − |
| |
| − | <p>Acquisition of images by confocal microscopy, control samples NCA, NCB, NCC, PCA, PCB and PCC (each in technical triplicates).<br>Analysis of images using quantitative image analysis based on ImageJ and MATLAB. </p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 29</h6>
| |
| − |
| |
| − | <p>Gad digestion with Bste2 and Nde1 enzymes.<br>PCR amplification.<br>Nanodrop measurement of concentration.<br>Sticky end ligation.<br>Gel run for confirmation.<br>Heat transformation of DH5α competent cells.<br>Plating cells on LA for overnight incubation.<br>OD measurement of overnight incubated <i>P. putida</i>.<br>Plate reader (Spectrophotometry) of <i>P. putida</i> with Kgu and Zwf promoters.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 30</h6>
| |
| − |
| |
| − | <p>Checked plates, they didn’t grow.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-dry">
| |
| − | <div>
| |
| − | <h6>August 30</h6>
| |
| − |
| |
| − | <p></p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="nb-week" id="week36entry">
| |
| − | <div class="twelve columns">
| |
| − | <h3>Week 36</h3>
| |
| − | <h6>(31/08 - 06/09)</h6>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>August 31</h6>
| |
| − |
| |
| − | <p>PCR of stock DNA (sent by company) with new Prefix/Suffix primers.<br>Digestion of backbone and insert with Pml1 and PshA1.<br>Gel run to check PCR products and digestion.<br>Measurement of PCR products and miniprep (GFP+pHH and mCherry+pHH) concentration on Nanodrop.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>August 31</h6>
| |
| − |
| |
| − | <p>Sterilisation of equipment and glassware for cell encapsulation by autoclavation.<br>Inoculation of <i>P. putida</i> pHH+GFP in LB + kan.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 01</h6>
| |
| − |
| |
| − | <p>Blunt ligation for Kgu and Zwf.<br>Heat transformation of DH5α competent cells.<br>Plating cells on LA for overnight incubation.<br>Enzyme digestion of new plasmid backbone and all four promoters.<br>Blunt ligation overnight.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>September 01</h6>
| |
| − |
| |
| − | <p>5 % inoculation of overnight culture in LB + kan + 2mM m-toluic acid (inducer).<br>Cell encapsulation in Alginate. Parameters: 1.8 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1 cm. <br>Capsule leakage tests (encapsulated <i>P. putida</i> pHH+GFP in PBS).</p>
| |
| − | </div>
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| − | </div>
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| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 02</h6>
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| − |
| |
| − | <p>Checked plates, they didn’t grow.<br>Heat transformation of DH5α competent cells with overnight ligation.<br>Plating cells on LA for overnight incubation.<br>New construction of two promoters on one plasmid.<br>Enzyme digestion with Xba1 and Spe1 of Zwf promoter plasmid as backbone and Kgu promoter as insert.<br>Blunt ligation overnight incubation.<br>M9 medium preparation.</p>
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| − | </div>
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| − | </div>
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| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>September 02</h6>
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| − |
| |
| − | <p>Checked plates from leakage tests, similar results to last time.<br>Analysis of results.</p>
| |
| − | </div>
| |
| − | </div>
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| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 03</h6>
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| − |
| |
| − | <p>Checked plates, they grew.<br>Single colony inoculation (5 colonies).<br>Inoculation of Kgu and Zwf <i>P. putida</i> in M9 medium.<br>Incubation for 2 hours.<br>Plate reader measurements.<br>Flow cytometry.</p>
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| − | </div>
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| − | </div>
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| − |
| |
| − | <div class="entry nb-dry">
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| − | <div>
| |
| − | <h6>September 04</h6>
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| − |
| |
| − | <p>Proposed information theoretical modelling to Team Warwick for predicting the probability of bonding of Brixells.</p>
| |
| − | </div>
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| − | </div>
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| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 04</h6>
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| − |
| |
| − | <p>Miniprep of all single colony inoculations.<br>PCR with prefix/suffix primers.<br>Gel run to confirm presence: confirmed.<br>Electrocompetent cells preparation <i>P. putida</i>.<br>Electroporation of wild type <i>P. putida</i>.<br>Plating <i>P. putida</i> cells on LA plates.</p>
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| − | </div>
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| − | </div>
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| − |
| |
| − | <div class="entry nb-wet">
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| − | <div>
| |
| − | <h6>September 05</h6>
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| − |
| |
| − | <p>Check on plates: they all grew!<br>Single colony inoculation.</p>
| |
| − | </div>
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| − | </div>
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| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>September 05</h6>
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| − |
| |
| − | <p>Preparation LA + kan + m-toluic acid plates for leakage tests.</p>
| |
| − | </div>
| |
| − | </div>
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| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 06</h6>
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| − |
| |
| − | <p>Standard assembly for new constructs Kgu as backbone and Zwf as insert.<br>Digestion with EcoR1, Spe1 and Xba1 enzymes.<br>Run on gel to confirm digestion: confirmed.<br>Gel extraction.<br>Ligation.<br>Heat transformation of DH5α competent cells.<br>Plating cells on LA for overnight incubation.</p>
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| − | </div>
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| − | </div>
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| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>September 06</h6>
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| − |
| |
| − | <p>Sterilisation of equipment and glassware for cell encapsulation by autoclavation.<br>Inoculation of <i>P. putida</i> pHH+GFP in LB + kan.</p>
| |
| − | </div>
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| − | </div>
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| − |
| |
| − | </div>
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| − | </div>
| |
| − |
| |
| − | <div class="nb-week" id="week37entry">
| |
| − | <div class="twelve columns">
| |
| − | <h3>Week 37</h3>
| |
| − | <h6>(07/09 - 13/09)</h6>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 07</h6>
| |
| − |
| |
| − | <p>Checked plates, they didn’t grow.<br>Miniprep plasmids preparation for sequencing.<br>Inoculation of <i>P. putida</i> with all promoters in different media.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>September 07</h6>
| |
| − |
| |
| − | <p>Cell encapsulation in Alginate. Parameters: 1.8 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1.5 cm. <br>Capsule leakage tests (encapsulated <i>P. putida</i> pHH+GFP in PBS).</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 08</h6>
| |
| − |
| |
| − | <p>Miniprep preparation for sequencing.<br>Check on wild type <i>P. putida</i> and new <i>P. putida</i> with pHH-100 mCherry plasmid.<br>Inoculation of wild type for control.<br>Plate reader of overnight incubation in LB and LB + Glucose medium.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>September 08</h6>
| |
| − |
| |
| − | <p>Checked plates from leakage tests, results were similar to first and second experiments in PBS.<br>Analysis of results.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 09</h6>
| |
| − |
| |
| − | <p>Controls of wild type and <i>P. putida</i> with pHH-100 mCherry plasmid were confirmed.<br>Inoculation of <i>P. putida</i> in LB, LB + glucose and different concentrations of M9 medium.<br>Overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 10</h6>
| |
| − |
| |
| − | <p>Plate reader of overnight samples.<br>Inoculation of <i>P. putida</i> in LB and different concentrations of M9 medium.<br>Overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 11</h6>
| |
| − |
| |
| − | <p>Plate reader of overnight inoculations.<br>Inoculation of <i>P. putida</i> in LB and different concentrations of M9 medium<br>Overnight incubation.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 12</h6>
| |
| − |
| |
| − | <p>Plate reader of of overnight inoculations.<br>Inoculation of <i>P. putida</i> in LB and different concentrations of M9 medium.<br>Overnight incubation.<br>RNA isolation of <i>P. putida</i> with our promoters for rtPCR.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 13</h6>
| |
| − |
| |
| − | <p>Plate reader of Edd, Kgu, Zwf, Gad samples (3 biological and 3 technical replicates each) of overnight inoculations in M9 + 5%, 10%, 20%.<br>Inoculation of <i>P. putida</i> in LB in different concentrations of M9 medium.<br>Incubation of 3 biological replicates of Edd, Kgu, Zwf, Gad in LB.<br>Measured RNA concentration.<br>DNA free kit procedure.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="nb-week" id="week38entry">
| |
| − | <div class="twelve columns">
| |
| − | <h3>Week 38</h3>
| |
| − | <h6>(14/09 - 20/09)</h6>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 14</h6>
| |
| − |
| |
| − | <p>Digest and Ligation of biobrick parts (BBa_K184005, BBa_K1840006, BBa_K1840007, BBa_K1840008) and pSB1C3<br>Heat transformation to competent E.coli</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-dry">
| |
| − | <div>
| |
| − | <h6>September 14</h6>
| |
| − |
| |
| − | <p>Deployed new iGEM Matchmaker version with keyword analysis and machine learning.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 15</h6>
| |
| − |
| |
| − | <p>Plate reader of Edd, Kgu, Zwf and Gad samples in different M9 media incubated for 12 hours (3 biological and 3 technical replicates each) in LB with 0%, 5%, 10%, or 20%<br>Flow cytometry of pHH+mCherry as positive control<br>Inoculation of one colony from each transformation in LB+Chloramphenicol</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-dry">
| |
| − | <div>
| |
| − | <h6>September 16</h6>
| |
| − |
| |
| − | <p>Proposed and simulated Brixells based on Poisson-Boltzmann theory.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 16</h6>
| |
| − |
| |
| − | <p>Inoculation of <i>P. putida</i> pHH + mCherry.<br>Inoculation of biological replicate A of Edd, Gad, Zwf + Kgu samples in M9 medium + glucose (1, 5, 10 + 20 %).<br>Miniprep of inoculated samples for biobrick part sending, digest with EcoR1 and Pst1, gelelectrophoresis shows only one band<br>Digest and Ligation of biobrick parts (BBa_K184005, BBa_K1840006, BBa_K1840007, BBa_K1840008) and pSB1C3<br>Heat transformation to competent E.coli</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 17</h6>
| |
| − |
| |
| − | <p>Plate reader measurement for 6 hours after 6 hours inoculation, and additionally after 12 hours inoculation of Edd, Kgu, Zwf and Gad samples in M9 media with 1%, 5%, 10%, and 20% glucose<br>Flow cytometry of Edd, Kgu, Zwf and Gad samples in M9 media with 1%, 5%, 10%, and 20% glucose<br>Acquisition of images of promoters Edd, Gad, Zwf + Kgu in M9 medium + glucose (1, 5, 10 + 20 %) by confocal microscopy.<br>Colony PCR with prefix/suffix primers to confirm the right contruct for biobrick part sending</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-anim">
| |
| − | <div>
| |
| − | <h6>September 17</h6>
| |
| − |
| |
| − | <p>Encapsulation of <i>P. putida</i> pHH+mCherry in alginate. Parameters: 1.8 % alginate solution, needle inner diameter 130 micron, voltage 5000V, 6 ml/h, distance needle to gelling bath 1.5 cm. <br>Acquisition of z-stack images by confocal microscopy.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-wet">
| |
| − | <div>
| |
| − | <h6>September 18</h6>
| |
| − |
| |
| − | <p>Miniprep of transformed cells with our Biobrick parts in the pSB1C3 backbone, preparation for sending</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-hprac">
| |
| − | <div>
| |
| − | <h6>September 18</h6>
| |
| − |
| |
| − | <p>Analysis of survey responses.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-dry">
| |
| − | <div>
| |
| − | <h6>September 18</h6>
| |
| − |
| |
| − | <p>3D Rendering of z-stack images obtained by confocal microscopy.</p>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="entry nb-dry">
| |
| − | <div>
| |
| − | <h6>September 18</h6>
| |
| − |
| |
| − | <p>Quantitative analysis of confocal microscopy images of <i>P. putida</i> pHH+mCherry.</p>
| |
| − | </div>
| |
| − | </div>
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| − | </div>
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| − | </div>
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| − | <p><!--NAVIGATION
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