Team:Evry/Notebook/w9

From 2013.igem.org

(Difference between revisions)
 
(34 intermediate revisions not shown)
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 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 55: 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 74: Line 572:
   <td align="center">
   <td align="center">
     0.68
     0.68
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 88: Line 589:
   <td align="center">
   <td align="center">
     0.44
     0.44
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 102: Line 606:
   <td align="center">
   <td align="center">
     0.13
     0.13
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 116: Line 623:
   <td align="center">
   <td align="center">
     0.72
     0.72
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 121: 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 134: Line 645:
   <td align="center">
   <td align="center">
     0.40
     0.40
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 148: Line 662:
   <td align="center">
   <td align="center">
     0.29
     0.29
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 162: Line 679:
   <td align="center">
   <td align="center">
     0.71
     0.71
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 176: Line 696:
   <td align="center">
   <td align="center">
     0.68
     0.68
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 181: 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 194: Line 718:
   <td align="center">
   <td align="center">
     0.78
     0.78
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 208: Line 735:
   <td align="center">
   <td align="center">
     0.78
     0.78
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 222: Line 752:
   <td align="center">
   <td align="center">
     1.21
     1.21
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 227: 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 240: Line 774:
   <td align="center">
   <td align="center">
     0.99
     0.99
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 254: Line 791:
   <td align="center">
   <td align="center">
     0.81
     0.81
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 268: Line 808:
   <td align="center">
   <td align="center">
     4.23
     4.23
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 273: 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 286: Line 830:
   <td align="center">
   <td align="center">
     2.16
     2.16
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 300: Line 847:
   <td align="center">
   <td align="center">
     1.21
     1.21
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 314: Line 864:
   <td align="center">
   <td align="center">
     1.52
     1.52
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 328: Line 881:
   <td align="center">
   <td align="center">
     1.65
     1.65
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 333: 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 346: Line 903:
   <td align="center">
   <td align="center">
     1.93
     1.93
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 360: Line 920:
   <td align="center">
   <td align="center">
     1.22
     1.22
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 374: Line 937:
   <td align="center">
   <td align="center">
     1.22
     1.22
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 379: 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 392: Line 959:
   <td align="center">
   <td align="center">
     0.84
     0.84
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 406: Line 976:
   <td align="center">
   <td align="center">
     1.10
     1.10
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 420: Line 993:
   <td align="center">
   <td align="center">
     1.43
     1.43
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 434: Line 1,010:
   <td align="center">
   <td align="center">
     1.61
     1.61
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 439: 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 452: Line 1,032:
   <td align="center">
   <td align="center">
     1.37
     1.37
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 466: Line 1,049:
   <td align="center">
   <td align="center">
     1.61
     1.61
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 480: Line 1,066:
   <td align="center">
   <td align="center">
     1.79
     1.79
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 494: Line 1,083:
   <td align="center">
   <td align="center">
     1.76
     1.76
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 499: 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 512: Line 1,105:
   <td align="center">
   <td align="center">
     1.22
     1.22
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 526: Line 1,122:
   <td align="center">
   <td align="center">
     1.67
     1.67
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 540: Line 1,139:
   <td align="center">
   <td align="center">
     1.62
     1.62
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 554: Line 1,156:
   <td align="center">
   <td align="center">
     1.44
     1.44
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 559: 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 572: Line 1,178:
   <td align="center">
   <td align="center">
     1.49
     1.49
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 586: Line 1,195:
   <td align="center">
   <td align="center">
     1.37
     1.37
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 600: Line 1,212:
   <td align="center">
   <td align="center">
     1.44
     1.44
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 614: Line 1,229:
   <td align="center">
   <td align="center">
     1.20
     1.20
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 619: 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 632: 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 646: Line 1,268:
   <td align="center">
   <td align="center">
     1.16
     1.16
 +
  </td>
 +
  <td align="center">
 +
    Bad
   </td>
   </td>
   </tr>
   </tr>
Line 660: 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 678: 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 691: 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 707: Line 1,548:
   <td align="center">
   <td align="center">
     2.29
     2.29
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 722: Line 1,566:
   <td align="center">
   <td align="center">
     1.78
     1.78
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
   </td>
   </td>
   </tr>
   </tr>
Line 737: 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>
 +
 +
<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>
</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.

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