Difference between revisions of "Part:BBa K3470013"
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| + | Constitutive Promoter – RBS – SoxR - Double terminator - SoxS promoter – GFP - Double Terminator | ||
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| + | SoxR is a transcription factor, activated in the conditions of oxidative stresses (presence of NO; pro-inflammatory signal) by reversible one-electron oxidation of its [2Fe-2S] cluster. In the active state, it activates the SoxS promoter by untwisting the promoter DNA by binding to the long spacer DNA (19- or 20-bp spacer between the -35 and -10 operator elements). (Miki et al., 2008). SoxS is a conditional promoter, regulated by the SoxR transcription factor (In Composite BioBrick 1). In the active state (when it is bound to NO; pro-inflammatory signal), SoxR activates the SoxS promoter and starts the transcription of the genes downstream to it. (Hidalgo et al., 1998) | ||
| + | Escherichia coli Nissle 1917 transformed with SoxR and SoxS genes is tested to analyse the relationship between the amount of oxidative stress and transcription rates of the genes in response to methyl viologen dichloride hydrate which acts as an inducer by measuring GFP intensity. The intensity of GFP increases with time indicating that even if the concentration of the oxidative stress inducer is constant, the rate of activation of SoxR and thus the rate of transcription of SoxS promoter increases and then stabilizes to a constant rate with a higher transcription rate of SoxS promoter with a higher inducer concentration. The team tested to understand the amount of oxidative stress required to activate SoxR and in turn activate SoxS promoter occurs even at low oxidative stress, preventing any unwanted inflammation due to our probiotic thus getting an anti-inflammatory response activation by composite bio-brick 2. | ||
| + | The team observed that the GFP intensity should increase with time for all the concentrations except the control and stabilize after a few hours. The culture fluorescence will be more for a higher concentration of paraquat, with the highest being for the 40μM.The intensity of GFP increases with time indicating that even if the concentration of the oxidative stress inducer is constant, the rate of activation of SoxR and thus the rate of transcription of SoxS promoter increases and then stabilizes to a constant rate. The more the concentration of the inducer, the more is the transcription rate of SoxS promoter. The most likely unexpected result would be that the GFP intensity for the 40μM concentration is not the highest which could be the result of a high amount of oxidative stress on the bacteria which reduces the growth rate. This is checked by the experiment (Measurement of growth rate of Escherichia coli Nissle 1917: Under oxidative stresses) and the dosage of the probiotic can be modified accordingly. | ||
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| + | References: | ||
| + | Hidalgo, E., Leautaud, V., & Demple, B. (1998). The redox-regulated SoxR protein acts from a single DNA site as a repressor and an allosteric activator. EMBO Journal, 17(9), 2629–2636. https://doi.org/10.1093/emboj/17.9.2629 | ||
| + | Miki, K., Watanabe, S., Kita, A., & Kobayashi, K. (2008). Crystal structure of the [2Fe-2S] transcriptional activator SoxR bound to DNA. Acta Crystallographica Section A Foundations of Crystallography, 64(a1), C89–C89. https://doi.org/10.1107/s0108767308097122 | ||
Revision as of 10:58, 17 October 2020
Constitutive Promoter – RBS – SoxR - Double terminator - SoxS promoter – GFP - Double Terminator
SoxR is a transcription factor, activated in the conditions of oxidative stresses (presence of NO; pro-inflammatory signal) by reversible one-electron oxidation of its [2Fe-2S] cluster. In the active state, it activates the SoxS promoter by untwisting the promoter DNA by binding to the long spacer DNA (19- or 20-bp spacer between the -35 and -10 operator elements). (Miki et al., 2008). SoxS is a conditional promoter, regulated by the SoxR transcription factor (In Composite BioBrick 1). In the active state (when it is bound to NO; pro-inflammatory signal), SoxR activates the SoxS promoter and starts the transcription of the genes downstream to it. (Hidalgo et al., 1998)
Escherichia coli Nissle 1917 transformed with SoxR and SoxS genes is tested to analyse the relationship between the amount of oxidative stress and transcription rates of the genes in response to methyl viologen dichloride hydrate which acts as an inducer by measuring GFP intensity. The intensity of GFP increases with time indicating that even if the concentration of the oxidative stress inducer is constant, the rate of activation of SoxR and thus the rate of transcription of SoxS promoter increases and then stabilizes to a constant rate with a higher transcription rate of SoxS promoter with a higher inducer concentration. The team tested to understand the amount of oxidative stress required to activate SoxR and in turn activate SoxS promoter occurs even at low oxidative stress, preventing any unwanted inflammation due to our probiotic thus getting an anti-inflammatory response activation by composite bio-brick 2.
The team observed that the GFP intensity should increase with time for all the concentrations except the control and stabilize after a few hours. The culture fluorescence will be more for a higher concentration of paraquat, with the highest being for the 40μM.The intensity of GFP increases with time indicating that even if the concentration of the oxidative stress inducer is constant, the rate of activation of SoxR and thus the rate of transcription of SoxS promoter increases and then stabilizes to a constant rate. The more the concentration of the inducer, the more is the transcription rate of SoxS promoter. The most likely unexpected result would be that the GFP intensity for the 40μM concentration is not the highest which could be the result of a high amount of oxidative stress on the bacteria which reduces the growth rate. This is checked by the experiment (Measurement of growth rate of Escherichia coli Nissle 1917: Under oxidative stresses) and the dosage of the probiotic can be modified accordingly.
References:
Hidalgo, E., Leautaud, V., & Demple, B. (1998). The redox-regulated SoxR protein acts from a single DNA site as a repressor and an allosteric activator. EMBO Journal, 17(9), 2629–2636. https://doi.org/10.1093/emboj/17.9.2629
Miki, K., Watanabe, S., Kita, A., & Kobayashi, K. (2008). Crystal structure of the [2Fe-2S] transcriptional activator SoxR bound to DNA. Acta Crystallographica Section A Foundations of Crystallography, 64(a1), C89–C89. https://doi.org/10.1107/s0108767308097122