Team:Evry/Notebook/w10

From 2013.igem.org

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   </td>
   </td>
   <td align="center">
   <td align="center">
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     No sequencing
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     -
   </td>
   </td>
   </tr>
   </tr>
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</table>
</table>
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<h3>Other</h3>
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 +
<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="4" align="center">
 +
    <p>Enterobactin construction
 +
    <br/>(Lac O with EntA, EntD, EntF)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    62.7 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    2.02
 +
  </td>
 +
  <td align="center">
 +
    0.84
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    86.8 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.97
 +
  </td>
 +
  <td align="center">
 +
    0.99
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    3
 +
  </td>
 +
  <td align="center">
 +
    112.2 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.98
 +
  </td>
 +
  <td align="center">
 +
    1.23
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    4
 +
  </td>
 +
  <td align="center">
 +
    78.7 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.97
 +
  </td>
 +
  <td align="center">
 +
    1.17
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  </tr>
 +
 
 +
  <tr>
 +
  <td rowspan="2" align="center">
 +
    <p>Enterobactin construction
 +
    <br/>(Lac O with EntB, EntC, EntE)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    80.1 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.92
 +
  </td>
 +
  <td align="center">
 +
    1.01
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    4
 +
  </td>
 +
  <td align="center">
 +
    330.3 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.91
 +
  </td>
 +
  <td align="center">
 +
    1.20
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  </tr>
 +
</tbody>
 +
</table>
 +
 
 +
<h2>Other</h2>
<p>
<p>
-
We prepared BL21 competent cells.<br/>
+
We prepared 20 tubes of BL21 competent cells.
-
We launched Golden Gate 3 again.<br/>
+
-
Isolation and culture of 4 colonies of Fur 1, 2, 3 with sfGFP or LacI.<br/>
+
</p>
</p>

Latest revision as of 08:30, 27 August 2013

Iron coli project

Week 10: 19th August - 25th August

2nd Construction

Primer hybridation

After sequencing analysis we observed that our Golden Gate results were not significant for the 2nd construction.
However, our sequences of natural promoter give us good results. Then we supposed that the problem of our Golden Gate come from the hybridation of our synthetic promoters.
We made new hybridations of:

  • Fur Binding Site 1 to 15 (5 µM)
  • Andersen promoter J23100 (5 µM)
  • RBS 0034 (5 µM)

Golden Gate

A new Golden Gate of our 2nd construction are made with:

  • Fur Binding Site 1, 2 or 3 + sfGFP
  • Fur Binding Site 1, 2 or 3 + LacI-LVA

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.

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 20.2 ng/µL 2.07 1.29 Bad

Fur Binding Site n°1
(with LacI-LVA)

 1 28.5 ng/µL 2.06 1.65 -
2 25.5 ng/µL 2.01 1.84 No sequencing
3 45.6 ng/µL 1.91 1.90 Bad
4 54.7 ng/µL 1.83 1.27 Bad

Fur Binding Site n°2
(with sfGFP)

 1 29.4 ng/µL 2.01 1.46 Bad
2 25.5 ng/µL 2.07 2.00 Bad
3 15.0 ng/µL 2.21 1.72 No sequencing
4 47.1 ng/µL 1.84 1.53 -

Fur Binding Site n°2
(with LacI-LVA)

 1 25.4 ng/µL 1.72 0.60 -
2 27.7 ng/µL 1.93 1.67 No sequencing
3 41.8 ng/µL 1.87 1.69 Bad
4 31.1 ng/µL 1.82 1.26 Bad

Fur Binding Site n°3
(with sfGFP)

 1 37.8 ng/µL 1.85 1.40 Bad
2 78.3 ng/µL 1.87 1.85 Bad
3 53.4 ng/µL 1.72 0.98 Bad
4 23.3 ng/µL 2.06 2.53 No sequencing

Fur Binding Site n°3
(with LacI-LVA)

 2 43.8 ng/µL 1.78 1.21 Bad
4 28.2 ng/µL 2.15 2.31 -

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

Enterobactin construction
(Lac O with EntA, EntD, EntF)

 1 62.7 ng/µL 2.02 0.84 -
2 86.8 ng/µL 1.97 0.99 -
3 112.2 ng/µL 1.98 1.23 -
4 78.7 ng/µL 1.97 1.17 -

Enterobactin construction
(Lac O with EntB, EntC, EntE)

 1 80.1 ng/µL 1.92 1.01 -
4 330.3 ng/µL 1.91 1.20 -

Other

We prepared 20 tubes of BL21 competent cells.

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