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Team:Evry/Notebook/w9
From 2013.igem.org
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).
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 |
