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Team:Evry/Notebook/w9
From 2013.igem.org
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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. | 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> | </p> | ||
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| + | <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"> | ||
| + | --- ng/µL | ||
| + | </td> | ||
| + | <td align="center"> | ||
| + | --- | ||
| + | </td> | ||
| + | <td align="center"> | ||
| + | --- | ||
| + | </td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td align="center"> | ||
| + | 2 | ||
| + | </td> | ||
| + | <td align="center"> | ||
| + | --- ng/µL | ||
| + | </td> | ||
| + | <td align="center"> | ||
| + | --- | ||
| + | </td> | ||
| + | <td align="center"> | ||
| + | --- | ||
| + | </td> | ||
| + | </tr> | ||
| + | |||
| + | <tr> | ||
| + | <td align="center"> | ||
| + | 3 | ||
| + | </td> | ||
| + | <td align="center"> | ||
| + | --- ng/µL | ||
| + | </td> | ||
| + | <td align="center"> | ||
| + | --- | ||
| + | </td> | ||
| + | <td align="center"> | ||
| + | --- | ||
| + | </td> | ||
| + | </tr> | ||
| + | </body> | ||
| + | </table> | ||
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Revision as of 20:22, 13 August 2013
Week 9: 12th August - 18th August
TECAN
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
2nd construction
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 |
|---|---|---|---|---|
| Fur Binding Site n°1 | 1 | 50.7 ng/µL | 1.62 | 0.68 |
| 2 | 21.1 ng/µL | 1.69 | 0.44 | |
| 3 | 21.6 ng/µL | 1.20 | 0.13 | |
| 4 | 70.8 ng/µL | 1.66 | 0.72 | |
| Fur Binding Site n°2 | 1 | 39.1 ng/µL | 1.23 | 0.40 |
| 2 | 25.1 ng/µL | 1.37 | 0.29 | |
| 3 | 61.1 ng/µL | 1.57 | 0.71 | |
| 4 | 54.7 ng/µL | 1.62 | 0.68 | |
| Fur Binding Site n°3 | 2 | 38.4 ng/µL | 1.84 | 0.78 |
| 3 | 114.6 ng/µL | 1.66 | 0.78 | |
| 4 | 39.2 ng/µL | 1.87 | 1.21 | |
| Fur Binding Site n°5 | 1 | 77.7 ng/µL | 1.76 | 0.99 |
| 3 | 78.4 ng/µL | 1.61 | 0.81 | |
| 4 | 72.7 ng/µL | 1.85 | 4.23 | |
| Fur Binding Site n°6 | 1 | 70.4 ng/µL | 1.76 | 2.16 |
| 2 | 91.5 ng/µL | 1.55 | 1.21 | |
| 3 | 65.4 ng/µL | 1.70 | 1.52 | |
| 4 | 85.6 ng/µL | 1.72 | 1.65 | |
| Fur Binding Site n°7 | 1 | 53.5 ng/µL | 1.76 | 1.93 |
| 2 | 77.6 ng/µL | 1.69 | 1.22 | |
| 3 | 72.3 ng/µL | 1.68 | 1.22 | |
| Fur Binding Site n°8 | 1 | 73.9 ng/µL | 1.58 | 0.84 |
| 2 | 51.3 ng/µL | 1.75 | 1.10 | |
| 3 | 45.9 ng/µL | 1.85 | 1.43 | |
| 4 | 46.1 ng/µL | 1.88 | 1.61 | |
| Fur Binding Site n°9 | 1 | 108.3 ng/µL | 1.78 | 1.37 |
| 2 | 67.0 ng/µL | 1.88 | 1.61 | |
| 3 | 103.5 ng/µL | 1.87 | 1.79 | |
| 4 | 28.2 ng/µL | 2.06 | 1.76 | |
| Fur Binding Site n°10 | 1 | 29.5 ng/µL | 1.95 | 1.22 |
| 2 | 70.0 ng/µL | 1.90 | 1.67 | |
| 3 | 46.0 ng/µL | 1.89 | 1.62 | |
| 4 | 55.8 ng/µL | 1.88 | 1.44 | |
| Fur Binding Site n°11 | 1 | 55.3 ng/µL | 1.85 | 1.49 |
| 2 | 87.1 ng/µL | 1.81 | 1.37 | |
| 3 | 57.7 ng/µL | 1.84 | 1.44 | |
| 4 | 21.7 ng/µL | 2.06 | 1.20 | |
| Fur Binding Site n°15 | 1 | 16.2 ng/µL | 2.13 | 1.20 |
| 3 | 47.4 ng/µL | 1.78 | 1.16 | |
| 4 | 63.2 ng/µL | 1.78 | 1.16 |
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.
| Clone | Concentration | 260/280 | 260/230 |
|---|---|---|---|
| 1 | 25.7 ng/µL | 2.02 | 2.29 |
| 2 | 27.0 ng/µL | 2.09 | 1.78 |
| 3 | 79.7 ng/µL | 1.86 | 1.60 |
Golden Gate
A new Golden Gate of our 3rd construction as been made for the two plasmids containing:
- Enterobactin biosynthesis genes (Ent A, D and F)
- Enterobactin biosynthesis genes (Ent B, C and E)
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 | --- ng/µL | --- | --- |
| 2 | --- ng/µL | --- | --- |
| 3 | --- ng/µL | --- | --- |
