Difference between revisions of "Team:Pasteur Paris/Week 10"

 
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               <p></p><br><br><br>
 
               <p></p><br><br><br>
 
                
 
                
−
<h3><em>Toxicity and solubility of TPA (Terephthalic acid)</em></h3><br>
+
<center><h3><em>Toxicity and solubility of TPA (Terephthalic acid)</em></h3></center></br>
 
<ol>
 
<ol>
−
   <li style="text-align:left">Dissolution of TPA in DMSO (Dimethyl sulfoxide). The best concentration obtained is 62 mg/l </li></br>
+
   <li style="text-align:left">Dissolution of TPA in DMSO (dimethyl sulfoxide). The best concentration obtained is 62 mg/l </li></br>
 
   <li style="text-align:left">Dilution of TPA stock in LB Broth =&gt; TPA  precipitates at a concentration of 3.9 g/l, maximum concentration in LB Broth is  0.9 mg/l </li></br>
 
   <li style="text-align:left">Dilution of TPA stock in LB Broth =&gt; TPA  precipitates at a concentration of 3.9 g/l, maximum concentration in LB Broth is  0.9 mg/l </li></br>
 
   <li style="text-align:left">Solubilization of TPA by NaOH</li></br>
 
   <li style="text-align:left">Solubilization of TPA by NaOH</li></br>
−
   <li style="text-align:left">Creation of a stock solution of soluble TPA in water at 0,5mol/L</li></br>
+
   <li style="text-align:left">Creation of a stock solution of soluble TPA in water at 0.5 mol/l</li></br>
 
   <li style="text-align:left">Test of the TPA Solubilization in LB Broth</li></br>
 
   <li style="text-align:left">Test of the TPA Solubilization in LB Broth</li></br>
−
   <li style="text-align:left"><u>Preparation of different media containing TPA and EG:</u></br>Media 1 to 10 are made in solid form:</li>
+
   <li style="text-align:left"><u>Preparation of different media containing terephthalic acid (TPA) and ethylene glycol (EG):</u></br>
 
   <ol>
 
   <ol>
 +
Media 1 to 10 are made in solid form:
 
     <li style="text-align:left">M9</li>
 
     <li style="text-align:left">M9</li>
−
     <li style="text-align:left">M9 + TPA (1.2mM)</li>
+
     <li style="text-align:left">M9 + TPA (1.2 mM)</li>
−
     <li style="text-align:left">M9 + TPA (6mM)</li>
+
     <li style="text-align:left">M9 + TPA (6.0 mM)</li>
−
     <li style="text-align:left">M9 + TPA (12mM)</li>
+
     <li style="text-align:left">M9 + TPA (12.0 mM)</li>
−
     <li style="text-align:left">M9 + sucrose (1.2mM)</li>
+
     <li style="text-align:left">M9 + sucrose (1.2 mM)</li>
−
     <li style="text-align:left">M9 + sucrose (6mM)</li>
+
     <li style="text-align:left">M9 + sucrose (6.0 mM)</li>
−
     <li style="text-align:left">M9 + sucrose (12mM)</li>
+
     <li style="text-align:left">M9 + sucrose (12.0 mM)</li>
−
     <li style="text-align:left">M9 + sucrose + TPA (6mM)</li>
+
     <li style="text-align:left">M9 + sucrose + TPA (6.0 mM)</li>
−
     <li style="text-align:left">M9 + sucrose + TPA (1,2mM)</li>
+
     <li style="text-align:left">M9 + sucrose + TPA (1.2 mM)</li>
 +
Media 11 to 15 are made in both solid and liquid form:
 +
    <li style="text-align:left">LB Broth</li>
 +
    <li style="text-align:left">LB + TPA (1.2 mM)</li>
 +
    <li style="text-align:left">LB + TPA (6.0 mM)</li>
 +
    <li style="text-align:left">LB + TPA (12.0 mM)</li>
 +
    <li style="text-align:left">LB + TPA (20.0 mM)</li>
 +
  </ol>
 +
  </li>
 +
  </br>
 +
  <li style="text-align:left">Growth curve of BAP 1 (E. coli, kindly provided by Blaine Pfeifer, U. Buffalo)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></br>
 
   </ol>
 
   </ol>
−
  <li style="text-align:left">Media 11 to 15 are made in both solid and liquid form:</li>
 
  
−
 
+
<center><h3><em>Enzymatic Assay of NB-Esterase</em></h3></center><br>
−
<!--  <u>3 solutions: </u><br />
+
−
<br />
+
−
  <u>Solution 1: (salts)</u><br />
+
−
  5.3 g of Potassium  Phosphate (dibasic) K<sub>2</sub>HPO<sub>4</sub><br />
+
−
  2 g of Potassium  Phosphate (monobasic) KH<sub>2</sub>PO<sub>4</sub><br />
+
−
  1 g of Ammonium  Sulfate (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>.<br />
+
−
  0.5 g of Sodium Citrate (tribasic, dihydrate) <br />
+
−
  Autoclave<br />
+
−
  Complete with  water to 333 ml</p>
+
−
<p style="text-align:left"><u>Solution 2: (agar)</u><br />
+
−
  16 g of agar<br />
+
−
  Complete with  water to 333 ml<br />
+
−
  Autoclave</p>
+
−
<p style="text-align:left"><u>Solution 3: (sugar)</u><br />
+
−
  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>
+
−
<p>&nbsp;</p>
+
−
<p style="text-align:left">When the three  parts have been autoclaved, 2 more ingredients must be added to the medium aseptically:<br />
+
−
  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 />
+
−
  1 ml of 0.2%  Thiamin (vitamin B1).</p>
+
−
<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>
+
−
<p style="text-align:center"><u>05-08-15 – Experiment: Preparation of M9  medium – VB, PV</u><br />
+
−
  <u>Redaction by VB</u></p>
+
−
<p style="text-align:left"><strong><u>Aim:</u></strong> Get culture medium for BAP 1 Pfeifer cells.</p>
+
−
<p style="text-align:left"><strong><u>Protocol:</u></strong><strong>&nbsp;</strong>(for 1 l medium)</p>
+
−
<p style="text-align:left">Agar was directly  put in a bottle and salts were dissolved in a beaker with water.</p>
+
−
<p style="text-align:left">6 g of Sodium  Phosphate Na<sub>2</sub>HPO<sub>4</sub>.H<sub>2</sub>O<br />
+
−
  3 g of Potassium  Phosphate KH<sub>2</sub>PO<sub>4</sub><br />
+
−
  0.5 g of Sodium  chloride NaCl<br />
+
−
  1 g of Ammonium  chloride NH<sub>4</sub>Cl<br />
+
−
  16 g of agar<br />
+
−
  Complete with water to 1l</p>
+
−
<p style="text-align:left">When this flask  was autoclaved some ingredients were added in aseptical conditions:</p>
+
−
<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 />
+
−
  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>
+
−
<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>
+
−
<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 />
+
−
  <u>Redaction by VB</u><u> </u></p>
+
−
<p style="text-align:left"><strong><u>Aim:</u></strong> Have prepared M9  medium and minimal medium for Petri dishes</p>
+
−
<p style="text-align:left">We restarted the last two protocols but this time we included the agar for the future Petri dishes.<br />
+
−
  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 />
+
−
  This time we  decided to make 1.5 l of minimal medium<br />
+
−
  <strong><u>Protocol:</u></strong><strong><u> </u></strong></p>
+
−
<p style="text-align:left"><strong><u>Solution 1: (salts)</u></strong><strong><u> </u></strong><br />
+
−
  8 g of Potassium Phosphate K<sub>2</sub>HPO<sub>4</sub> <br />
+
−
  3 g of Potassium Phosphate KH<sub>2</sub>PO<sub>4</sub> <br />
+
−
  1.5 g of Ammonium Sulfate (NH<sub>4</sub>)2SO<sub>4</sub> <br />
+
−
  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 />
+
−
  Complete with water to 1 l </p>
+
−
<p style="text-align:left"><strong><u>Solution 2: (agar)</u></strong><strong><u> </u></strong><br />
+
−
  24g of Agar <br />
+
−
  Complete with water to 500 ml </p>
+
−
<p style="text-align:left"><strong><u>Solution 3: </u></strong><strong><u> </u></strong><br />
+
−
  500 ml of H<sub>2</sub>O </p>
+
−
<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>
+
−
<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>
+
−
<p style="text-align:left">&nbsp;</p>-->
+
−
 
+
−
<h3><em>Enzymatic Assay of NB-Esterase</em></h3><br>
+
 
<ol>
 
<ol>
−
<li style="text-align:left">XbaI ans PstI enzymatic digest of the Biobrick BBa_K808030 and pDG011 plasmid.</li>
+
<li style="text-align:left">XbaI ans PstI enzymatic digestion of the Biobrick BBa_K808030 and pDG011 plasmid.</li></br>
−
<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></br>
−
<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></br>
−
<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>
−
 
+
</ol>
−
<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 />
+
<center><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><u>Figure 8:</u></strong> Gel for verification of ligation. <u> </u><br />
+
<strong><u>Figure 8:</u></strong> Gel for verification of the ligation of BBa_K808030 and pDG011 plasmid.<br />
−
There is no band for the ligation sample.
+
There is no band for the ligation sample.</center>
 
</p>
 
</p>
 
</br>
 
</br>
−
<h3><em>Gibson Assembly</em></h3>
+
 
 +
<center><h3><em>Gibson Assembly</em></h3></center>
 
</br>
 
</br>
−
   <li style="text-align:left">Ligation of ThpA1,  Transp_, 1392932_, using T4 DNA ligase. </li>
+
<ol>
 +
   <li style="text-align:left">Ligation of TphA1/,  Transp_, BBa_K1392932_, using T4 DNA ligase. </li></br>
 
   <li style="text-align:left">DNA concentration  measurement using a spectrophotometer. </li>
 
   <li style="text-align:left">DNA concentration  measurement using a spectrophotometer. </li>
 
</ol>
 
</ol>
 
<table border="1" cellspacing="0" cellpadding="0" width="643" align="center">
 
<table border="1" cellspacing="0" cellpadding="0" width="643" align="center">
 
   <tr>
 
   <tr>
−
     <td width="74" valign="center"><p><strong>Biobrick</strong></p></td>
+
     <td width="90" valign="center"><p><strong>Biobrick</strong></p></td>
−
     <td width="106" valign="top"><p>DNA   Concentration (ng/µl)</p></td>
+
     <td width="106" valign="top"><p><strong>DNA Concentration (ng/µl)</strong></p></td>
−
     <td width="88" valign="top"><p>OD (230 nm)</strong></p></td>
+
     <td width="88" valign="top"><p><strong>OD (230 nm)</strong></p></td>
−
     <td width="88" valign="top"><p>OD (260 nm)</strong></p></td>
+
     <td width="88" valign="top"><p><strong>OD (260 nm)</strong></p></td>
−
     <td width="71" valign="top"><p>OD (280 nm)</strong></p></td>
+
     <td width="71" valign="top"><p><strong>OD (280 nm)</strong></p></td>
−
     <td width="106" valign="top"><p>OD (260 nm) / OD (280 nm)</strong></p></td>
+
     <td width="106" valign="top"><p><strong>OD (260 nm) / OD (280 nm)</strong></p></td>
−
     <td width="109" valign="top"><p>OD (260 nm) / OD (230 nm)</strong></p></td>
+
     <td width="109" valign="top"><p><strong>OD (260 nm) / OD (230 nm)</strong></p></td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="74" valign="top"><p><strong>808010</strong></p></td>
+
     <td width="90" valign="top"><p><strong>808010</strong></p></td>
 
     <td width="106" valign="top"><p>30.00</p></td>
 
     <td width="106" valign="top"><p>30.00</p></td>
−
     <td width="88" valign="top"><p>9.33</p></td>
+
     <td width="90" valign="top"><p>9.33</p></td>
−
     <td width="88" valign="top"><p>0.75</p></td>
+
     <td width="90" valign="top"><p>0.75</p></td>
−
     <td width="71" valign="top"><p>0,30</p></td>
+
     <td width="90" valign="top"><p>0,30</p></td>
 
     <td width="106" valign="top"><p>1.87</p></td>
 
     <td width="106" valign="top"><p>1.87</p></td>
 
     <td width="109" valign="top"><p>0.06</p></td>
 
     <td width="109" valign="top"><p>0.06</p></td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="74" valign="top"><p><strong>808011</strong></p></td>
+
     <td width="90" valign="top"><p><strong>808011</strong></p></td>
 
     <td width="106" valign="top"><p>50.00</p></td>
 
     <td width="106" valign="top"><p>50.00</p></td>
−
     <td width="88" valign="top"><p>11.78</p></td>
+
     <td width="90" valign="top"><p>11.78</p></td>
−
     <td width="88" valign="top"><p>1.00</p></td>
+
     <td width="90" valign="top"><p>1.00</p></td>
−
     <td width="71" valign="top"><p>0.63</p></td>
+
     <td width="90" valign="top"><p>0.63</p></td>
−
     <td width="106" valign="top"><p>1.61</p></td>
+
     <td width="115" valign="top"><p>1.61</p></td>
−
     <td width="109" valign="top"><p>0.09</p></td>
+
     <td width="115" valign="top"><p>0.09</p></td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="74" valign="top"><p><strong>808012</strong></p></td>
+
     <td width="90" valign="top"><p><strong>808012</strong></p></td>
 
     <td width="106" valign="top"><p>17.50</p></td>
 
     <td width="106" valign="top"><p>17.50</p></td>
−
     <td width="88" valign="top"><p>10.93</p></td>
+
     <td width="90" valign="top"><p>10.93</p></td>
−
     <td width="88" valign="top"><p>0.33</p></td>
+
     <td width="90" valign="top"><p>0.33</p></td>
−
     <td width="71" valign="top"><p>0.18</p></td>
+
     <td width="90" valign="top"><p>0.18</p></td>
−
     <td width="106" valign="top"><p>0.84</p></td>
+
     <td width="115" valign="top"><p>0.84</p></td>
−
     <td width="109" valign="top"><p>0.03</p></td>
+
     <td width="115" valign="top"><p>0.03</p></td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="74" valign="top"><p><strong>808013</strong></p></td>
+
     <td width="90" valign="top"><p><strong>808013</strong></p></td>
 
     <td width="106" valign="top"><p>5.00</p></td>
 
     <td width="106" valign="top"><p>5.00</p></td>
−
     <td width="88" valign="top"><p>10.55</p></td>
+
     <td width="90" valign="top"><p>10.55</p></td>
−
     <td width="88" valign="top"><p>0.10</p></td>
+
     <td width="90" valign="top"><p>0.10</p></td>
−
     <td width="71" valign="top"><p>0.06</p></td>
+
     <td width="90" valign="top"><p>0.06</p></td>
−
     <td width="106" valign="top"><p>1.56</p></td>
+
     <td width="115" valign="top"><p>1.56</p></td>
−
     <td width="109" valign="top"><p>0.01</p></td>
+
     <td width="115" valign="top"><p>0.01</p></td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="74" valign="top"><p><strong>80801</strong>v</p></td>
+
     <td width="90" valign="top"><p><strong>808014</strong></p></td>
 
     <td width="106" valign="top"><p>22.5</p></td>
 
     <td width="106" valign="top"><p>22.5</p></td>
−
     <td width="88" valign="top"><p>12.13</p></td>
+
     <td width="90" valign="top"><p>12.13</p></td>
−
     <td width="88" valign="top"><p>0.47</p></td>
+
     <td width="90" valign="top"><p>0.47</p></td>
−
     <td width="71" valign="top"><p>0.30</p></td>
+
     <td width="90" valign="top"><p>0.30</p></td>
−
     <td width="106" valign="top"><p>1.54</p></td>
+
     <td width="115" valign="top"><p>1.54</p></td>
−
     <td width="109" valign="top"><p>0.04</p></td>
+
     <td width="115" valign="top"><p>0.04</p></td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="74" valign="top"><p><strong>1095000</strong></p></td>
+
     <td width="90" valign="top"><p><strong>1095000</strong></p></td>
 
     <td width="106" valign="top"><p>37.50</p></td>
 
     <td width="106" valign="top"><p>37.50</p></td>
−
     <td width="88" valign="top"><p>18.48</p></td>
+
     <td width="90" valign="top"><p>18.48</p></td>
−
     <td width="88" valign="top"><p>0.78</p></td>
+
     <td width="90" valign="top"><p>0.78</p></td>
−
     <td width="71" valign="top"><p>0.43</p></td>
+
     <td width="90" valign="top"><p>0.43</p></td>
−
     <td width="106" valign="top"><p>1.83</p></td>
+
     <td width="115" valign="top"><p>1.83</p></td>
−
     <td width="109" valign="top"><p>0.04</p></td>
+
     <td width="115" valign="top"><p>0.04</p></td>
 
   </tr>
 
   </tr>
 
</table>
 
</table>
Line 269: Line 225:
 
<br>
 
<br>
 
</div>
 
</div>
 +
 +
 
<style type="text/css">
 
<style type="text/css">
 
.hautdepage {
 
.hautdepage {

Latest revision as of 02:15, 19 September 2015

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week 10

08/03 - 08/07




Toxicity and solubility of TPA (Terephthalic acid)


  1. Dissolution of TPA in DMSO (dimethyl sulfoxide). The best concentration obtained is 62 mg/l

  2. Dilution of TPA stock in LB Broth => TPA precipitates at a concentration of 3.9 g/l, maximum concentration in LB Broth is 0.9 mg/l

  3. Solubilization of TPA by NaOH

  4. Creation of a stock solution of soluble TPA in water at 0.5 mol/l

  5. Test of the TPA Solubilization in LB Broth

  6. Preparation of different media containing terephthalic acid (TPA) and ethylene glycol (EG):
      Media 1 to 10 are made in solid form:
    1. M9
    2. M9 + TPA (1.2 mM)
    3. M9 + TPA (6.0 mM)
    4. M9 + TPA (12.0 mM)
    5. M9 + sucrose (1.2 mM)
    6. M9 + sucrose (6.0 mM)
    7. M9 + sucrose (12.0 mM)
    8. M9 + sucrose + TPA (6.0 mM)
    9. M9 + sucrose + TPA (1.2 mM)
      10. Media 11 to 15 are made in both solid and liquid form:
    10. LB Broth
    11. LB + TPA (1.2 mM)
    12. LB + TPA (6.0 mM)
    13. LB + TPA (12.0 mM)
    14. LB + TPA (20.0 mM)

  7. Growth curve of BAP 1 (E. coli, kindly provided by Blaine Pfeifer, U. Buffalo)in media 11 to 14 in order to assess TPA toxicity.

  8. 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.

Enzymatic Assay of NB-Esterase


  1. XbaI ans PstI enzymatic digestion of the Biobrick BBa_K808030 and pDG011 plasmid.

  2. Gel purification of the digested fragments on 0.8% agarose gel.

  3. Ligation of pDG011 plasmid and BBa_K808030 using T4 DNA ligase.

  4. Gel migration on 0.8% agarose gel.

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Figure 8: Gel for verification of the ligation of BBa_K808030 and pDG011 plasmid.
There is no band for the ligation sample.


Gibson Assembly


  1. Ligation of TphA1/, Transp_, BBa_K1392932_, using T4 DNA ligase.

  2. DNA concentration measurement using a spectrophotometer.

Biobrick

DNA Concentration (ng/µl)

OD (230 nm)

OD (260 nm)

OD (280 nm)

OD (260 nm) / OD (280 nm)

OD (260 nm) / OD (230 nm)

808010

30.00

9.33

0.75

0,30

1.87

0.06

808011

50.00

11.78

1.00

0.63

1.61

0.09

808012

17.50

10.93

0.33

0.18

0.84

0.03

808013

5.00

10.55

0.10

0.06

1.56

0.01

808014

22.5

12.13

0.47

0.30

1.54

0.04

1095000

37.50

18.48

0.78

0.43

1.83

0.04