Team:Evry/Notebook/w9

From 2013.igem.org

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<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 29: Line 154:
After one night of culture, time the precultures have been refreshed by diluting them 200 times in M9 medium (with iron and carbenicillin).
After one night of culture, time the precultures have been refreshed by diluting them 200 times in M9 medium (with iron and carbenicillin).
</p>
</p>
 +
 +
<p>
 +
After 8 hours of culture, the 96 wells plate has been prepare (see following scheme).
 +
</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>
 +
 +
 +
 +
<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 38: Line 531:
</p>
</p>
-
<table>
+
<table cellpadding="10" cellspacing="0" align='center' border="1">
  <thead>
  <thead>
   <tr>
   <tr>
Line 48: Line 541:
   </th>
   </th>
   <th align="center">
   <th align="center">
-
     Concentration<br/>
+
     Concentration
-
    (ng/µL)
+
   </th>
   </th>
   <th align="center">
   <th align="center">
Line 56: 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>
   <td rowspan="4" align="center">
   <td rowspan="4" align="center">
-
     Fur Binding Site n°1
+
     <p>Fur Binding Site n°1
 +
    <br/>(with sfGFP)</p>
   </td>
   </td>
   <td align="center">
   <td align="center">
Line 68: Line 565:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     50.7 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.62
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     0.68
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     Bad
   </td>
   </td>
   </tr>
   </tr>
Line 82: Line 582:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     21.1 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.69
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     0.44
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 96: Line 599:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     21.6 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.20
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     0.13
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 110: Line 616:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     70.8 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.66
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     0.72
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 122: Line 631:
   <tr>
   <tr>
   <td rowspan="4" align="center">
   <td rowspan="4" align="center">
-
     Fur Binding Site n°2
+
     <p>Fur Binding Site n°2
 +
    <br/>(with sfGFP)</p>
   </td>
   </td>
   <td align="center">
   <td align="center">
Line 128: Line 638:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     39.1 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.23
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     0.40
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 142: Line 655:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     25.1 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.37
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     0.29
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 156: Line 672:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     61.1 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.57
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     0.71
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     Bad
   </td>
   </td>
   </tr>
   </tr>
Line 170: Line 689:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     54.7 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.62
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     0.68
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 182: Line 704:
   <tr>
   <tr>
   <td rowspan="3" align="center">
   <td rowspan="3" align="center">
-
     Fur Binding Site n°3
+
     <p>Fur Binding Site n°3
 +
    <br/>(with sfGFP)</p>
   </td>
   </td>
   <td align="center">
   <td align="center">
Line 188: Line 711:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     38.4 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.84
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     0.78
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 202: Line 728:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     114.6 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.66
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     0.78
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 216: Line 745:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     39.2 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.87
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.21
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 228: Line 760:
   <tr>
   <tr>
   <td rowspan="3" align="center">
   <td rowspan="3" align="center">
-
     Fur Binding Site n°5
+
     <p>Fur Binding Site n°5
 +
    <br/>(with sfGFP)</p>
   </td>
   </td>
   <td align="center">
   <td align="center">
Line 234: Line 767:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     77.7 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.76
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     0.99
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     Bad
   </td>
   </td>
   </tr>
   </tr>
Line 248: Line 784:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     78.4 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.61
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     0.81
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 262: Line 801:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     72.7 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.85
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     4.23
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 274: Line 816:
   <tr>
   <tr>
   <td rowspan="4" align="center">
   <td rowspan="4" align="center">
-
     Fur Binding Site n°6
+
     <p>Fur Binding Site n°6
 +
    <br/>(with sfGFP)</p>
   </td>
   </td>
   <td align="center">
   <td align="center">
Line 280: Line 823:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     70.4 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.76
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     2.16
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 294: Line 840:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     91.5 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.55
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.21
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 308: Line 857:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     65.4 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.70
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.52
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 322: Line 874:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     85.6 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.72
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.65
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     Bad
   </td>
   </td>
   </tr>
   </tr>
Line 334: Line 889:
   <tr>
   <tr>
   <td rowspan="3" align="center">
   <td rowspan="3" align="center">
-
     Fur Binding Site n°7
+
     <p>Fur Binding Site n°7
 +
    <br/>(with sfGFP)</p>
   </td>
   </td>
   <td align="center">
   <td align="center">
Line 340: Line 896:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     53.5 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.76
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.93
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 354: Line 913:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     77.6 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.69
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.22
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 368: Line 930:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     72.3 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.68
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.22
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 380: Line 945:
   <tr>
   <tr>
   <td rowspan="4" align="center">
   <td rowspan="4" align="center">
-
     Fur Binding Site n°8
+
     <p>Fur Binding Site n°8
 +
    <br/>(with sfGFP)</p>
   </td>
   </td>
   <td align="center">
   <td align="center">
Line 386: Line 952:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     73.9 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.58
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     0.84
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 400: Line 969:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     51.3 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.75
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.10
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     Bad
   </td>
   </td>
   </tr>
   </tr>
Line 414: Line 986:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     45.9 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.85
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.43
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 428: Line 1,003:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     46.1 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.88
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.61
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 440: Line 1,018:
   <tr>
   <tr>
   <td rowspan="4" align="center">
   <td rowspan="4" align="center">
-
     Fur Binding Site n°9
+
     <p>Fur Binding Site n°9
 +
    <br/>(with sfGFP)</p>
   </td>
   </td>
   <td align="center">
   <td align="center">
Line 446: Line 1,025:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     108.3 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.78
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.37
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 460: Line 1,042:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     67.0 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.88
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.61
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 474: Line 1,059:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     103.5 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.87
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.79
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 488: Line 1,076:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     28.2 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    2.06
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.76
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 500: Line 1,091:
   <tr>
   <tr>
   <td rowspan="4" align="center">
   <td rowspan="4" align="center">
-
     Fur Binding Site n°10
+
     <p>Fur Binding Site n°10
 +
    <br/>(with sfGFP)</p>
   </td>
   </td>
   <td align="center">
   <td align="center">
Line 506: Line 1,098:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     29.5 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.95
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.22
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 520: Line 1,115:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     70.0 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.90
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.67
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 534: Line 1,132:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     46.0 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.89
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.62
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 548: Line 1,149:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     55.8 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.88
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.44
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 560: Line 1,164:
   <tr>
   <tr>
   <td rowspan="4" align="center">
   <td rowspan="4" align="center">
-
     Fur Binding Site n°11
+
     <p>Fur Binding Site n°11
 +
    <br/>(with sfGFP)</p>
   </td>
   </td>
   <td align="center">
   <td align="center">
Line 566: Line 1,171:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     55.3 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.85
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.49
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     Bad
   </td>
   </td>
   </tr>
   </tr>
Line 580: Line 1,188:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     87.1 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.81
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.37
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 594: Line 1,205:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     57.7 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.84
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.44
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 608: Line 1,222:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     21.7 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     2.06
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.20
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 620: Line 1,237:
   <tr>
   <tr>
   <td rowspan="3" align="center">
   <td rowspan="3" align="center">
-
     Fur Binding Site n°15
+
     <p>Fur Binding Site n°15
 +
    <br/>(with sfGFP)</p>
   </td>
   </td>
   <td align="center">
   <td align="center">
Line 626: Line 1,244:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     16.2 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     2.13
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.20
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    47.4 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.78
 +
  </td>
 +
  <td align="center">
 +
    1.16
 +
  </td>
 +
  <td align="center">
 +
    Bad
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    4
 +
  </td>
 +
  <td align="center">
 +
    63.2 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.78
 +
  </td>
 +
  <td align="center">
 +
    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>
   </td>
   </tr>
   </tr>
Line 640: Line 1,334:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     55.8 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.87
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.75
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 654: Line 1,351:
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     35.3 ng/µL
   </td>
   </td>
   <td align="center">
   <td align="center">
-
     --
+
     1.84
   </td>
   </td>
   <td align="center">
   <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 668: Line 1,497:
<p>
<p>
<b>Note:</b> Fur Binding Sites n°4, 12, 13 and 14 are missing.
<b>Note:</b> Fur Binding Sites n°4, 12, 13 and 14 are missing.
 +
</p>
 +
 +
<h2>3<sup>rd</sup> construction</h2>
 +
 +
<h3>Sequencing preparation</h3>
 +
 +
<p>
 +
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.
 +
</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="3" align="center">
 +
    <p>Testing construction
 +
    <br/>(Lac O with sfGFP)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    25.7 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    2.02
 +
  </td>
 +
  <td align="center">
 +
    2.29
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    27.0 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    2.09
 +
  </td>
 +
  <td align="center">
 +
    1.78
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td align="center">
 +
    3
 +
  </td>
 +
  <td align="center">
 +
    79.7 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.86
 +
  </td>
 +
  <td align="center">
 +
    1.60
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
</tbody>
 +
</table>
 +
 +
<h3>Golden Gate</h3>
 +
 +
<p>
 +
A new Golden Gate of our 3<sup>rd</sup> construction as been made for the two plasmids containing:
 +
</p>
 +
<ol>
 +
<li>Enterobactin biosynthesis genes (Ent A, D and F)
 +
<li>Enterobactin biosynthesis genes (Ent B, C and E)
 +
</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>
 +
 +
<h3>Miniprep of Plasmid 1A3 with RFP designed for Golden Gates</h3>
 +
 +
<p>
 +
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.
 +
</p>
 +
 +
<table cellpadding="10" cellspacing="0" align='center' border="1">
 +
<thead>
 +
  <tr>
 +
  <th align="center">
 +
    Sample
 +
  </th>
 +
  <th align="center">
 +
    Concentration
 +
  </th>
 +
  <th align="center">
 +
    260/280
 +
  </th>
 +
  <th align="center">
 +
    260/230
 +
  </th>
 +
  </tr>
 +
</thead>
 +
<tbody>
 +
  <tr>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    265 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.84
 +
  </td>
 +
  <td align="center">
 +
    1.78
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    237.8 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.82
 +
  </td>
 +
  <td align="center">
 +
    1.67
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td align="center">
 +
    3
 +
  </td>
 +
  <td align="center">
 +
    240.8 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.84
 +
  </td>
 +
  <td align="center">
 +
    1.81
 +
  </td>
 +
  </tr>
 +
</tbody>
 +
</table>
 +
 +
<p>
 +
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.
 +
</p>
 +
 +
<h3>Competent cells</h3>
 +
 +
<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>
</p>

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.

Retrieved from "http://2013.igem.org/Team:Evry/Notebook/w9"