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| <li style="text-align:left">Growth curve of BAP 1 in media 11 to 14 in order to assess TPA toxicity.</li></br> | | <li style="text-align:left">Growth curve of BAP 1 in media 11 to 14 in order to assess TPA toxicity.</li></br> |
− | <li style="text-align:left">Plating of BAP 1 bacteria on all solid media to assess TPA toxicity and the ability of TPA to be used as a carbon source.</br> | + | <li style="text-align:left">Plating of BAP 1 bacteria on all solid media to assess TPA toxicity and the ability of TPA to be used as a carbon source.</br></br> |
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− | <!-- <u>3 solutions: </u><br />
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− | <br />
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− | <u>Solution 1: (salts)</u><br />
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− | 5.3 g of Potassium Phosphate (dibasic) K<sub>2</sub>HPO<sub>4</sub><br />
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− | 2 g of Potassium Phosphate (monobasic) KH<sub>2</sub>PO<sub>4</sub><br />
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− | 1 g of Ammonium Sulfate (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>.<br />
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− | 0.5 g of Sodium Citrate (tribasic, dihydrate) <br />
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− | Autoclave<br />
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− | Complete with water to 333 ml</p>
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− | <p style="text-align:left"><u>Solution 2: (agar)</u><br />
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− | 16 g of agar<br />
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− | Complete with water to 333 ml<br />
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− | Autoclave</p>
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− | <p style="text-align:left"><u>Solution 3: (sugar)</u><br />
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− | In fact we have to put 4 g of sugar in 333 ml of H<sub>2</sub>O. We wanted to see if <i>E. coli</i> bacteria took TPA as a carbon source. So just the water was added. Next, the flask was autoclaved.</p>
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− | <p> </p>
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− | <p style="text-align:left">When the three parts have been autoclaved, 2 more ingredients must be added to the medium aseptically:<br />
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− | 1 ml of 10% magnesium sulfate MgSO<sub>4</sub> (autoclaved too) (1.5 g in 15 ml of H<sub>2</sub>O).<br />
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− | 1 ml of 0.2% Thiamin (vitamin B1).</p>
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− | <p style="text-align:left">At the end the volume of Agar was exceeded (350 ml and not 333 ml). So we had to redo this experiment the next day.</p>
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− | <p style="text-align:center"><u>05-08-15 – Experiment: Preparation of M9 medium – VB, PV</u><br />
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− | <u>Redaction by VB</u></p>
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− | <p style="text-align:left"><strong><u>Aim:</u></strong> Get culture medium for BAP 1 Pfeifer cells.</p>
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− | <p style="text-align:left"><strong><u>Protocol:</u></strong><strong> </strong>(for 1 l medium)</p>
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− | <p style="text-align:left">Agar was directly put in a bottle and salts were dissolved in a beaker with water.</p>
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− | <p style="text-align:left">6 g of Sodium Phosphate Na<sub>2</sub>HPO<sub>4</sub>.H<sub>2</sub>O<br />
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− | 3 g of Potassium Phosphate KH<sub>2</sub>PO<sub>4</sub><br />
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− | 0.5 g of Sodium chloride NaCl<br />
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− | 1 g of Ammonium chloride NH<sub>4</sub>Cl<br />
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− | 16 g of agar<br />
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− | Complete with water to 1l</p>
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− | <p style="text-align:left">When this flask was autoclaved some ingredients were added in aseptical conditions:</p>
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− | <p style="text-align:left">1 ml of 1M Magnesium sulphate MgSO<sub>4</sub> (6.02 g in 50 ml H<sub>2</sub>O)<br />
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− | 1 ml of 0.1 M of Calcium Chloride CaCl<sub>2</sub> (0.55 g in 50 ml of H<sub>2</sub>O)</p>
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− | <p style="text-align:left">A mistake was done yesterday but this time an other did too: The last ingredients were added but they were not sterile so the medium failed again.</p>
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− | <p align="center"><u>06-08-2015 </u><u>–</u><u> </u><u>Experiment: Minimal and M9 medium for TPA Toxicity and BAP 1</u><u> </u><u>–</u><u> </u><u>VB, PV</u><u> </u><br />
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− | <u>Redaction by VB</u><u> </u></p>
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− | <p style="text-align:left"><strong><u>Aim:</u></strong> Have prepared M9 medium and minimal medium for Petri dishes</p>
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− | <p style="text-align:left">We restarted the last two protocols but this time we included the agar for the future Petri dishes.<br />
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− | For the future experiments every medium will have the same concentration of sugar for the bacteria growth, and the test of the TPA toxicity. We began by a theoretical concentration for our Petri dishes: to see the saturation concentration of TPA we decided to do dilutions at 1/10 and 1/20. <br />
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− | This time we decided to make 1.5 l of minimal medium<br />
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− | <strong><u>Protocol:</u></strong><strong><u> </u></strong></p>
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− | <p style="text-align:left"><strong><u>Solution 1: (salts)</u></strong><strong><u> </u></strong><br />
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− | 8 g of Potassium Phosphate K<sub>2</sub>HPO<sub>4</sub> <br />
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− | 3 g of Potassium Phosphate KH<sub>2</sub>PO<sub>4</sub> <br />
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− | 1.5 g of Ammonium Sulfate (NH<sub>4</sub>)2SO<sub>4</sub> <br />
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− | 0.75 g of Sodium Citrate (tribasic, dihydrate) Na<sub>3</sub>C<sub>6</sub>H<sub>5</sub>O<sub>7</sub>.(H<sub>2</sub>O) 2 <br />
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− | Complete with water to 1 l </p>
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− | <p style="text-align:left"><strong><u>Solution 2: (agar)</u></strong><strong><u> </u></strong><br />
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− | 24g of Agar <br />
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− | Complete with water to 500 ml </p>
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− | <p style="text-align:left"><strong><u>Solution 3: </u></strong><strong><u> </u></strong><br />
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− | 500 ml of H<sub>2</sub>O </p>
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− | <p style="text-align:left">We underestimated the time for agar to solidify, so we had solid agar in the <strong><u>test-tube</u></strong>. Moreover one of the flask was not sterile so we contaminated our medium. We decided to return at the first protocol (for 1l). </p>
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− | <p style="text-align:left">At the end of this journey the two media were ready but they did not have the last ingredients.</p>
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− | <p style="text-align:left"> </p>-->
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| <center><h3><em>Enzymatic Assay of NB-Esterase</em></h3></center><br> | | <center><h3><em>Enzymatic Assay of NB-Esterase</em></h3></center><br> |
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| <li style="text-align:left">Gel purification of the digested fragments on 0.8% agarose gel. </li> | | <li style="text-align:left">Gel purification of the digested fragments on 0.8% agarose gel. </li> |
| <li style="text-align:left">Ligation of pDG011 plasmid and BBa_K808030 using T4 DNA ligase. </li> | | <li style="text-align:left">Ligation of pDG011 plasmid and BBa_K808030 using T4 DNA ligase. </li> |
− | <li style="text-align:left">Gel migration on 0.8% agarose gel. </li> | + | <li style="text-align:left">Gel migration on 0.8% agarose gel. </li></br> |
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| <strong><img src="https://static.igem.org/mediawiki/2015/9/91/IGEM_Pasteur-gel--week-10.png" alt="k" width="292" height="455" /></strong><u> </u><br /> | | <strong><img src="https://static.igem.org/mediawiki/2015/9/91/IGEM_Pasteur-gel--week-10.png" alt="k" width="292" height="455" /></strong><u> </u><br /> |