Difference between revisions of "Team:NTNU Trondheim/Notebook"

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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>
+
−
            <h6>(22/06 - 28/06)</h6>
+
  
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            <div class="entry nb-anim">
+
    <div class="nb-week" id="week23entry">   
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                <div>
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        <div class="twelve columns">
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                    <h6>June 3</h6>
+
            <h3>Week 23</h3>
 +
            <h6>(02/02 - 08/02)</h6>
 +
 
 +
            <div class="entry nb-dry">
 +
                <div>
 +
                    <h6>February 1</h6>
 
 
−
Introduction to alginate encapsulation + demo on how to operate the electrostatic capsule generator
+
The iGEM Matchmaker (V2.0) is deployed for 2015.
−
                </div>
+
                </div>
−
            </div>
+
            </div>
 +
 
 +
        </div>
 +
    </div>
 +
 
 +
    <div class="nb-week" id="week24entry">   
 +
        <div class="twelve columns">
 +
            <h3>Week 24</h3>
 +
            <h6>(08/06 - 14/06)</h6>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>June 4</h6>
 +
 +
Discussed <i>Escherichia coli</i> glucose uptake and possible promoters in E. coli.
 +
                </div>
 +
            </div>
 +
 
 +
        </div>
 +
    </div>
 +
 
 +
    <div class="nb-week" id="week25entry">   
 +
        <div class="twelve columns">
 +
            <h3>Week 25</h3>
 +
            <h6>(15/06 - 21/06)</h6>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>June 1</h6>
 +
 +
Discussed <i>Pseudomonas putida</i> as a candidate microorganism.<br>Discussion of a system for glucose uptake in <i>P. putida</i>.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>June 1</h6>
 +
 +
Design of promoter regions for <i>P. putida</i>.<br>Ordered promoter regions for <i>P. putida</i>.
 +
                </div>
 +
            </div>
 +
 
 +
        </div>
 +
    </div>
 +
 
 +
    <div class="nb-week" id="week26entry">   
 +
        <div class="twelve columns">
 +
            <h3>Week 26</h3>
 +
            <h6>(22/06 - 28/06)</h6>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>June 3</h6>
 +
 +
Introduction to alginate encapsulation + demo on how to operate the electrostatic capsule generator.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>June 3</h6>
 +
 +
Playing around with the electrostatic bead generator, making capsules of different sizes by varying the different parameters of the instrument.
 +
                </div>
 +
            </div>
 +
 
 +
        </div>
 +
    </div>
 +
 
 +
    <div class="nb-week" id="week27entry">   
 +
        <div class="twelve columns">
 +
            <h3>Week 27</h3>
 +
            <h6>(29/06 - 05/07)</h6>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>June 1</h6>
 +
 +
Moved the cell encapsulation equipment into our lab, so it was ready for the GM bacteria.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>June 1</h6>
 +
 +
Preparation of alginate solution, gelling solution and washing solution.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>June 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>June 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-dry">
 +
                <div>
 +
                    <h6>June 1</h6>
 +
 +
Created the website design using Bootstrap, and set up the Notebook and Protocols pages.
 +
                </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-anim">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Preparation of alginate solution, and put this in the 4 <sup>0</sup>C fridge for future use.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Checked plates, they did not grow.<br>Inoculation of <i>P. putida</i>.
 +
                </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 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-dry">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Written first version of the image analysis tool.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Overnight inoculated cultures grew.<br>OD measurement: 0.3 to 0.5.<br>Plated them again to obtain single colonies.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Plates grew.<br>Single colony inoculation in LB + Kan.<br>Overnight incubation 37 <sup>0</sup>C.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Cells grew well in LB + Kan.<br>OD measurement: 0.2344.<br>2 % inoculation of cells.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </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 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Preparation of gelling solution, washing solution + MQ water with NaCl for tomorrow’s alginate solution.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Preparation of alginate solution. Reduced the alginate concentration from 4.0% to 3.6% (1.8% in the finished encapsulation mixture).
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Checked the electroporation plates, they didn’t grow.<br>PCR of KguE.<br>Gel run.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Checked plates, they didn’t grow.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Heat transformation of <i>P. putida</i> cells with Edd promoter.<br>Plating <i>P. putida</i> cells with Edd promoter for overnight incubation.
 +
                </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 1</h6>
 +
 +
Checked plates, they didn’t grow.<br>Inoculation of wild type <i>P. putida</i> from -80 <sup>0</sup>C and overnight incubation.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Enzyme digest of KguE (EcoR1 and Pst1).<br>Backbone preparation.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Checked plates, they didn’t grow.<br>Gel preparation (agarose).
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Prepared LA + kan plates for capsule leakage tests.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Checked plates, they didn’t grow.<br>Inoculation of <i>P. putida</i> with Edd promoter in new medium.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>July 1</h6>
 +
 +
Inoculation of <i>P. putida</i> pHH+GFP in LB + kan.<br>Sterilisation of equipment and glassware for cell encapsulation by autoclavation.
 +
                </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 1</h6>
 +
 +
PCR of KguE with different annealing temperature.<br>Enzyme digest of KguE.<br>Miniprep of DH5α Edd and KguE.<br>Gel run.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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).
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
PCR of edd promoter.<br>Gel run.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Checked plates from leakage tests, substantial leakage observed.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Confocal microscopy after 1 day incubation of capsules.
 +
                </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 1</h6>
 +
 +
Inoculation of pHH+mCherry DH5α cells to new medium + Kan.<br>Miniprep.<br>Gel run.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Prepared LA + kan plates.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Inoculation of pHH+mCherry DH5α cells to new medium + Kan.<br>Ordered new primers.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Inoculation of <i>P. putida</i> PHH+GFP in LB + kan.<br>Sterilisation of equipment and glassware for cell encapsulation by autoclavation.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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).
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Looked at BioBrick protocol and preparations.<br>New primers arrived.<br>PCR of all promoters: Edd, KguE, Zwf and Gad.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Checked plates from leakage tests, substantial leakage observed. No improvement with poly-L-lysine HBr coating.<br>Analysis of results.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Prepared LA + kan plates for capsule leakage tests.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Checked plates, they didn’t grow.<br>Inoculation of new pHH+mCherry Dh5α cells to medium + Kan from -80 <sup>0</sup>C.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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.
 +
                </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 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Inoculation of <i>P. putida</i> pHH+GFP in LB + kan.<br>Sterilisation of equipment and glassware for cell encapsulation by autoclavation.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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>.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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).
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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).
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Checked plates from capsule leakage tests, substantial leakage observed. No improvement with poly-L-lysine HCl coating.<br>Analysis of results.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Blunt ligation of Gad overnight.<br>Lig1 - PshA1, Lig2 - Pml1 and Lig3 - HPA1.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Heat transformation of Dh5α competent cells with overnight ligated plasmids.<br>Plating cells on LA for overnight incubation.
 +
                </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 1</h6>
 +
 +
<i>P. putida</i> with Kgu and Zwf promoters are ready.<br>Checked plates, they grew (Gad promoter).<br>Single colony inoculation.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.<br>Inoculation of <i>P. putida</i> pHH+GFP in LB + kan.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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).
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Checked plates from leakage tests, much better results in PBS compared to LB.<br>Analysis of results.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Prepared LA + kan + 2mM m-Toluic acid plates for leakage tests.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Checked plates, they didn’t grow.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-dry">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
 +
                </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 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.<br>Inoculation of <i>P. putida</i> pHH+GFP in LB + kan.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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).
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Checked plates from leakage tests, similar results to last time.<br>Analysis of results.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-dry">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Proposed information theoretical modelling to Team Warwick for predicting the probability of bonding of Brixells.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Check on plates: they all grew!<br>Single colony inoculation.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Preparation LA + kan + m-toluic acid plates for leakage tests.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>August 1</h6>
 +
 +
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.<br>Inoculation of <i>P. putida</i> pHH+GFP in LB + kan.
 +
                </div>
 +
            </div>
 +
 
 +
        </div>
 +
    </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 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
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).
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
Checked plates from leakage tests, results were similar to first and second experiments in PBS.<br>Analysis of results.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
Plate reader of overnight samples.<br>Inoculation of <i>P. putida</i> in LB and different concentrations of M9 medium.<br>Overnight incubation.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
Plate reader of overnight inoculations.<br>Inoculation of <i>P. putida</i> in LB and different concentrations of M9 medium<br>Overnight incubation.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
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.
 +
                </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 1</h6>
 +
 +
Digest and Ligation of biobrick parts (BBa_K184005, BBa_K1840006, BBa_K1840007, BBa_K1840008) and pSB1C3<br>Heat transformation to competent E.coli
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-dry">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
Deployed new iGEM Matchmaker version with keyword analysis and machine learning.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
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
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-dry">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
Proposed and simulated Brixells based on Poisson-Boltzmann theory.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
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
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
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
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-anim">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
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.
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-wet">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
Miniprep of transformed cells with our Biobrick parts in the pSB1C3 backbone, preparation for sending
 +
                </div>
 +
            </div>
 +
 
 +
            <div class="entry nb-dry">
 +
                <div>
 +
                    <h6>September 1</h6>
 +
 +
3D Rendering of z-stack images obtained by confocal microscopy.
 +
                </div>
 +
            </div>
 +
 
 +
        </div>
 +
    </div>
 +
 
 +
    <div class="nb-week" id="week39entry">   
 +
        <div class="twelve columns">
 +
            <h3>Week 39</h3>
 +
            <h6>(14/09 - 20/09)</h6>
  
−
            <div class="entry nb-anim">
+
            <div class="entry nb-dry">
−
                <div>
+
                <div>
−
                    <h6>June 3</h6>
+
                    <h6>September 5</h6>
 
 
−
Playing around with the electrostatic bead generator, making capsules of different sizes by varying the different parameters of the instrument.
+
Quantitative analysis of confocal microscopy images of <i>P. putida</i> pHH+mCherry.
−
                </div>
+
                </div>
−
            </div>
+
            </div>
  
−
        </div>
+
        </div>
−
    </div>
+
    </div>
 +
 +
 +
 +
 
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             <p><!--NAVIGATION
 
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             <div class="nav" onclick="weekFilter(this)">

Revision as of 21:49, 18 September 2015

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Week 23

(02/02 - 08/02)
February 1
The iGEM Matchmaker (V2.0) is deployed for 2015.

Week 24

(08/06 - 14/06)
June 4
Discussed Escherichia coli glucose uptake and possible promoters in E. coli.

Week 25

(15/06 - 21/06)
June 1
Discussed Pseudomonas putida as a candidate microorganism.
Discussion of a system for glucose uptake in P. putida.
June 1
Design of promoter regions for P. putida.
Ordered promoter regions for P. putida.

Week 26

(22/06 - 28/06)
June 3
Introduction to alginate encapsulation + demo on how to operate the electrostatic capsule generator.
June 3
Playing around with the electrostatic bead generator, making capsules of different sizes by varying the different parameters of the instrument.

Week 27

(29/06 - 05/07)
June 1
Moved the cell encapsulation equipment into our lab, so it was ready for the GM bacteria.
June 1
Preparation of alginate solution, gelling solution and washing solution.
June 1
Inoculation of Pseudomonas putida pHH+GFP from -80 0C in 3 ml LB + Kan medium. Incubation at 30 0C.
June 1
5 % inoculation of overnight culture in fresh LB + Kan.
Incubation at 30 0C for two hours. Induction of P. putida pHH+GFP with m-Toluic acid.
June 1
Created the website design using Bootstrap, and set up the Notebook and Protocols pages.

Week 28

(06/07 - 12/07)
July 1
Inoculation of Pseudomonas putida pHH+GFP from -80 0C in 3 ml LB + kan medium. Incubation at 30 0C.
July 1
Preparation of LB + kan medium.
1 % inoculation of overnight culture in fresh LB + kan. Incubation at 30 0C for 6 hours. Measurement of the OD every 2h and observation of the bacteria number to the microscopy.
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.
July 1
Preparation of alginate solution, and put this in the 4 0C fridge for future use.
July 1
Prepared TE buffer.
Added TE buffer to DNA.
HIFI (0.5 μl backbone DNA, 1 μl insert DNA Edd promoter), once with each of the purified backbones.
July 1
Heat transformation of E. coliDH5α competent cells with Edd promoter (HIFI).
Electroporation of P. putida with cells with Edd promoter.
Plating P. putida for overnight incubation.
July 1
Checked plates, they did not grow.
Inoculation of P. putida.

Week 29

(13/07 - 19/07)
July 1
HIFI (0.5 μl backbone DNA, 1 μl insert DNA KguE1 and KguE2 promoter parts), once with each of the purified backbones.
Heat transformation of E. coliDH5α competent cell with KguE promoter (HIFI).
HIFI (0.5 μl backbone DNA, 1 μl Insert DNA edd promoter), once with each of the purified backbones, again.
Heat transformation of E. coliDH5α competent cell with Edd promoter (HIFI).
Preparation of LA plates (+ Kan).
Plating E. coliDH5α and ET12567 competent cells for overnight incubation.
July 1
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.
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.
July 1
Checked plates, they didn’t grow (only few colonies of edd promoter cells on some plates).
Inoculations of these colonies in LB for few hours and plating on LA for overnight.
Since we received very low amount of ordered DNA we decide to order primers to amplify it.
Design of primer sequence for KguE1, KguE2 and Kdd promoters.
Left yesterday’s plates for another day.
July 1
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.
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.
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.
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.
July 1
Yesterday’s plates grew, but very few like before.
2 days inoculated cells grew more.
No control plates did grow as expected.
ET12567 competent cells grew better than E. coliDH5α cells.
Inoculation of a single (doubt of taking only one) colony of edd1 and edd2 to LB + Kan medium.
July 1
Written first version of the image analysis tool.
July 1
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.
The capsules are bigger than expected with diameters such as 286, 283, 263 microns.
Inoculation of a P. putida pHH+GFP that was stored in the 4 0C fridge since last week, 1 % in LB + kan medium. Incubation at 30 0C for 3 hours and OD check. After 3 hours of incubation, OD = 0.308.
Encapsulation of P. putida 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.
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.
Inoculation of P. putida PHH+GFP from -80 0C in 3 ml LB + kan medium. Incubation at 30 0C.
July 1
Overnight inoculated cultures grew.
OD measurement: 0.3 to 0.5.
Plated them again to obtain single colonies.
July 1
1 % inoculation of overnight culture in fresh LB + kan. Incubation at 30 0C for 2h15. OD = 0.132.
Encapsulation of P. putida 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.
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.
July 1
Plates grew.
Single colony inoculation in LB + Kan.
Overnight incubation 37 0C.
July 1
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.
July 1
Cells grew well in LB + Kan.
OD measurement: 0.2344.
2 % inoculation of cells.
July 1
Gel results were not clear.
Cells grew well in LB + Kan.
OD measurement: 0.2344.
Miniprep of Edd1 and Edd2.
Gel run to see sizes.
1 % inoculation of cells with Edd.
July 1
Inoculation of P. putida pHH+GFP from -80 0C in 3 ml LB + kan medium. Incubation at 30 0C.

Week 30

(20/07 - 26/07)
July 1
Gel run to see sizes – results were not clear.
Gel run again – bands clearly visible, but ladder was not clear.
OD measurement: Edd1 – 1.9858, Edd2 – 1.8782.
HIFI (1 μl Backbone DNA, 2 μl Insert DNA KguE1 and KguE2 promoter parts), once with each of the purified backbones.
Heat transformation of E. coliDH5α competent cells with KguE promoter (HIFI).
Plating cells with KguE promoter for overnight incubation.
July 1
Preparation of gelling solution, washing solution + MQ water with NaCl for tomorrow’s alginate solution.
July 1
Checked plates, they didn’t grow.
PCR for KguE promoter, because our new primers arrived.
Gel run.
Inoculation of wild type P. putida from -80 0C and overnight incubation.
July 1
Preparation of alginate solution. Reduced the alginate concentration from 4.0% to 3.6% (1.8% in the finished encapsulation mixture).
July 1
Inoculation of wild type P. putida from overnight incubation.
Incubation for 2h.
OD measurement: 0.3 to 0.4.
Electrocompetent cells preparation.
Electroporation of P. putida with cells with Edd promoter.
Plating P. putida for overnight incubation.
Gel run to see sizes of Edd again – results were better this time, the band was between 3500 - 4000 bp.
July 1
Checked the electroporation plates, they didn’t grow.
Electrocompetent cells preparation, again.
Electroporation of P. putida with cells with KguE promoter (Last time something was wrong with volt shock).
Plating P. putida for overnight incubation.
July 1
Checked the electroporation plates, they didn’t grow.
PCR of KguE.
Gel run.
July 1
Checked plates, they didn’t grow.
July 1
Heat transformation of P. putida cells with Edd promoter.
Plating P. putida cells with Edd promoter for overnight incubation.

Week 31

(27/07 - 02/08)
July 1
Checked plates, they didn’t grow.
Inoculation of wild type P. putida from -80 0C and overnight incubation.
July 1
Enzyme digest of KguE (EcoR1 and Pst1).
Backbone preparation.
July 1
PCR of KguE without primers.
Enzyme digestion.
Gel run.
Electrocompetent cells preparation.
Electroporation of P. putida with cells with Edd promoter.
Plating P. putida for overnight incubation.
July 1
Checked electroporation plates, they grew.
Overnight single colony inoculation (P. putida).
Enzyme digestion of KguE (EcoR1 and Pst1) again with 4x concentration.
Preparation of new LA plates (+ Kan).
Gel run, results in no band.
Ligation of backbone and KguE insert.
Heat transformation of DH5α with KguE.
Plating cells with KguE promoter for overnight incubation.
July 1
Checked plates, they didn’t grow.
Gel preparation (agarose).
July 1
Glucose preparation for medium.
P. putida incubation with new glucose medium.
Plate reading of P. putida with and without glucose in medium.
Results showed no fluorescence (mCherry).
HIFI and heat transformation for KguE promoter with DH5α competent cells.
Plating cells with KguE promoter for overnight incubation.
July 1
Prepared LA + kan plates for capsule leakage tests.
July 1
Checked plates, they didn’t grow.
Inoculation of P. putida with Edd promoter in new medium.
July 1
Inoculation of P. putida pHH+GFP in LB + kan.
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.

Week 32

(03/08 - 09/08)
August 1
PCR of KguE with different annealing temperature.
Enzyme digest of KguE.
Miniprep of DH5α Edd and KguE.
Gel run.
August 1
5 % Inoculation of overnight culture in LB + kan.
Encapsulation of P. putida 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.
Capsule leakage tests (encapsulated P. putida pHH+GFP stored in LB).
August 1
PCR of edd promoter.
Gel run.
August 1
Checked plates from leakage tests, substantial leakage observed.
August 1
Confocal microscopy after 1 day incubation of capsules.

Week 33

(10/08 - 16/08)
August 1
Inoculation of pHH+mCherry DH5α cells to new medium + Kan.
Miniprep.
Gel run.
August 1
Prepared LA + kan plates.
August 1
Inoculation of pHH+mCherry DH5α cells to new medium + Kan.
Ordered new primers.
August 1
Inoculation of P. putida PHH+GFP in LB + kan.
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.
August 1
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.
Capsule leakage tests (encapsulated P. putida pHH+GFP with poly-L-lysine HBr coating stored in LB).
August 1
Looked at BioBrick protocol and preparations.
New primers arrived.
PCR of all promoters: Edd, KguE, Zwf and Gad.
August 1
Checked plates from leakage tests, substantial leakage observed. No improvement with poly-L-lysine HBr coating.
Analysis of results.
August 1
Measurements of PCR products and miniprep (backbone plasmid) concentrations on Nanodrop.
Digestion of backbone with Nde1 and Bste2 enzymes.
Digestion of PCR products (promoters).
PCR purification of all promoters (from PCR) and backbone.
Gel run to check sizes – results showed multiple bands (we needed only 2 bands).
Gel cut of highest 3 bands (ladder on gel was not so clear).
Gel cut DNA extraction.
Backbone plasmid separately digested with Nde1 and Bste2 enzymes.
Gel run again with all digested products and 2nd digestions.
Gel results showed that the Bste2 enzyme was cutting in many places.
Ligation of three gel cut DNA and one insert (Gad promoter).
Heat transformation of Dh5α competent cells with Gad promoter.
Plated cells on LA with Gad promoter for overnight incubation.
August 1
Prepared LA + kan plates for capsule leakage tests.
August 1
Checked plates, they didn’t grow.
Inoculation of new pHH+mCherry Dh5α cells to medium + Kan from -80 0C.
August 1
Miniprep of overnight inoculation pHH+mCherry Dh5α cells (backbone plasmid).
Measurements of miniprep (backbone plasmid) concentrations on Nanodrop.
Digestion of backbone with Nde1 and Bste2 enzymes (backbone preparation).
Gel run with digested plasmid backbone to see size.
Gel results showed Bste2 was cutting in many places again but this time less.
Ligation of plasmid backbone and one insert (Zwf promoter).
Heat transformation of Dh5α competent cells with Zwf promoter.
Plating cells on LA with Zwf promoter for overnight incubation.

Week 34

(17/08 - 23/08)
August 1
Checked plates, they grew.
Single colony inoculation.
Digestion of all promoters (PCR products) with Nde1 and Bste2 enzymes (insert preparation).
PCR purification of digested products.
Ligation of backbone and other promoters.
Heat transformation of Dh5α competent cells with other promoter.
Plating cells on LA with other promoter for overnight incubation.
August 1
Inoculation of P. putida pHH+GFP in LB + kan.
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.
August 1
Checked plates, they grew (Edd, Kgu and Zwf).
Single colony inoculation (5 colonies from each plate).
Miniprep of overnight single inoculation (Zwf promoter plasmid).
Measurements of miniprep (Zwf promoter plasmid) concentrations on Nanodrop.
PCR it with our primers to check presence of promoter sequence.
Gel run to see PCR product (confirmation).
Gel results confirmed.
Electrocompetent cells preparation (Wild type P. putida with no plasmid).
Electroporation of WT P. putida.
August 1
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.
Capsule leakage tests (encapsulated P. putida pHH+GFP + poly-L-lysine HCl coating stored in LB).
August 1
Checked plates: P. putida didn’t grow, electroporation didn’t work.
Miniprep of overnight single inoculation (Kgu and Edd).
Measurements of miniprep (Kgu and Edd) concentrations on Nanodrop.
PCR it with our primers to check presence of promoter sequence.
Gel run to see PCR product (confirmation).
August 1
Checked plates from capsule leakage tests, substantial leakage observed. No improvement with poly-L-lysine HCl coating.
Analysis of results.
August 1
Blunt ligation of Gad overnight.
Lig1 - PshA1, Lig2 - Pml1 and Lig3 - HPA1.
August 1
Heat transformation of Dh5α competent cells with overnight ligated plasmids.
Plating cells on LA for overnight incubation.

Week 35

(24/08 - 30/08)
August 1
P. putida with Kgu and Zwf promoters are ready.
Checked plates, they grew (Gad promoter).
Single colony inoculation.
August 1
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.
Inoculation of P. putida pHH+GFP in LB + kan.
August 1
Miniprep of overnight single inoculation (Gad).
Measurements of miniprep (Gad) concentrations on Nanodrop.
PCR it with our primers to check presence of promoter sequence.
Gel run to see PCR product (confirmation). Result of gel: was not confirmed.
Glucose medium preparation.
Incubation of P. putida with Kgu and Zwf promoters with glucose medium.
Digestion of Edd and Gad PCR products with PshA1, Pml1 and HPA1.
Gel run to see digestion result.
Gel extraction.
August 1
5 % inoculation of overnight culture in LB + kan + m-toluic acid (inducer).
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.
Capsule leakage tests (encapsulated P. putida pHH+GFP in PBS).
August 1
OD measurement of overnight incubated P. putida.
Plate reader (Spectrophotometry) of P. putida with Kgu and Zwf promoters.
Enzyme digestion.
Blunt ligation overnight.
Analysis of results.
August 1
Checked plates from leakage tests, much better results in PBS compared to LB.
Analysis of results.
August 1
Heat transformation of DH5α competent cells.
Plating cells on LA for overnight incubation.
Inoculation of P. putida in glucose medium.
2 hour incubation.
OD measurement of overnight incubated P. putida.
Plate reader (Spectrophotometry) of P. putida with Kgu and Zwf promoters.
August 1
Checked plates, they didn’t grow.
PCR amplification of all four promoters.
Inoculation of P. putida in different glucose media.
OD measurement of overnight incubated P. putida.
2 hour incubation.
Plate reader (Spectrophotometry) of P. putida with Kgu and Zwf promoters.
PCR of miniprep DH5α Kgu and Zwf plasmids with Prefix/Suffix primers.
August 1
Prepared LA + kan + 2mM m-Toluic acid plates for leakage tests.
August 1
Gad digestion with Bste2 and Nde1 enzymes.
PCR amplification.
Nanodrop measurement of concentration.
Sticky end ligation.
Gel run for confirmation.
Heat transformation of DH5α competent cells.
Plating cells on LA for overnight incubation.
OD measurement of overnight incubated P. putida.
Plate reader (Spectrophotometry) of P. putida with Kgu and Zwf promoters.
August 1
Checked plates, they didn’t grow.
August 1

Week 36

(31/08 - 06/09)
August 1
PCR of stock DNA (sent by company) with new Prefix/Suffix primers.
Digestion of backbone and insert with Pml1 and PshA1.
Gel run to check PCR products and digestion.
Measurement of PCR products and miniprep (GFP+pHH and mCherry+pHH) concentration on Nanodrop.
August 1
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.
Inoculation of P. putida pHH+GFP in LB + kan.
August 1
Blunt ligation for Kgu and Zwf.
Heat transformation of DH5α competent cells.
Plating cells on LA for overnight incubation.
Enzyme digestion of new plasmid backbone and all four promoters.
Blunt ligation overnight.
August 1
5 % inoculation of overnight culture in LB + kan + 2mM m-toluic acid (inducer).
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.
Capsule leakage tests (encapsulated P. putida pHH+GFP in PBS).
August 1
Checked plates, they didn’t grow.
Heat transformation of DH5α competent cells with overnight ligation.
Plating cells on LA for overnight incubation.
New construction of two promoters on one plasmid.
Enzyme digestion with Xba1 and Spe1 of Zwf promoter plasmid as backbone and Kgu promoter as insert.
Blunt ligation overnight incubation.
M9 medium preparation.
August 1
Checked plates from leakage tests, similar results to last time.
Analysis of results.
August 1
Checked plates, they grew.
Single colony inoculation (5 colonies).
Inoculation of Kgu and Zwf P. putida in M9 medium.
Incubation for 2 hours.
Plate reader measurements.
Flow cytometry.
August 1
Proposed information theoretical modelling to Team Warwick for predicting the probability of bonding of Brixells.
August 1
Miniprep of all single colony inoculations.
PCR with prefix/suffix primers.
Gel run to confirm presence: confirmed.
Electrocompetent cells preparation P. putida.
Electroporation of wild type P. putida.
Plating P. putida cells on LA plates.
August 1
Check on plates: they all grew!
Single colony inoculation.
August 1
Preparation LA + kan + m-toluic acid plates for leakage tests.
August 1
Standard assembly for new constructs Kgu as backbone and Zwf as insert.
Digestion with EcoR1, Spe1 and Xba1 enzymes.
Run on gel to confirm digestion: confirmed.
Gel extraction.
Ligation.
Heat transformation of DH5α competent cells.
Plating cells on LA for overnight incubation.
August 1
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.
Inoculation of P. putida pHH+GFP in LB + kan.

Week 37

(07/09 - 13/09)
September 1
Checked plates, they didn’t grow.
Miniprep plasmids preparation for sequencing.
Inoculation of P. putida with all promoters in different media.
September 1
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.
Capsule leakage tests (encapsulated P. putida pHH+GFP in PBS).
September 1
Miniprep preparation for sequencing.
Check on wild type P. putida and new P. putida with pHH-100 mCherry plasmid.
Inoculation of wild type for control.
Plate reader of overnight incubation in LB and LB + Glucose medium.
September 1
Checked plates from leakage tests, results were similar to first and second experiments in PBS.
Analysis of results.
September 1
Controls of wild type and P. putida with pHH-100 mCherry plasmid were confirmed.
Inoculation of P. putida in LB, LB + glucose and different concentrations of M9 medium.
Overnight incubation.
September 1
Plate reader of overnight samples.
Inoculation of P. putida in LB and different concentrations of M9 medium.
Overnight incubation.
September 1
Plate reader of overnight inoculations.
Inoculation of P. putida in LB and different concentrations of M9 medium
Overnight incubation.
September 1
Plate reader of of overnight inoculations.
Inoculation of P. putida in LB and different concentrations of M9 medium.
Overnight incubation.
RNA isolation of P. putida with our promoters for rtPCR.
September 1
Plate reader of Edd, Kgu, Zwf, Gad samples (3 biological and 3 technical replicates each) of overnight inoculations in M9 + 5%, 10%, 20%.
Inoculation of P. putida in LB in different concentrations of M9 medium.
Incubation of 3 biological replicates of Edd, Kgu, Zwf, Gad in LB.
Measured RNA concentration.
DNA free kit procedure.

Week 38

(14/09 - 20/09)
September 1
Digest and Ligation of biobrick parts (BBa_K184005, BBa_K1840006, BBa_K1840007, BBa_K1840008) and pSB1C3
Heat transformation to competent E.coli
September 1
Deployed new iGEM Matchmaker version with keyword analysis and machine learning.
September 1
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%
Flow cytometry of pHH+mCherry as positive control
Inoculation of one colony from each transformation in LB+Chloramphenicol
September 1
Proposed and simulated Brixells based on Poisson-Boltzmann theory.
September 1
Inoculation of P. putida pHH + mCherry.
Inoculation of biological replicate A of Edd, Gad, Zwf + Kgu samples in M9 medium + glucose (1, 5, 10 + 20 %).
Miniprep of inoculated samples for biobrick part sending, digest with EcoR1 and Pst1, gelelectrophoresis shows only one band
Digest and Ligation of biobrick parts (BBa_K184005, BBa_K1840006, BBa_K1840007, BBa_K1840008) and pSB1C3
Heat transformation to competent E.coli
September 1
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
Flow cytometry of Edd, Kgu, Zwf and Gad samples in M9 media with 1%, 5%, 10%, and 20% glucose
Acquisition of images of promoters Edd, Gad, Zwf + Kgu in M9 medium + glucose (1, 5, 10 + 20 %) by confocal microscopy.
Colony PCR with prefix/suffix primers to confirm the right contruct for biobrick part sending
September 1
Encapsulation of P. putida 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.
Acquisition of z-stack images by confocal microscopy.
September 1
Miniprep of transformed cells with our Biobrick parts in the pSB1C3 backbone, preparation for sending
September 1
3D Rendering of z-stack images obtained by confocal microscopy.

Week 39

(14/09 - 20/09)
September 5
Quantitative analysis of confocal microscopy images of P. putida pHH+mCherry.

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