Template:IONIS Paris/Notebook/15-09-15
Measurement of the OD600 of liquid culture
When the OD600 has reached 0.6 or more, the volume of culture has been divided into 3 erlenmeyers:
1 erlenmeyer without light exposition, 37°C
1 erlenmeyer with light exposition, 37°C
1 erlenmeyer with light exposition, room temperature
Measurement of the OD600 every hour
3 culture on 5have shown a bacterial development
Colony - A6
Colony - A8
Colony - F1
All colonies exhibits same profile under same condition of light exposition and temperature. When the temperature is favorable for a bacterial development, the light activation of pDawn leading to the production of toxin is weak and do not impact a lot the growth of the population of bacteria. But at room temperature (RT°), the induction for protein synthesis is high enough to stop bacterial development
Two things lack to this experiment:
- A longer time to measure if the population of bacteria could really decrease
- A control at room temperature without light exposition to compare it evolution of OD 600 to the grey curve
Digestion
| Tube | Miniprep | pSB1C3 iGEM |
|---|---|---|
| H2O MQ | 4 μL | - |
| Buffer 2.1 | 2 μL | 4 μL |
| DNA | 3 μL | 30 μL |
| EcoRI | 0,5 μL | 3 μL |
| PstI | 0,5 μL | 3 μL |
37°C, 1h heat kill: 80°C, 20min
Ligation
| Tube | pSB1C3-nanoluc (1:3) | pSB1C3-nanoluc (1:5) |
|---|---|---|
| H2O MQ | 9 μL | 9,5 μL |
| T4 buffer | 2 μL | 2 μL |
| pSB1C3 | 5 μL | 5 μL |
| NanoLuc | 3 µL (35ng.µL-1) | 2,5 µl (70 ng.µL-1) |
| T4 ligase | 1 μL | 1 μL |
Room temperature, 30min heat kill: 65°C, 20min
- Transformation of competent cells with 2 µL of ligation products
Liquid culture
4 colonies from transformation with pSB1C3-Holin/Endolysin
2 colonies from transformation with pSB1C3-pDawn
Transformation
Transformation of competent BL 21(DE3) cells with BBa_K325909, BBa_K1159001 and T7-nanoluc plasmids
15 Sept. 15