Difference between revisions of "Team:NTNU Trondheim/Notebook"
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<p> | <p> | ||
</p> | </p> | ||
+ | |||
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<div class="entry nb-dry"> | <div class="entry nb-dry"> | ||
<div> | <div> | ||
− | <h6>February | + | <h6>February 02</h6> |
− | The iGEM Matchmaker (V2.0) is deployed for 2015. | + | <p>The iGEM Matchmaker (V2.0) is deployed for 2015.</p> |
</div> | </div> | ||
</div> | </div> | ||
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<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>June | + | <h6>June 11</h6> |
− | Discussed <i>Escherichia coli</i> glucose uptake and possible promoters in E. coli. | + | <p>Discussed <i>Escherichia coli</i> glucose uptake and possible promoters in E. coli.</p> |
</div> | </div> | ||
</div> | </div> | ||
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<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>June | + | <h6>June 15</h6> |
− | Discussed <i>Pseudomonas putida</i> as a candidate microorganism.<br>Discussion of a system for glucose uptake in <i>P. putida</i>. | + | <p>Discussed <i>Pseudomonas putida</i> as a candidate microorganism.<br>Discussion of a system for glucose uptake in <i>P. putida</i>.</p> |
</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-hprac"> | ||
+ | <div> | ||
+ | <h6>June 15</h6> | ||
+ | |||
+ | <p>Interviews.</p> | ||
+ | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>June | + | <h6>June 17</h6> |
+ | |||
+ | <p>Design of promoter regions for <i>P. putida</i>.<br>Ordered promoter regions for <i>P. putida</i>.</p> | ||
+ | </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-meas"> | ||
+ | <div> | ||
+ | <h6>June 18</h6> | ||
+ | |||
+ | <p>Discussion of InterLab Study.<br>Preparations for the InterLab Study</p> | ||
+ | </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-meas"> | ||
+ | <div> | ||
+ | <h6>June 19</h6> | ||
+ | |||
+ | <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> | ||
+ | </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-hprac"> | ||
+ | <div> | ||
+ | <h6>June 20</h6> | ||
+ | |||
+ | <p>Created survey for students and employees at NTNU.<br>Interviews with diabetic patients.</p> | ||
+ | </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-meas"> | ||
+ | <div> | ||
+ | <h6>June 21</h6> | ||
− | + | <p>Inoculation of DH5α in 3 ml, incubation over night at 37 <sup>0</sup>C.</p> | |
</div> | </div> | ||
</div> | </div> | ||
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</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-meas"> | ||
+ | <div> | ||
+ | <h6>June 22</h6> | ||
+ | |||
+ | <p>Making <i>E. coli</i>electrocompetent (after 3h incubation, OD<SUB>600</SUB> = 0.44).<br>Electroporation of E. coli.</p> | ||
+ | </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-meas"> | ||
+ | <div> | ||
+ | <h6>June 23</h6> | ||
+ | |||
+ | <p>Transformation.</p> | ||
+ | </div> | ||
+ | </div> | ||
<div class="nb-week" id="week26entry"> | <div class="nb-week" id="week26entry"> | ||
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<div class="entry nb-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>June | + | <h6>June 24</h6> |
− | Introduction to alginate encapsulation + demo on how to operate the electrostatic capsule generator. | + | <p>Introduction to alginate encapsulation + demo on how to operate the electrostatic capsule generator.</p> |
</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-meas"> | ||
+ | <div> | ||
+ | <h6>June 24</h6> | ||
+ | |||
+ | <p>Inoculation of a colony from plate in 3 ml LB.</p> | ||
+ | </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 class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>June | + | <h6>June 25</h6> |
− | Playing around with the electrostatic bead generator, making capsules of different sizes by varying the different parameters of the instrument. | + | <p>Playing around with the electrostatic bead generator, making capsules of different sizes by varying the different parameters of the instrument.</p> |
+ | </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-meas"> | ||
+ | <div> | ||
+ | <h6>June 25</h6> | ||
+ | |||
+ | <p>Miniprep of transformed E. coli.<br>Nanodrop measurement of DNA concentration.</p> | ||
+ | </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-meas"> | ||
+ | <div> | ||
+ | <h6>June 26</h6> | ||
+ | |||
+ | <p>Enzyme digestion.<br>Ligation.<br>Transformation.<br>Prepared LA plates.</p> | ||
+ | </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-meas"> | ||
+ | <div> | ||
+ | <h6>June 27</h6> | ||
+ | |||
+ | <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> | ||
+ | </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-meas"> | ||
+ | <div> | ||
+ | <h6>June 28</h6> | ||
+ | |||
+ | <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> | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="entry nb-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>June | + | <h6>June 29</h6> |
− | Moved the cell encapsulation equipment into our lab, so it was ready for the GM bacteria. | + | <p>Moved the cell encapsulation equipment into our lab, so it was ready for the GM bacteria.</p> |
</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-meas"> | ||
+ | <div> | ||
+ | <h6>July 01</h6> | ||
+ | |||
+ | <p>Miniprep.<br>Nanodrop measurement after miniprep (58.38 + 64.98 + 70.48 ~ 64.63).</p> | ||
+ | </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 class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>July 01</h6> |
− | Preparation of alginate solution, gelling solution and washing solution. | + | <p>Preparation of alginate solution, gelling solution and washing solution.</p> |
</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 class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>July 02</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. | + | <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> | </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 class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>July 03</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. | + | <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> | </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-meas"> | ||
+ | <div> | ||
+ | <h6>July 03</h6> | ||
+ | |||
+ | <p>Introduction to confocal microscopy.</p> | ||
+ | </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-dry"> | <div class="entry nb-dry"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>July 05</h6> |
− | Created the website design using Bootstrap, and set up the Notebook and Protocols pages. | + | <p>Created the website design using Bootstrap, and set up the Notebook and Protocols pages.</p> |
</div> | </div> | ||
</div> | </div> | ||
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</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="nb-week" id="week28entry"> | <div class="nb-week" id="week28entry"> | ||
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<div class="entry nb-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 06</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. | + | <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> | </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 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="nb-week" id="week28entry"> | ||
+ | <div class="twelve columns"> | ||
+ | <h3>Week 28</h3> | ||
+ | <h6>(06/07 - 12/07)</h6> | ||
<div class="entry nb-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 07</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. | + | <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> | </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 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="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 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="nb-week" id="week28entry"> | ||
+ | <div class="twelve columns"> | ||
+ | <h3>Week 28</h3> | ||
+ | <h6>(06/07 - 12/07)</h6> | ||
<div class="entry nb-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 09</h6> |
− | Preparation of alginate solution, and put this in the 4 <sup>0</sup>C fridge for future use. | + | <p>Preparation of alginate solution, and put this in the 4 <sup>0</sup>C fridge for future use.</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-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 10</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. | + | <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> | </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-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 11</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. | + | <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> | </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 11</h6> | ||
+ | |||
+ | <p>Plate reader measurement of InterLab samples.</p> | ||
+ | </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-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 12</h6> |
+ | |||
+ | <p>Checked plates, they did not grow.<br>Inoculation of <i>P. putida</i>.</p> | ||
+ | </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 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> | ||
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<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 13</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. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 13</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. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 14</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. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 14</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. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 15</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. | + | <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> | </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-meas"> | ||
+ | <div> | ||
+ | <h6>July 15</h6> | ||
+ | |||
+ | <p>Changed the medium of the Devices.</p> | ||
+ | </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-dry"> | <div class="entry nb-dry"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 15</h6> |
− | Written first version of the image analysis tool. | + | <p>Written first version of the image analysis tool.</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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 15</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. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 16</h6> |
− | Overnight inoculated cultures grew.<br>OD measurement: 0.3 to 0.5.<br>Plated them again to obtain single colonies. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 16</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. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 17</h6> |
− | Plates grew.<br>Single colony inoculation in LB + Kan.<br>Overnight incubation 37 <sup>0</sup>C. | + | <p>Plates grew.<br>Single colony inoculation in LB + Kan.<br>Overnight incubation 37 <sup>0</sup>C.</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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 17</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. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 18</h6> |
− | Cells grew well in LB + Kan.<br>OD measurement: 0.2344.<br>2 % inoculation of cells. | + | <p>Cells grew well in LB + Kan.<br>OD measurement: 0.2344.<br>2 % inoculation of cells. </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 19</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. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 19</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. | + | <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> | ||
Line 468: | Line 862: | ||
<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 20</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. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 20</h6> |
− | Preparation of gelling solution, washing solution + MQ water with NaCl for tomorrow’s alginate solution. | + | <p>Preparation of gelling solution, washing solution + MQ water with NaCl for tomorrow’s alginate solution.</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 21</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. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 21</h6> |
− | Preparation of alginate solution. Reduced the alginate concentration from 4.0% to 3.6% (1.8% in the finished encapsulation mixture). | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 22</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. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 23</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. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 24</h6> |
− | Checked the electroporation plates, they didn’t grow.<br>PCR of KguE.<br>Gel run. | + | <p>Checked the electroporation plates, they didn’t grow.<br>PCR of KguE.<br>Gel run.</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 25</h6> |
− | Checked plates, they didn’t grow. | + | <p>Checked plates, they didn’t grow.</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 26</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. | + | <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> | ||
Line 548: | Line 982: | ||
<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 27</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. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 28</h6> |
− | Enzyme digest of KguE (EcoR1 and Pst1).<br>Backbone preparation. | + | <p>Enzyme digest of KguE (EcoR1 and Pst1).<br>Backbone preparation.</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 29</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. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 30</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. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>July | + | <h6>July 31</h6> |
− | Checked plates, they didn’t grow.<br>Gel preparation (agarose). | + | <p>Checked plates, they didn’t grow.<br>Gel preparation (agarose).</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>August 01</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. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>August 01</h6> |
− | Prepared LA + kan plates for capsule leakage tests. | + | <p>Prepared LA + kan plates for capsule leakage tests.</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>August 02</h6> |
− | Checked plates, they didn’t grow.<br>Inoculation of <i>P. putida</i> with Edd promoter in new medium. | + | <p>Checked plates, they didn’t grow.<br>Inoculation of <i>P. putida</i> with Edd promoter in new medium.</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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>August 02</h6> |
− | Inoculation of <i>P. putida</i> pHH+GFP in LB + kan.<br>Sterilisation of equipment and glassware for cell encapsulation by autoclavation. | + | <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> | ||
Line 628: | Line 1,102: | ||
<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 03</h6> |
− | PCR of KguE with different annealing temperature.<br>Enzyme digest of KguE.<br>Miniprep of DH5α Edd and KguE.<br>Gel run. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 03</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). | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 04</h6> |
− | PCR of edd promoter.<br>Gel run. | + | <p>PCR of edd promoter.<br>Gel run.</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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 04</h6> |
− | Checked plates from leakage tests, substantial leakage observed. | + | <p>Checked plates from leakage tests, substantial leakage observed.</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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 05</h6> |
− | Confocal microscopy after 1 day incubation of capsules. | + | <p>Confocal microscopy after 1 day incubation of capsules.</p> |
</div> | </div> | ||
</div> | </div> | ||
Line 676: | Line 1,170: | ||
<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 10</h6> |
− | Inoculation of pHH+mCherry DH5α cells to new medium + Kan.<br>Miniprep.<br>Gel run. | + | <p>Inoculation of pHH+mCherry DH5α cells to new medium + Kan.<br>Miniprep.<br>Gel run.</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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 10</h6> |
− | Prepared LA + kan plates. | + | <p>Prepared LA + kan plates.</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 11</h6> |
− | Inoculation of pHH+mCherry DH5α cells to new medium + Kan.<br>Ordered new primers. | + | <p>Inoculation of pHH+mCherry DH5α cells to new medium + Kan.<br>Ordered new primers.</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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 11</h6> |
− | Inoculation of <i>P. putida</i> PHH+GFP in LB + kan.<br>Sterilisation of equipment and glassware for cell encapsulation by autoclavation. | + | <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="week33entry"> | ||
+ | <div class="twelve columns"> | ||
+ | <h3>Week 33</h3> | ||
+ | <h6>(10/08 - 16/08)</h6> | ||
<div class="entry nb-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 12</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). | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 13</h6> |
− | Looked at BioBrick protocol and preparations.<br>New primers arrived.<br>PCR of all promoters: Edd, KguE, Zwf and Gad. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 13</h6> |
− | Checked plates from leakage tests, substantial leakage observed. No improvement with poly-L-lysine HBr coating.<br>Analysis of results. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 14</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. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 14</h6> |
− | Prepared LA + kan plates for capsule leakage tests. | + | <p>Prepared LA + kan plates for capsule leakage tests.</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 15</h6> |
− | Checked plates, they didn’t grow.<br>Inoculation of new pHH+mCherry Dh5α cells to medium + Kan from -80 <sup>0</sup>C. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 16</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. | + | <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> | ||
Line 772: | Line 1,316: | ||
<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 17</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. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 17</h6> |
− | Inoculation of <i>P. putida</i> pHH+GFP in LB + kan.<br>Sterilisation of equipment and glassware for cell encapsulation by autoclavation. | + | <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="week34entry"> | ||
+ | <div class="twelve columns"> | ||
+ | <h3>Week 34</h3> | ||
+ | <h6>(17/08 - 23/08)</h6> | ||
<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 18</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>. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 18</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). | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 19</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). | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 19</h6> |
− | Checked plates from capsule leakage tests, substantial leakage observed. No improvement with poly-L-lysine HCl coating.<br>Analysis of results. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 22</h6> |
− | Blunt ligation of Gad overnight.<br>Lig1 - PshA1, Lig2 - Pml1 and Lig3 - HPA1. | + | <p>Blunt ligation of Gad overnight.<br>Lig1 - PshA1, Lig2 - Pml1 and Lig3 - HPA1.</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 23</h6> |
− | Heat transformation of Dh5α competent cells with overnight ligated plasmids.<br>Plating cells on LA for overnight incubation. | + | <p>Heat transformation of Dh5α competent cells with overnight ligated plasmids.<br>Plating cells on LA for overnight incubation.</p> |
</div> | </div> | ||
</div> | </div> | ||
Line 844: | Line 1,423: | ||
<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 24</h6> |
− | <i>P. putida</i> with Kgu and Zwf promoters are ready.<br>Checked plates, they grew (Gad promoter).<br>Single colony inoculation. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 24</h6> |
− | Sterilisation of equipment and glassware for cell encapsulation by autoclavation.<br>Inoculation of <i>P. putida</i> pHH+GFP in LB + kan. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 25</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. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 25</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). | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 26</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. | + | <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> | </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-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="nb-week" id="week35entry"> | ||
+ | <div class="twelve columns"> | ||
+ | <h3>Week 35</h3> | ||
+ | <h6>(24/08 - 30/08)</h6> | ||
<div class="entry nb-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 26</h6> |
− | Checked plates from leakage tests, much better results in PBS compared to LB.<br>Analysis of results. | + | <p>Checked plates from leakage tests, much better results in PBS compared to LB.<br>Analysis of results.</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 27</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. | + | <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> | </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-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="nb-week" id="week35entry"> | ||
+ | <div class="twelve columns"> | ||
+ | <h3>Week 35</h3> | ||
+ | <h6>(24/08 - 30/08)</h6> | ||
<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 28</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. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 28</h6> |
− | Prepared LA + kan + 2mM m-Toluic acid plates for leakage tests. | + | <p>Prepared LA + kan + 2mM m-Toluic acid plates for leakage tests.</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-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="nb-week" id="week35entry"> | ||
+ | <div class="twelve columns"> | ||
+ | <h3>Week 35</h3> | ||
+ | <h6>(24/08 - 30/08)</h6> | ||
<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 29</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. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 30</h6> |
− | Checked plates, they didn’t grow. | + | <p>Checked plates, they didn’t grow.</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-dry"> | <div class="entry nb-dry"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 30</h6> |
− | + | ||
+ | <p></p> | ||
</div> | </div> | ||
</div> | </div> | ||
Line 948: | Line 1,621: | ||
<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 31</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. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>August | + | <h6>August 31</h6> |
− | Sterilisation of equipment and glassware for cell encapsulation by autoclavation.<br>Inoculation of <i>P. putida</i> pHH+GFP in LB + kan. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>September 01</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. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>September 01</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). | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>September 02</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. | + | <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> |
</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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>September 02</h6> |
− | Checked plates from leakage tests, similar results to last time.<br>Analysis of results. | + | <p>Checked plates from leakage tests, similar results to last time.<br>Analysis of results.</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>September 03</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. | + | <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> |
</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-dry"> | <div class="entry nb-dry"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>September 04</h6> |
− | Proposed information theoretical modelling to Team Warwick for predicting the probability of bonding of Brixells. | + | <p>Proposed information theoretical modelling to Team Warwick for predicting the probability of bonding of Brixells.</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>September 04</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. | + | <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> |
</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>September 05</h6> |
− | Check on plates: they all grew!<br>Single colony inoculation. | + | <p>Check on plates: they all grew!<br>Single colony inoculation.</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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>September 05</h6> |
− | Preparation LA + kan + m-toluic acid plates for leakage tests. | + | <p>Preparation LA + kan + m-toluic acid plates for leakage tests.</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>September 06</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. | + | <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> |
</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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6> | + | <h6>September 06</h6> |
− | Sterilisation of equipment and glassware for cell encapsulation by autoclavation.<br>Inoculation of <i>P. putida</i> pHH+GFP in LB + kan. | + | <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> | </div> | ||
Line 1,060: | Line 1,793: | ||
<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 07</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. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 07</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). | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 08</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. | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 08</h6> |
− | Checked plates from leakage tests, results were similar to first and second experiments in PBS.<br>Analysis of results. | + | <p>Checked plates from leakage tests, results were similar to first and second experiments in PBS.<br>Analysis of results.</p> |
</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 09</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. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 10</h6> |
− | Plate reader of overnight samples.<br>Inoculation of <i>P. putida</i> in LB and different concentrations of M9 medium.<br>Overnight incubation. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 11</h6> |
− | Plate reader of overnight inoculations.<br>Inoculation of <i>P. putida</i> in LB and different concentrations of M9 medium<br>Overnight incubation. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 12</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. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 13</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. | + | <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> | ||
Line 1,140: | Line 1,913: | ||
<div class="entry nb-wet"> | <div class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 14</h6> |
− | Digest and Ligation of biobrick parts (BBa_K184005, BBa_K1840006, BBa_K1840007, BBa_K1840008) and pSB1C3<br>Heat transformation to competent E.coli | + | <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> | </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-dry"> | <div class="entry nb-dry"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 14</h6> |
− | Deployed new iGEM Matchmaker version with keyword analysis and machine learning. | + | <p>Deployed new iGEM Matchmaker version with keyword analysis and machine learning.</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 15</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 | + | <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> | </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-dry"> | <div class="entry nb-dry"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 16</h6> |
− | Proposed and simulated Brixells based on Poisson-Boltzmann theory. | + | <p>Proposed and simulated Brixells based on Poisson-Boltzmann theory.</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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 16</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 | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 17</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 | + | <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> | </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-anim"> | <div class="entry nb-anim"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 17</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. | + | <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> | </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 class="entry nb-wet"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 18</h6> |
− | Miniprep of transformed cells with our Biobrick parts in the pSB1C3 backbone, preparation for sending | + | <p>Miniprep of transformed cells with our Biobrick parts in the pSB1C3 backbone, preparation for sending</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-hprac"> | ||
+ | <div> | ||
+ | <h6>September 18</h6> | ||
+ | |||
+ | <p>Analysis of survey responses.</p> | ||
+ | </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-dry"> | <div class="entry nb-dry"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 18</h6> |
− | 3D Rendering of z-stack images obtained by confocal microscopy. | + | <p>3D Rendering of z-stack images obtained by confocal microscopy.</p> |
</div> | </div> | ||
</div> | </div> | ||
Line 1,220: | Line 2,046: | ||
<div class="entry nb-dry"> | <div class="entry nb-dry"> | ||
<div> | <div> | ||
− | <h6>September | + | <h6>September 18</h6> |
− | Quantitative analysis of confocal microscopy images of <i>P. putida</i> pHH+mCherry. | + | <p>Quantitative analysis of confocal microscopy images of <i>P. putida</i> pHH+mCherry.</p> |
</div> | </div> | ||
</div> | </div> |
Revision as of 22:08, 18 September 2015
Notebook
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Week 23
(02/02 - 08/02)
February 02
The iGEM Matchmaker (V2.0) is deployed for 2015.
Week 24
(08/06 - 14/06)
June 11
Discussed Escherichia coli glucose uptake and possible promoters in E. coli.
Week 25
(15/06 - 21/06)
June 15
Discussed Pseudomonas putida as a candidate microorganism.
Discussion of a system for glucose uptake in P. putida.
Week 25
(15/06 - 21/06)
June 15
Interviews.
Week 25
(15/06 - 21/06)
June 17
Design of promoter regions for P. putida.
Ordered promoter regions for P. putida.
Week 25
(15/06 - 21/06)
June 18
Discussion of InterLab Study.
Preparations for the InterLab Study
Week 25
(15/06 - 21/06)
June 19
Prepared and autoclaved LB medium 300 ml, LA medium 3 x 500 ml, Eppendorf tubes, distilled water and pipette tips.
Prepared antibiotic stock solutions (Ampicillin, Chloramphenicol and Kanamycin).
Week 25
(15/06 - 21/06)
June 20
Created survey for students and employees at NTNU.
Interviews with diabetic patients.
Week 25
(15/06 - 21/06)
June 21
Inoculation of DH5α in 3 ml, incubation over night at 37 0C.
Week 26
(22/06 - 28/06)
June 22
Making E. colielectrocompetent (after 3h incubation, OD600 = 0.44).
Electroporation of E. coli.
Week 26
(22/06 - 28/06)
June 23
Transformation.
Week 26
(22/06 - 28/06)
June 24
Introduction to alginate encapsulation + demo on how to operate the electrostatic capsule generator.
Week 26
(22/06 - 28/06)
June 24
Inoculation of a colony from plate in 3 ml LB.
Week 26
(22/06 - 28/06)
June 25
Playing around with the electrostatic bead generator, making capsules of different sizes by varying the different parameters of the instrument.
Week 26
(22/06 - 28/06)
June 25
Miniprep of transformed E. coli.
Nanodrop measurement of DNA concentration.
Week 26
(22/06 - 28/06)
June 26
Enzyme digestion.
Ligation.
Transformation.
Prepared LA plates.
Week 26
(22/06 - 28/06)
June 27
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).
Week 26
(22/06 - 28/06)
June 28
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).
Week 27
(29/06 - 05/07)
June 29
Moved the cell encapsulation equipment into our lab, so it was ready for the GM bacteria.
Week 27
(29/06 - 05/07)
July 01
Miniprep.
Nanodrop measurement after miniprep (58.38 + 64.98 + 70.48 ~ 64.63).
Week 27
(29/06 - 05/07)
July 01
Preparation of alginate solution, gelling solution and washing solution.
Week 27
(29/06 - 05/07)
July 02
Inoculation of Pseudomonas putida pHH+GFP from -80 0C in 3 ml LB + Kan medium. Incubation at 30 0C.
Week 27
(29/06 - 05/07)
July 03
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.
Week 27
(29/06 - 05/07)
July 03
Introduction to confocal microscopy.
Week 27
(29/06 - 05/07)
July 05
Created the website design using Bootstrap, and set up the Notebook and Protocols pages.
Week 28
(06/07 - 12/07)
July 06
PCR (1 μl template DNA = miniprepped from P. putida pHH+GFP, annealing temperature = 60 0C).
Prepared LB (200 ml with Kan, 100 ml with Chl).
GelRed to test the PCR. It did not work.
PCR repeated but with Rahmi’s DNA, did not work either.
PCR repeated again and gel shows the right band (but also an unknown second band).
Week 28
(06/07 - 12/07)
July 06
Inoculation of Pseudomonas putida pHH+GFP from -80 0C in 3 ml LB + kan medium. Incubation at 30 0C.
Week 28
(06/07 - 12/07)
July 07
Repeated PCR because of the unknown second band. This time annealing temperature is 61 °C, annealing time 10 sec.
OD measurement on devices (to make a standard curve with the plate reader, but the plate reader did not work).
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).
Week 28
(06/07 - 12/07)
July 07
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.
Week 28
(06/07 - 12/07)
July 08
Inoculation of InterLab samples in 3 ml LB (+ Kan/Chl).
Prepared 400 ml PBS.
Plate reader measurement of InterLab samples (undiluted from ON culture).
Week 28
(06/07 - 12/07)
July 09
1 % inoculation of samples Dev 1A – Dev 3B in 3 ml LB + Kan for flow cytometry.
1 % inoculation of samples Dev 3C – NCC in 3 ml LB (+ Kan/Chl) in the same way.
Purification of backbone PCR.
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.
Week 28
(06/07 - 12/07)
July 09
Preparation of alginate solution, and put this in the 4 0C fridge for future use.
Week 28
(06/07 - 12/07)
July 10
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.
Week 28
(06/07 - 12/07)
July 11
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.
Week 28
(06/07 - 12/07)
July 11
Plate reader measurement of InterLab samples.
Week 28
(06/07 - 12/07)
July 12
Checked plates, they did not grow.
Inoculation of P. putida.
Week 28
(06/07 - 12/07)
July 12
Inoculation of InterLab samples in 3 ml LB (+Kan/+Chl).
Plate reader calibration.
Plate reader measurement of InterLab samples for 5 hours.
Week 29
(13/07 - 19/07)
July 13
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.
Week 29
(13/07 - 19/07)
July 13
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.
Week 29
(13/07 - 19/07)
July 14
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.
Week 29
(13/07 - 19/07)
July 14
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.
Week 29
(13/07 - 19/07)
July 15
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.
Week 29
(13/07 - 19/07)
July 15
Changed the medium of the Devices.
Week 29
(13/07 - 19/07)
July 15
Written first version of the image analysis tool.
Week 29
(13/07 - 19/07)
July 15
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.
Week 29
(13/07 - 19/07)
July 16
Overnight inoculated cultures grew.
OD measurement: 0.3 to 0.5.
Plated them again to obtain single colonies.
Week 29
(13/07 - 19/07)
July 16
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.
Week 29
(13/07 - 19/07)
July 17
Plates grew.
Single colony inoculation in LB + Kan.
Overnight incubation 37 0C.
Week 29
(13/07 - 19/07)
July 17
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.
Week 29
(13/07 - 19/07)
July 18
Cells grew well in LB + Kan.
OD measurement: 0.2344.
2 % inoculation of cells.
Week 29
(13/07 - 19/07)
July 19
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.
Week 29
(13/07 - 19/07)
July 19
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 20
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.
Week 30
(20/07 - 26/07)
July 20
Preparation of gelling solution, washing solution + MQ water with NaCl for tomorrow’s alginate solution.
Week 30
(20/07 - 26/07)
July 21
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.
Week 30
(20/07 - 26/07)
July 21
Preparation of alginate solution. Reduced the alginate concentration from 4.0% to 3.6% (1.8% in the finished encapsulation mixture).
Week 30
(20/07 - 26/07)
July 22
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.
Week 30
(20/07 - 26/07)
July 23
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.
Week 30
(20/07 - 26/07)
July 24
Checked the electroporation plates, they didn’t grow.
PCR of KguE.
Gel run.
Week 30
(20/07 - 26/07)
July 25
Checked plates, they didn’t grow.
Week 30
(20/07 - 26/07)
July 26
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 27
Checked plates, they didn’t grow.
Inoculation of wild type P. putida from -80 0C and overnight incubation.
Week 31
(27/07 - 02/08)
July 28
Enzyme digest of KguE (EcoR1 and Pst1).
Backbone preparation.
Week 31
(27/07 - 02/08)
July 29
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.
Week 31
(27/07 - 02/08)
July 30
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.
Week 31
(27/07 - 02/08)
July 31
Checked plates, they didn’t grow.
Gel preparation (agarose).
Week 31
(27/07 - 02/08)
August 01
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.
Week 31
(27/07 - 02/08)
August 01
Prepared LA + kan plates for capsule leakage tests.
Week 31
(27/07 - 02/08)
August 02
Checked plates, they didn’t grow.
Inoculation of P. putida with Edd promoter in new medium.
Week 31
(27/07 - 02/08)
August 02
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 03
PCR of KguE with different annealing temperature.
Enzyme digest of KguE.
Miniprep of DH5α Edd and KguE.
Gel run.
Week 32
(03/08 - 09/08)
August 03
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).
Week 32
(03/08 - 09/08)
August 04
PCR of edd promoter.
Gel run.
Week 32
(03/08 - 09/08)
August 04
Checked plates from leakage tests, substantial leakage observed.
Week 32
(03/08 - 09/08)
August 05
Confocal microscopy after 1 day incubation of capsules.
Week 33
(10/08 - 16/08)
August 10
Inoculation of pHH+mCherry DH5α cells to new medium + Kan.
Miniprep.
Gel run.
Week 33
(10/08 - 16/08)
August 10
Prepared LA + kan plates.
Week 33
(10/08 - 16/08)
August 11
Inoculation of pHH+mCherry DH5α cells to new medium + Kan.
Ordered new primers.
Week 33
(10/08 - 16/08)
August 11
Inoculation of P. putida PHH+GFP in LB + kan.
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.
Week 33
(10/08 - 16/08)
August 12
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).
Week 33
(10/08 - 16/08)
August 13
Looked at BioBrick protocol and preparations.
New primers arrived.
PCR of all promoters: Edd, KguE, Zwf and Gad.
Week 33
(10/08 - 16/08)
August 13
Checked plates from leakage tests, substantial leakage observed. No improvement with poly-L-lysine HBr coating.
Analysis of results.
Week 33
(10/08 - 16/08)
August 14
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.
Week 33
(10/08 - 16/08)
August 14
Prepared LA + kan plates for capsule leakage tests.
Week 33
(10/08 - 16/08)
August 15
Checked plates, they didn’t grow.
Inoculation of new pHH+mCherry Dh5α cells to medium + Kan from -80 0C.
Week 33
(10/08 - 16/08)
August 16
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 17
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.
Week 34
(17/08 - 23/08)
August 17
Inoculation of P. putida pHH+GFP in LB + kan.
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.
Week 34
(17/08 - 23/08)
August 18
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.
Week 34
(17/08 - 23/08)
August 18
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).
Week 34
(17/08 - 23/08)
August 19
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).
Week 34
(17/08 - 23/08)
August 19
Checked plates from capsule leakage tests, substantial leakage observed. No improvement with poly-L-lysine HCl coating.
Analysis of results.
Week 34
(17/08 - 23/08)
August 22
Blunt ligation of Gad overnight.
Lig1 - PshA1, Lig2 - Pml1 and Lig3 - HPA1.
Week 34
(17/08 - 23/08)
August 23
Heat transformation of Dh5α competent cells with overnight ligated plasmids.
Plating cells on LA for overnight incubation.
Week 35
(24/08 - 30/08)
August 24
P. putida with Kgu and Zwf promoters are ready.
Checked plates, they grew (Gad promoter).
Single colony inoculation.
Week 35
(24/08 - 30/08)
August 24
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.
Inoculation of P. putida pHH+GFP in LB + kan.
Week 35
(24/08 - 30/08)
August 25
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.
Week 35
(24/08 - 30/08)
August 25
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).
Week 35
(24/08 - 30/08)
August 26
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.
Week 35
(24/08 - 30/08)
August 26
Overnight inoculation of samples 1A, 1B, 1C, 2A, 2B, 2C, 3A, 3B and 3C.
Week 35
(24/08 - 30/08)
August 26
Checked plates from leakage tests, much better results in PBS compared to LB.
Analysis of results.
Week 35
(24/08 - 30/08)
August 27
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.
Week 35
(24/08 - 30/08)
August 27
Acquisition of images by confocal microscopy, devices 1A, 1B, 1C, 2A, 2B, 2C, 3A, 3B and 3C (each in technical triplicates).
Week 35
(24/08 - 30/08)
August 28
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.
Week 35
(24/08 - 30/08)
August 28
Prepared LA + kan + 2mM m-Toluic acid plates for leakage tests.
Week 35
(24/08 - 30/08)
August 28
Acquisition of images by confocal microscopy, control samples NCA, NCB, NCC, PCA, PCB and PCC (each in technical triplicates).
Analysis of images using quantitative image analysis based on ImageJ and MATLAB.
Week 35
(24/08 - 30/08)
August 29
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.
Week 35
(24/08 - 30/08)
August 30
Checked plates, they didn’t grow.
Week 35
(24/08 - 30/08)
August 30
Week 36
(31/08 - 06/09)
August 31
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.
Week 36
(31/08 - 06/09)
August 31
Sterilisation of equipment and glassware for cell encapsulation by autoclavation.
Inoculation of P. putida pHH+GFP in LB + kan.
Week 36
(31/08 - 06/09)
September 01
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.
Week 36
(31/08 - 06/09)
September 01
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).
Week 36
(31/08 - 06/09)
September 02
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.
Week 36
(31/08 - 06/09)
September 02
Checked plates from leakage tests, similar results to last time.
Analysis of results.
Week 36
(31/08 - 06/09)
September 03
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.
Week 36
(31/08 - 06/09)
September 04
Proposed information theoretical modelling to Team Warwick for predicting the probability of bonding of Brixells.
Week 36
(31/08 - 06/09)
September 04
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.
Week 36
(31/08 - 06/09)
September 05
Check on plates: they all grew!
Single colony inoculation.
Week 36
(31/08 - 06/09)
September 05
Preparation LA + kan + m-toluic acid plates for leakage tests.
Week 36
(31/08 - 06/09)
September 06
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.
Week 36
(31/08 - 06/09)
September 06
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 07
Checked plates, they didn’t grow.
Miniprep plasmids preparation for sequencing.
Inoculation of P. putida with all promoters in different media.
Week 37
(07/09 - 13/09)
September 07
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).
Week 37
(07/09 - 13/09)
September 08
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.
Week 37
(07/09 - 13/09)
September 08
Checked plates from leakage tests, results were similar to first and second experiments in PBS.
Analysis of results.
Week 37
(07/09 - 13/09)
September 09
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.
Week 37
(07/09 - 13/09)
September 10
Plate reader of overnight samples.
Inoculation of P. putida in LB and different concentrations of M9 medium.
Overnight incubation.
Week 37
(07/09 - 13/09)
September 11
Plate reader of overnight inoculations.
Inoculation of P. putida in LB and different concentrations of M9 medium
Overnight incubation.
Week 37
(07/09 - 13/09)
September 12
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.
Week 37
(07/09 - 13/09)
September 13
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 14
Digest and Ligation of biobrick parts (BBa_K184005, BBa_K1840006, BBa_K1840007, BBa_K1840008) and pSB1C3
Heat transformation to competent E.coli
Week 38
(14/09 - 20/09)
September 14
Deployed new iGEM Matchmaker version with keyword analysis and machine learning.
Week 38
(14/09 - 20/09)
September 15
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
Week 38
(14/09 - 20/09)
September 16
Proposed and simulated Brixells based on Poisson-Boltzmann theory.
Week 38
(14/09 - 20/09)
September 16
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
Week 38
(14/09 - 20/09)
September 17
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