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Team:INSA-Lyon/Project/Stage3/Results
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<h3>Characterization Curli</h3><br> | <h3>Characterization Curli</h3><br> | ||
| - | <p>We studied the Curli promoter with the PHL1273 chassis which | + | <p>We studied the Curli promoter with the PHL1273 chassis which contains 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 is unstable and has a half-life of 40 min. 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 | + | We chose to study the influence of shaking speed, temperature and osmotic pressure on the activity of this promoter. For all the experiments, we used the same protocoles, (the GFP was quantified by spectrophotometry, see Protocols). We were three operators to do the same experiment at the same time.</p> |
<p> | <p> | ||
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. | 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. | ||
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| - | <p>First of all | + | <p>First of all, cultures were in a waterbath at 28°C and only the shaking speed was changed. We have tried 9 different shaking speeds from 50 to 200 rpm.</p> |
<div style="text-align:center;"> | <div style="text-align:center;"> | ||
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</div> | </div> | ||
| - | <br><p> | + | <br><p>The curve shows that our system presents the nice property to have an optimal shaking speed at 100 rpm when the temperature is 28°C. </p> |
<br> | <br> | ||
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</ol></p> | </ol></p> | ||
<br> | <br> | ||
| - | <p>The next series of | + | <p>The next series of cultures 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.</p> |
<br> | <br> | ||
<div style="text-align:center;"> | <div style="text-align:center;"> | ||
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<p> | <p> | ||
| - | The optimal temperature seems to be 28°C. | + | The optimal temperature seems to be 28°C. So our system presents also a optimal response according to the temperature. |
| - | For | + | For both series of measurements, fluorescence was very low when the shaking speed and the temperature were high. Notably, when the temperature is higher than 36°C, the fluorescence is the same as the negative control. |
| - | The Curli promoter is very responsive at these two conditions of culture: shaking speed and temperature. Indeed, | + | The Curli promoter is very responsive at these two conditions of culture: shaking speed and temperature. Indeed, a gene under the Curli promoter control can be switched ON or OFF by acting on temperature or speed shaking. |
</p> | </p> | ||
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<p> | <p> | ||
| - | We | + | We then studied the influence of osmotic pressure on our promoter. We modified the osmotic pressure by changing the concentration in sucrose of the culture media. We chose 5 different concentrations in sucrose : 0M - 0,01M - 0,05M - 0,1M – 0,5M. The samples were incubated at 28°C with a shaking speed of 100rpm (previous optimal conditions). |
</p> | </p> | ||
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</div> | </div> | ||
| - | <p>We | + | <p>We deduced from this curve that when the osmotic pressure is increased, the promoter Curli is less expressed. |
</p> | </p> | ||
Revision as of 17:04, 26 October 2010
Results
Characterization Curli
We studied the Curli promoter with the PHL1273 chassis which contains 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 is unstable and has a half-life of 40 min. 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 on the activity of this promoter. For all the experiments, we used the same protocoles, (the GFP was quantified by spectrophotometry, see Protocols). 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.
Influence of the shaking speed
First of all, cultures were in a waterbath at 28°C and only the shaking speed was changed. We have tried 9 different shaking speeds from 50 to 200 rpm.
The curve shows that our system presents the nice property to have an optimal shaking speed at 100 rpm when the temperature is 28°C.
Influence of the temperature
The next series of cultures 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. So our system presents also a optimal response according to the temperature. For both series of measurements, fluorescence was very low when the shaking speed and the temperature were high. Notably, when the temperature is higher than 36°C, the fluorescence is the same as the negative control. The Curli promoter is very responsive at these two conditions of culture: shaking speed and temperature. Indeed, a gene under the Curli promoter control can be switched ON or OFF by acting on temperature or speed shaking.
Influence of the osmotic pressure
We then studied the influence of osmotic pressure on our promoter. We modified the osmotic pressure by changing the concentration in sucrose of the culture media. We chose 5 different concentrations in sucrose : 0M - 0,01M - 0,05M - 0,1M – 0,5M. The samples were incubated at 28°C with a shaking speed of 100rpm (previous optimal conditions).
We deduced from this curve that when the osmotic pressure is increased, the promoter Curli is less expressed.
