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Team:Edinburgh/biology(results)
From 2009.igem.org
Biology - Results
The OmpC promoter was characterised using procaine. Procaine acts as an antagonist to phosphorylated omp-R, the inherent activator of this promoter. The first thing to notice from the characterisation results, is that this promoter is very leaky. Our team has found a way to overcome this problem by using an EnvZ – E. coli strain which we have constructed ourselves. Using this strain as a chassis for our construct would decrease background activation of this promoter, and thus ensure more precise expression of EYFP and onr in response to the presence of TNT.
Miller Test
The first test done on the YeaR promoter was the famous miller test. As you can see this test shows that pYeaR is more sensitive to Nitrates than to Nitrites, and the sensitivity increases with increased concentration. In order to get a more detailed representation to the response to nitrates, the main activator of this promoter, the Jason Kelly assay was performed.
Jason Kelly Assay
From the Jason Kelly Assay we got a higher resolution response curve to the presence of Nitrates, that will hopefully be useful for further work with pYeaR. As you see, as the concentration goes beyond 30 mM, the Fluorescence decreases, most likely due to poisoning of the cells with excess Nitrates.
Personal Note
The OmpC promoter was characterised using procaine. Procaine acts as an antagonist to phosphorylated omp-R, the inherent activator of this promoter. The first thing to notice from the characterisation results, is that this promoter is very leaky. Our team has found a way to overcome this problem by using an EnvZ – E. coli strain which we have constructed ourselves. Using this strain as a chassis for our construct would decrease background activation of this promoter, and thus ensure more precise expression of EYFP and onr in response to the presence of TNT.
Miller Test
The first test done on the YeaR promoter was the famous miller test. As you can see this test shows that pYeaR is more sensitive to Nitrates than to Nitrites, and the sensitivity increases with increased concentration. In order to get a more detailed representation to the response to nitrates, the main activator of this promoter, the Jason Kelly assay was performed.
Jason Kelly Assay
From the Jason Kelly Assay we got a higher resolution response curve to the presence of Nitrates, that will hopefully be useful for further work with pYeaR. As you see, as the concentration goes beyond 30 mM, the Fluorescence decreases, most likely due to poisoning of the cells with excess Nitrates.
Edinburgh University iGEM Team 2009
