Difference between revisions of "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

Mix digestion preparation:

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

Mix ligation preparation:

Tube	pSB1C3-nanoluc (1:3)	pSB1C3-nanoluc (1:5)	pSB1C3-pDawn I-V (2:1)
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

15 Sept. 15