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Team:INSA-Lyon/Project/Stage3/Results
From 2010.igem.org
Results
Caracterisation Curli
We studied the Curli promoter with the PHL1273 chassis which contain Curli promoter and the mutation ompR234. This mutation is required for the high expression of the Curli promoter. A modified GFP is behind the promoter. This GFP can’t be catabolized by the usual enzyme, so in a period of 24 hours all the produced GFP remained. The fluorescence of the culture is proportionnal to the quantity of GFP and thus, to the activity of the Curli promoter. We chose to study the influence of shaking speed, temperature and osmotic pressure. For all the experiment, we used the same protocoles (Mesurement of fluo and ….). We were three operators to do the same experiment at the same time.
The culture of reference was a culture of PHL818, which also contains the mutation OmpR234. The Curli promoter and the GFP gene was introduced in this chassis in order to create PHL1273.
We deduced from this curve that the optimal shaking speed at 28°C was 100 rpm.
- Influence of the shaking speed
First of all the culture were in a waterbath at 28°C and only the shaking speed changed. We try 9 shaking speeds from 50 to 200 rpm.
We deduced from this curve that the optimal shaking speed at 28°C was 100 rpm.
- Influence of the temperature
The next series of culture were in a waterbath with a shaking speed of 100 rpm and we changed the temperature from 27°C to 37°C degree by degree.
The optimal temperature seems to be 28°C. For the two series of measure, the fluorescence is very low when the shaking speed or the temperature was too high. Especially when the temperature is higher than 36°C, the fluorescence is the same as a culture of PHL818. The Curli promoter is very responsive at these two conditions of culture: shaking speed and temperature. Indeed, the gene under the Curli promoter control can be completely extinguished or expressed a lot.
- Influence of the osmotic pressure
We study the influence of osmotic pressure changing the concentration in sucrose of the culture media. We chose 5 concentrations in sucrose different: 0M - 0,01M - 0,05M - 0,1M – 0,5M. The samples are incubated at 28°C with a shaking speed of 100rpm.
We deduce from this curve that when the osmotic pressure increase, the promoter Curli is inactivated that’s why the measure of fluorescence decrease until zero.
