Team:Evry/Notebook/w9

From 2013.igem.org

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Line 5: Line 5:
<div id="mainTextcontainer">
<div id="mainTextcontainer">
-
<h1>Week 9: 12th August - 18th August</h1>
+
<h1>Week 9: 12<sup>th</sup> August - 18<sup>th</sup> August</h1>
<h2>TECAN</h2>
<h2>TECAN</h2>
 +
 +
 +
<h3>Medium preparation</h3>
 +
 +
<p>
 +
For the Tecan analysis we must use M9 medium and not a classical LB medium because turbidity and fluorescence of our sample are measured. Then, in order to obtain good results, the medium must not emit a side signal, that is why we use the M9 medium.
 +
</p>
 +
 +
<p>
 +
<u>Composition for 50 mL of:</u>
 +
</p>
 +
 +
<table cellpadding="10" cellspacing="0" align='center' border="1">
 +
<thead>
 +
  <tr>
 +
  <th align="center">
 +
    Reagent
 +
  </th>
 +
  <th align="center">
 +
    M9 medium (without iron)
 +
  </th>
 +
  <th align="center">
 +
    M9 medium (with iron)
 +
  </th>
 +
  </tr>
 +
</thead>
 +
 +
<tbody>
 +
  <tr>
 +
  <td align='center'>
 +
    M9 salt (5X)
 +
  </td>
 +
  <td colspan="2" align='center'>
 +
    10 mL
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td align='center'>
 +
    CaCl<sub>2</sub> (1M)
 +
  </td>
 +
  <td colspan="2" align='center'>
 +
    5 µL
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td align='center'>
 +
    MgSO<sub>4</sub> (1M)
 +
  </td>
 +
  <td colspan="2" align='center'>
 +
    100 µL
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td align='center'>
 +
    Glycerol (50%)
 +
  </td>
 +
  <td colspan="2" align='center'>
 +
    800 µL
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td align='center'>
 +
    Thiamine
 +
  </td>
 +
  <td colspan="2" align='center'>
 +
    5 µL
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td align='center'>
 +
    NaOH (pH 7.4)
 +
  </td>
 +
  <td colspan="2" align='center'>
 +
    12.5 µL
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td align='center'>
 +
    H<sub>2</sub>O
 +
  </td>
 +
  <td align='center'>
 +
    40 mL
 +
  </td>
 +
  <td align='center'>
 +
    39 mL
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td align='center'>
 +
    FeSO<sub>4</sub> (10mM)
 +
  </td>
 +
  <td align='center'>
 +
    -
 +
  </td>
 +
  <td align='center'>
 +
    50 µL
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td align='center'>
 +
    Casamino acids (0.2%)
 +
  </td>
 +
  <td align='center'>
 +
    -
 +
  </td>
 +
  <td align='center'>
 +
    1 mL
 +
  </td>
 +
  </tr>
 +
</tbody>
 +
</table>
 +
 +
<p>
 +
Once the mixture is prepared, the medium must be filtered to be sterilised using 0.22 µm filter.
 +
</p>
 +
 +
<h3>TECAN analysis</h3>
<p>
<p>
Line 33: Line 158:
After 8 hours of culture, the 96 wells plate has been prepare (see following scheme).
After 8 hours of culture, the 96 wells plate has been prepare (see following scheme).
</p>
</p>
 +
 +
<div align="center"><img src='https://static.igem.org/mediawiki/2013/1/1a/Plan_Tecan_12.08.2013.png' width="500px"/></div>
<h1>AJOUTER DETAIL DES CYCLES</h1>
<h1>AJOUTER DETAIL DES CYCLES</h1>
 +
 +
 +
 +
<h2>1<sup>st</sup> construction</h2>
 +
 +
<h3>Sequencing preparation</h3>
 +
 +
<p>
 +
Preculture with LB medium (with carbenicillin) have been launched for Top10 transformed with our 1<sup>st</sup> construction, then minipreps have been realized using the miniprep kit from Machrey Nagel.
 +
</p>
 +
 +
<table cellpadding="10" cellspacing="0" align='center' border="1">
 +
<thead>
 +
  <tr>
 +
  <th align="center">
 +
    Name
 +
  </th>
 +
  <th align="center">
 +
    Clone
 +
  </th>
 +
  <th align="center">
 +
    Concentration
 +
  </th>
 +
  <th align="center">
 +
    260/280
 +
  </th>
 +
  <th align="center">
 +
    260/230
 +
  </th>
 +
  <th align="center">
 +
    Sequencing
 +
  </th>
 +
  </tr>
 +
</thead>
 +
 +
<tbody>
 +
  <tr>
 +
  <td rowspan="4" align="center">
 +
    <p>AceB Fur Binding Site
 +
    <br/>(with LacI-LVA)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    66.4 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.89
 +
  </td>
 +
  <td align="center">
 +
    1.92
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    53.8 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.85
 +
  </td>
 +
  <td align="center">
 +
    1.61
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    3
 +
  </td>
 +
  <td align="center">
 +
    64.9 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.89
 +
  </td>
 +
  <td align="center">
 +
    1.87
 +
  </td>
 +
  <td align="center">
 +
    Bad
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    4
 +
  </td>
 +
  <td align="center">
 +
    71.8 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.84
 +
  </td>
 +
  <td align="center">
 +
    1.66
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td rowspan="4" align="center">
 +
    <p>ybiL Fur Binding Site
 +
    <br/>(with LacI-LVA)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    49.1 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.94
 +
  </td>
 +
  <td align="center">
 +
    1.80
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    58.6 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.99
 +
  </td>
 +
  <td align="center">
 +
    2.32
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    3
 +
  </td>
 +
  <td align="center">
 +
    41.2 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.97
 +
  </td>
 +
  <td align="center">
 +
    2.44
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    4
 +
  </td>
 +
  <td align="center">
 +
    31.8 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.93
 +
  </td>
 +
  <td align="center">
 +
    2.16
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td rowspan="4" align="center">
 +
    <p>yncE Fur Binding Site
 +
    <br/>(with LacI-LVA)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    43.7 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.95
 +
  </td>
 +
  <td align="center">
 +
    2.17
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    45.6 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.97
 +
  </td>
 +
  <td align="center">
 +
    2.22
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    3
 +
  </td>
 +
  <td align="center">
 +
    34.8 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    2.00
 +
  </td>
 +
  <td align="center">
 +
    2.41
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    4
 +
  </td>
 +
  <td align="center">
 +
    42.5 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    2.00
 +
  </td>
 +
  <td align="center">
 +
    2.28
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td rowspan="3" align="center">
 +
    <p>Fes Fur Binding Site
 +
    <br/>(with LacI-LVA)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    39.4 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.89
 +
  </td>
 +
  <td align="center">
 +
    1.82
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    36.6 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    4
 +
  </td>
 +
  <td align="center">
 +
    35.3 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.81
 +
  </td>
 +
  <td align="center">
 +
    1.13
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td rowspan="2" align="center">
 +
    <p>FepA Fur Binding Site
 +
    <br/>(with LacI-LVA)</p>
 +
  </td>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    50.7 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.90
 +
  </td>
 +
  <td align="center">
 +
    1.61
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    4
 +
  </td>
 +
  <td align="center">
 +
    57.4 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.92
 +
  </td>
 +
  <td align="center">
 +
    1.88
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
</tbody>
 +
</table>
<h2>2<sup>nd</sup> construction</h2>
<h2>2<sup>nd</sup> construction</h2>
 +
 +
<h3>Bacterial isolation</h3>
 +
 +
<p>
 +
4 clones of a Golden Gate plate are isolated for each construction. It allows us to save the clone on a plate.
 +
</p>
 +
 +
<div align="center"><img src='https://static.igem.org/mediawiki/2013/f/f4/Isolement.png' width="250px"/><br/><i>Top 10 transformed with the synthetic Fur Binding<br/>Site n°1, 2 and 3 isolated on a plate.</i></div>
 +
<h3>Sequencing preparation</h3>
<h3>Sequencing preparation</h3>
Line 61: Line 548:
   <th align="center">
   <th align="center">
     260/230
     260/230
 +
  </th>
 +
  <th align="center">
 +
    Sequencing
   </th>
   </th>
   </tr>
   </tr>
  </thead>
  </thead>
 +
  <tbody>
  <tbody>
   <tr>
   <tr>
Line 81: Line 572:
   <td align="center">
   <td align="center">
     0.68
     0.68
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 95: Line 589:
   <td align="center">
   <td align="center">
     0.44
     0.44
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 109: Line 606:
   <td align="center">
   <td align="center">
     0.13
     0.13
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 123: Line 623:
   <td align="center">
   <td align="center">
     0.72
     0.72
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 142: Line 645:
   <td align="center">
   <td align="center">
     0.40
     0.40
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 156: Line 662:
   <td align="center">
   <td align="center">
     0.29
     0.29
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 170: Line 679:
   <td align="center">
   <td align="center">
     0.71
     0.71
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 184: Line 696:
   <td align="center">
   <td align="center">
     0.68
     0.68
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 203: Line 718:
   <td align="center">
   <td align="center">
     0.78
     0.78
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 217: Line 735:
   <td align="center">
   <td align="center">
     0.78
     0.78
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 231: Line 752:
   <td align="center">
   <td align="center">
     1.21
     1.21
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 250: Line 774:
   <td align="center">
   <td align="center">
     0.99
     0.99
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 264: Line 791:
   <td align="center">
   <td align="center">
     0.81
     0.81
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 278: Line 808:
   <td align="center">
   <td align="center">
     4.23
     4.23
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 297: Line 830:
   <td align="center">
   <td align="center">
     2.16
     2.16
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 311: Line 847:
   <td align="center">
   <td align="center">
     1.21
     1.21
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 325: Line 864:
   <td align="center">
   <td align="center">
     1.52
     1.52
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 339: Line 881:
   <td align="center">
   <td align="center">
     1.65
     1.65
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 358: Line 903:
   <td align="center">
   <td align="center">
     1.93
     1.93
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 372: Line 920:
   <td align="center">
   <td align="center">
     1.22
     1.22
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 386: Line 937:
   <td align="center">
   <td align="center">
     1.22
     1.22
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 405: Line 959:
   <td align="center">
   <td align="center">
     0.84
     0.84
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 419: Line 976:
   <td align="center">
   <td align="center">
     1.10
     1.10
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 433: Line 993:
   <td align="center">
   <td align="center">
     1.43
     1.43
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 447: Line 1,010:
   <td align="center">
   <td align="center">
     1.61
     1.61
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 466: Line 1,032:
   <td align="center">
   <td align="center">
     1.37
     1.37
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 480: Line 1,049:
   <td align="center">
   <td align="center">
     1.61
     1.61
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 494: Line 1,066:
   <td align="center">
   <td align="center">
     1.79
     1.79
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 508: Line 1,083:
   <td align="center">
   <td align="center">
     1.76
     1.76
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 527: Line 1,105:
   <td align="center">
   <td align="center">
     1.22
     1.22
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 541: Line 1,122:
   <td align="center">
   <td align="center">
     1.67
     1.67
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 555: Line 1,139:
   <td align="center">
   <td align="center">
     1.62
     1.62
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 569: Line 1,156:
   <td align="center">
   <td align="center">
     1.44
     1.44
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 588: Line 1,178:
   <td align="center">
   <td align="center">
     1.49
     1.49
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 602: Line 1,195:
   <td align="center">
   <td align="center">
     1.37
     1.37
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 616: Line 1,212:
   <td align="center">
   <td align="center">
     1.44
     1.44
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 630: Line 1,229:
   <td align="center">
   <td align="center">
     1.20
     1.20
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 649: Line 1,251:
   <td align="center">
   <td align="center">
     1.20
     1.20
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
   <tr>
   <tr>
   <td align="center">
   <td align="center">
-
     3
+
     2
   </td>
   </td>
   <td align="center">
   <td align="center">
Line 663: Line 1,268:
   <td align="center">
   <td align="center">
     1.16
     1.16
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 677: Line 1,285:
   <td align="center">
   <td align="center">
     1.16
     1.16
 +
  </td>
 +
  <td align="center">
 +
    Bad
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td rowspan="4" align="center">
 +
    <p>Fur Binding Site n°1
 +
    <br/>(with LacI-LVA)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    99.7 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.73
 +
  </td>
 +
  <td align="center">
 +
    0.99
 +
  </td>
 +
  <td align="center">
 +
    Bad
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    46.9 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.78
 +
  </td>
 +
  <td align="center">
 +
    0.90
 +
  </td>
 +
  <td align="center">
 +
    Bad
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    3
 +
  </td>
 +
  <td align="center">
 +
    55.8 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.87
 +
  </td>
 +
  <td align="center">
 +
    1.75
 +
  </td>
 +
  <td align="center">
 +
    Bad
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    4
 +
  </td>
 +
  <td align="center">
 +
    35.3 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.84
 +
  </td>
 +
  <td align="center">
 +
    0.99
 +
  </td>
 +
  <td align="center">
 +
    Bad
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td rowspan="3" align="center">
 +
    <p>Fur Binding Site n°2
 +
    <br/>(with LacI-LVA)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    43.8 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.83
 +
  </td>
 +
  <td align="center">
 +
    1.01
 +
  </td>
 +
  <td align="center">
 +
    Bad
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    47.0 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.77
 +
  </td>
 +
  <td align="center">
 +
    0.92
 +
  </td>
 +
  <td align="center">
 +
    Bad
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    3
 +
  </td>
 +
  <td align="center">
 +
    32.4 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.78
 +
  </td>
 +
  <td align="center">
 +
    1.09
 +
  </td>
 +
  <td align="center">
 +
    Bad
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td rowspan="4" align="center">
 +
    <p>Fur Binding Site n°3
 +
    <br/>(with LacI-LVA)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    57.8 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.84
 +
  </td>
 +
  <td align="center">
 +
    1.45
 +
  </td>
 +
  <td align="center">
 +
    Bad
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    51.3 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.79
 +
  </td>
 +
  <td align="center">
 +
    1.04
 +
  </td>
 +
  <td align="center">
 +
    Bad
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    3
 +
  </td>
 +
  <td align="center">
 +
    41.6 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.88
 +
  </td>
 +
  <td align="center">
 +
    1.75
 +
  </td>
 +
  <td align="center">
 +
    Bad
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    4
 +
  </td>
 +
  <td align="center">
 +
    112.6 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.86
 +
  </td>
 +
  <td align="center">
 +
    1.81
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 695: Line 1,508:
<table cellpadding="10" cellspacing="0" align='center' border="1">
<table cellpadding="10" cellspacing="0" align='center' border="1">
-
<thead>
+
<thead>
   <tr>
   <tr>
 +
  <th align="center">
 +
    Name
 +
  </th>
   <th align="center">
   <th align="center">
     Clone
     Clone
Line 708: Line 1,524:
   <th align="center">
   <th align="center">
     260/230
     260/230
 +
  </th>
 +
  <th align="center">
 +
    Sequencing
   </th>
   </th>
   </tr>
   </tr>
  </thead>
  </thead>
 +
  <tbody>
  <tbody>
   <tr>
   <tr>
 +
  <td rowspan="3" align="center">
 +
    <p>Testing construction
 +
    <br/>(Lac O with sfGFP)</p>
 +
  </td>
   <td align="center">
   <td align="center">
     1
     1
Line 724: Line 1,548:
   <td align="center">
   <td align="center">
     2.29
     2.29
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 739: Line 1,566:
   <td align="center">
   <td align="center">
     1.78
     1.78
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 754: Line 1,584:
   <td align="center">
   <td align="center">
     1.60
     1.60
 +
  </td>
 +
  <td align="center">
 +
    Good
   </td>
   </td>
   </tr>
   </tr>
-
  </body>
+
  </tbody>
</table>
</table>
Line 768: Line 1,601:
<li>Enterobactin biosynthesis genes (Ent B, C and E)
<li>Enterobactin biosynthesis genes (Ent B, C and E)
</ol>
</ol>
 +
 +
<p>
 +
5 µL of each Golden Gate product are transformed into Top10 competent cells. To controlled the quality of the transformation, a negative controle (transformation procedure without plasmid) and a positive controle (transformation procedure with plasmid 1A3) are made.
 +
</p>
 +
 +
<p>
 +
Unfortunatly, the Golden Gate reactions did not work.
 +
</p>
<h2>Others</h2>
<h2>Others</h2>
Line 778: Line 1,619:
<table cellpadding="10" cellspacing="0" align='center' border="1">
<table cellpadding="10" cellspacing="0" align='center' border="1">
-
<thead>
+
<thead>
   <tr>
   <tr>
   <th align="center">
   <th align="center">
Line 800: Line 1,641:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --- ng/µL
+
     265 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     ---
+
     1.84
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     ---
+
     1.78
   </td>
   </td>
   </tr>
   </tr>
Line 815: Line 1,656:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --- ng/µL
+
     237.8 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     ---
+
     1.82
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     ---
+
     1.67
   </td>
   </td>
   </tr>
   </tr>
Line 830: Line 1,671:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --- ng/µL
+
     240.8 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     ---
+
     1.84
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     ---
+
     1.81
   </td>
   </td>
   </tr>
   </tr>
-
  </body>
+
  </tbody>
</table>
</table>
Line 846: Line 1,687:
</p>
</p>
-
<h1>AJOUTER LES RESULTATS DE MINIPREPS</h1>
+
<h3>Competent cells</h3>
-
 
+
-
<div align="center"><img src='https://static.igem.org/mediawiki/2013/f/f4/Isolement.png' width="200px"/></div>
+
 +
<p>
 +
Top10 competent cells had been made last week and we tested them on different antibiotics (Carbenicillin, Kanamycin, Chloramphenicol). After incubation at 37°C, overnight, no contaminant had been observed.
 +
</p>
</div>
</div>

Latest revision as of 10:25, 28 August 2013

Iron coli project

Week 9: 12th August - 18th August

TECAN

Medium preparation

For the Tecan analysis we must use M9 medium and not a classical LB medium because turbidity and fluorescence of our sample are measured. Then, in order to obtain good results, the medium must not emit a side signal, that is why we use the M9 medium.

Composition for 50 mL of:

Reagent M9 medium (without iron) M9 medium (with iron)
M9 salt (5X) 10 mL
CaCl2 (1M) 5 µL
MgSO4 (1M) 100 µL
Glycerol (50%) 800 µL
Thiamine 5 µL
NaOH (pH 7.4) 12.5 µL
H2O 40 mL 39 mL
FeSO4 (10mM) - 50 µL
Casamino acids (0.2%) - 1 mL

Once the mixture is prepared, the medium must be filtered to be sterilised using 0.22 µm filter.

TECAN analysis

Using the TECAN analysis, we are measuring the capacity of repression of the natural binding site that we extract from the E.coli's genome.

Preculture with M9 medium (with carbenicillin) have been launched for BL21 transformed with our 1st construction:

  • Natural Fur Binding Site of Fec A + sfGFP (clone 1, 2, 3)
  • Natural Fur Binding Site of Fep A + sfGFP (clone 1, 2, 3)
  • Natural Fur Binding Site of Ace B + sfGFP (clone 1, 2, 3)

Note: Preculture have been made in M9 medium with iron in oder to inhibit the expression of sfGFP.

After one night of culture, time the precultures have been refreshed by diluting them 200 times in M9 medium (with iron and carbenicillin).

After 8 hours of culture, the 96 wells plate has been prepare (see following scheme).

AJOUTER DETAIL DES CYCLES

1st construction

Sequencing preparation

Preculture with LB medium (with carbenicillin) have been launched for Top10 transformed with our 1st construction, then minipreps have been realized using the miniprep kit from Machrey Nagel.

Name Clone Concentration 260/280 260/230 Sequencing

AceB Fur Binding Site
(with LacI-LVA)

1 66.4 ng/µL 1.89 1.92 Good
2 53.8 ng/µL 1.85 1.61 Good
3 64.9 ng/µL 1.89 1.87 Bad
4 71.8 ng/µL 1.84 1.66 Good

ybiL Fur Binding Site
(with LacI-LVA)

1 49.1 ng/µL 1.94 1.80 Good
2 58.6 ng/µL 1.99 2.32 Good
3 41.2 ng/µL 1.97 2.44 Good
4 31.8 ng/µL 1.93 2.16 Good

yncE Fur Binding Site
(with LacI-LVA)

1 43.7 ng/µL 1.95 2.17 Good
2 45.6 ng/µL 1.97 2.22 Good
3 34.8 ng/µL 2.00 2.41 Good
4 42.5 ng/µL 2.00 2.28 Good

Fes Fur Binding Site
(with LacI-LVA)

1 39.4 ng/µL 1.89 1.82 Good
2 36.6 ng/µL - - Good
4 35.3 ng/µL 1.81 1.13 Good

FepA Fur Binding Site
(with LacI-LVA)

2 50.7 ng/µL 1.90 1.61 Good
4 57.4 ng/µL 1.92 1.88 Good

2nd construction

Bacterial isolation

4 clones of a Golden Gate plate are isolated for each construction. It allows us to save the clone on a plate.


Top 10 transformed with the synthetic Fur Binding
Site n°1, 2 and 3 isolated on a plate.

Sequencing preparation

Preculture with LB medium (with carbenicillin) have been launched for Top10 transformed with our 2nd construction, then minipreps have been realized using the miniprep kit from Machrey Nagel.

Name Clone Concentration 260/280 260/230 Sequencing

Fur Binding Site n°1
(with sfGFP)

1 50.7 ng/µL 1.62 0.68 Bad
2 21.1 ng/µL 1.69 0.44 No sequencing
3 21.6 ng/µL 1.20 0.13 No sequencing
4 70.8 ng/µL 1.66 0.72 Bad

Fur Binding Site n°2
(with sfGFP)

1 39.1 ng/µL 1.23 0.40 No sequencing
2 25.1 ng/µL 1.37 0.29 No sequencing
3 61.1 ng/µL 1.57 0.71 Bad
4 54.7 ng/µL 1.62 0.68 Bad

Fur Binding Site n°3
(with sfGFP)

2 38.4 ng/µL 1.84 0.78 No sequencing
3 114.6 ng/µL 1.66 0.78 Bad
4 39.2 ng/µL 1.87 1.21 No sequencing

Fur Binding Site n°5
(with sfGFP)

1 77.7 ng/µL 1.76 0.99 Bad
3 78.4 ng/µL 1.61 0.81 Bad
4 72.7 ng/µL 1.85 4.23 Bad

Fur Binding Site n°6
(with sfGFP)

1 70.4 ng/µL 1.76 2.16 Bad
2 91.5 ng/µL 1.55 1.21 Bad
3 65.4 ng/µL 1.70 1.52 Bad
4 85.6 ng/µL 1.72 1.65 Bad

Fur Binding Site n°7
(with sfGFP)

1 53.5 ng/µL 1.76 1.93 Bad
2 77.6 ng/µL 1.69 1.22 Bad
3 72.3 ng/µL 1.68 1.22 Bad

Fur Binding Site n°8
(with sfGFP)

1 73.9 ng/µL 1.58 0.84 No sequencing
2 51.3 ng/µL 1.75 1.10 Bad
3 45.9 ng/µL 1.85 1.43 Bad
4 46.1 ng/µL 1.88 1.61 Bad

Fur Binding Site n°9
(with sfGFP)

1 108.3 ng/µL 1.78 1.37 Bad
2 67.0 ng/µL 1.88 1.61 Bad
3 103.5 ng/µL 1.87 1.79 Bad
4 28.2 ng/µL 2.06 1.76 No sequencing

Fur Binding Site n°10
(with sfGFP)

1 29.5 ng/µL 1.95 1.22 No sequencing
2 70.0 ng/µL 1.90 1.67 Bad
3 46.0 ng/µL 1.89 1.62 Bad
4 55.8 ng/µL 1.88 1.44 Bad

Fur Binding Site n°11
(with sfGFP)

1 55.3 ng/µL 1.85 1.49 Bad
2 87.1 ng/µL 1.81 1.37 Bad
3 57.7 ng/µL 1.84 1.44 Bad
4 21.7 ng/µL 2.06 1.20 No sequencing

Fur Binding Site n°15
(with sfGFP)

1 16.2 ng/µL 2.13 1.20 No sequencing
2 47.4 ng/µL 1.78 1.16 Bad
4 63.2 ng/µL 1.78 1.16 Bad

Fur Binding Site n°1
(with LacI-LVA)

1 99.7 ng/µL 1.73 0.99 Bad
2 46.9 ng/µL 1.78 0.90 Bad
3 55.8 ng/µL 1.87 1.75 Bad
4 35.3 ng/µL 1.84 0.99 Bad

Fur Binding Site n°2
(with LacI-LVA)

1 43.8 ng/µL 1.83 1.01 Bad
2 47.0 ng/µL 1.77 0.92 Bad
3 32.4 ng/µL 1.78 1.09 Bad

Fur Binding Site n°3
(with LacI-LVA)

1 57.8 ng/µL 1.84 1.45 Bad
2 51.3 ng/µL 1.79 1.04 Bad
3 41.6 ng/µL 1.88 1.75 Bad
4 112.6 ng/µL 1.86 1.81 Bad

Note: Fur Binding Sites n°4, 12, 13 and 14 are missing.

3rd construction

Sequencing preparation

Preculture with LB medium (with carbenicillin) have been launched for Top10 transformed with our plasmid which contains the sfGFP under the control of Lac O promoter. After one night of culture minipreps of 3 different clones have been realized using the miniprep kit from Machrey Nagel.

Name Clone Concentration 260/280 260/230 Sequencing

Testing construction
(Lac O with sfGFP)

1 25.7 ng/µL 2.02 2.29 No sequencing
2 27.0 ng/µL 2.09 1.78 No sequencing
3 79.7 ng/µL 1.86 1.60 Good

Golden Gate

A new Golden Gate of our 3rd construction as been made for the two plasmids containing:

  1. Enterobactin biosynthesis genes (Ent A, D and F)
  2. Enterobactin biosynthesis genes (Ent B, C and E)

5 µL of each Golden Gate product are transformed into Top10 competent cells. To controlled the quality of the transformation, a negative controle (transformation procedure without plasmid) and a positive controle (transformation procedure with plasmid 1A3) are made.

Unfortunatly, the Golden Gate reactions did not work.

Others

Miniprep of Plasmid 1A3 with RFP designed for Golden Gates

Top 10 transformed with the plasmid 1A3 designed for Golden Gate have been cultivated into 10 mL of LB medium (with carbenicillin). After an overnight culture, plasmid has been minipreped using the Machrey Nagel kit.

Sample Concentration 260/280 260/230
1 265 ng/µL 1.84 1.78
2 237.8 ng/µL 1.82 1.67
3 240.8 ng/µL 1.84 1.81

The plasmid has been diluted as a working solution with a concenration of 80 ng/µL. Then the plasmid has been aliquoted in 50 µL into 9 different tubes.

Competent cells

Top10 competent cells had been made last week and we tested them on different antibiotics (Carbenicillin, Kanamycin, Chloramphenicol). After incubation at 37°C, overnight, no contaminant had been observed.