Team:INSA-Lyon/Results
From 2014.igem.org
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As a follow-up to the exploration of curli production and nickel chelation, we want to know the kinetics behind the 70 base-pair long promoter sequence that we used during the whole summer. | As a follow-up to the exploration of curli production and nickel chelation, we want to know the kinetics behind the 70 base-pair long promoter sequence that we used during the whole summer. | ||
In fact, it has the interesting property of being activated at 37°C instead of the 30°C of the natural 750 base-pair CsgA promoter from where it is originally isolated. However, we explored these two promoters' kinetics at 30 and 37°C by inserting a GFP downstream. | In fact, it has the interesting property of being activated at 37°C instead of the 30°C of the natural 750 base-pair CsgA promoter from where it is originally isolated. However, we explored these two promoters' kinetics at 30 and 37°C by inserting a GFP downstream. | ||
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+ | <img src= "https://static.igem.org/mediawiki/parts/f/fb/2.png" alt="<b>Figure 1 : GFP expression plasmid for promoter characterization. The promoter sequence can either be the 70 (P70) or the 750 base-pair promoter sequence.</b>"/></br> | ||
+ | </div> | ||
On a pKK backbone, two essential parts have been assembled: a promoter and a reporter gene. The reporter gene in this case is always the same: GFP. However, the promoter is different for each construction. | On a pKK backbone, two essential parts have been assembled: a promoter and a reporter gene. The reporter gene in this case is always the same: GFP. However, the promoter is different for each construction. | ||
On the one hand, P70 is the 70 base-pair long promoter sequence and when combined to the reporter GFP, the construction is called p70:GFP. | On the one hand, P70 is the 70 base-pair long promoter sequence and when combined to the reporter GFP, the construction is called p70:GFP. | ||
On the other hand, P750 is the 750 base-pair long promoter sequence coding for the inter-genic regulation region of curli production and combined to the reporter GFP, the construction is called P750:GFP.</br> | On the other hand, P750 is the 750 base-pair long promoter sequence coding for the inter-genic regulation region of curli production and combined to the reporter GFP, the construction is called P750:GFP.</br> | ||
- | The idea behind the two constructions is | + | The idea behind the two constructions is the correlate the GFP expression to the promoter activity. |
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<div align="center"> | <div align="center"> | ||
- | <img src= "https://static.igem.org/mediawiki/ | + | <img width="80%" src="https://static.igem.org/mediawiki/2014/5/58/PromoterExpression_EarlyStage.png" alt="<b>Figure 2 : Promoter expression as a function of the Optical Density. At early growth stage, P70 has a higher expression rate at 37°C relatively to the P750.</b>"></br> |
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<b>Early growth stage promoter kinetics</b></br> | <b>Early growth stage promoter kinetics</b></br> | ||
- | <p align="justify">During the early growth stages at 37°C, we can observe that the P70 (orange) has a higher | + | <p align="justify">During the early growth stages at 37°C, we can observe that the P70 (orange) has a higher expression level compared to the P750 (red). |
- | However at 30°C, both P70 (light blue) and P750 (dark blue) have low | + | However at 30°C, both P70 (light blue) and P750 (dark blue) have low expression levels. |
We conclude that P70 has the ability to prematurely activate downstream expression at 37°C. | We conclude that P70 has the ability to prematurely activate downstream expression at 37°C. | ||
</p></p> | </p></p> | ||
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<div align="center"> | <div align="center"> | ||
- | <img width="80%" src="https://static.igem.org/mediawiki/2014/ | + | <img width="80%" src="https://static.igem.org/mediawiki/2014/f/f9/PromoterExpression_LateStage.png" alt="<b>Figure 3 : Promoter expression as a function of the Optical Density. At mid/late growth stage, P750 has a delayed but higher expression rate at 30°C relatively to the P70.</b>">></br> |
</div> | </div> | ||
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<p> | <p> | ||
- | <b> | + | <b>Mid/late growth stage promoter kinetics</b></br> |
- | <p align="justify">During the late growth stages at 37°C, we can observe that both P70 (orange) and P750 (red) have a | + | <p align="justify">During the mid/late growth stages at 37°C, we can observe that both P70 (orange) and P750 (red) have a decreased promoter expression and are moving towards lower rates. |
- | However at 30°C, P70 (light blue) stabilizes within the range of low | + | However at 30°C, P70 (light blue) stabilizes within the range of low expression levels and P750 (dark blue) reaches the highest promoter expression rates. |
- | We conclude that P750 has a delayed, albeit extremely high-leveled, | + | We conclude that P750 has a delayed, albeit extremely high-leveled, promoter expression at 30°C. |
</p></p> | </p></p> | ||
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</ul> | </ul> |
Revision as of 02:27, 18 October 2014