From 2014.igem.org
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| - | <h1>Part B: Biosensor Development</h1>
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| - | <p>Glen and Fran will be tackling this area of the project in the lab while Oliver and Matt will be modelling this system to allow us to design the ideal biosensor. In the native bacterium, Methylobacterium Extorquens DM4, the DCM sensing protein (DCMR) is encoded by a dcmR gene that is opposite to the DCM degrading enzyme DCMA-encoding gene, dcmA.
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| - | [[File:oxfordigem_dcmrdcma.png|700px|centre]]<BR>
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| - | dcmR is thought to encode a repressor protein that binds to the region of DNA between dcmR and dcmA genes. Upon addition of DCM the repression is thought to be relieved and transcription of dcmA can begin. To test this hypothesis we have extracted this inter-gene region and put sfGFP downstream in place of dcmA. In this way we can induce expression of dcmR on another plasmid and then using varying levels of DCM we can investigate the dynamics of dcmR repression in this region. Thus confirming an activation or repression set-up.
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| - | [[File:oxfordigem_sfGFP.png|400px|centre]]<BR>
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| - | This construct will first generate data on the basal level of transcription. A second plasmid was designed to allow the levels of dcmR to be varied. We achieved this with a tetracycline-inducible promoter. Tagged to dcmR is mCherry which will allow us to characterise expression levels and cellular localisation. However, in the literature dcmR is only assumed to act as a repressor whereby DCM relieves repression and thus activates expression of downstream genes (natively dcmA, in our case - sfGFP). <BR><BR>
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| - | Characterisation of this regulatory network has never been done before; we will be the first to fully characterise the mode of action of dcmR. To do this we suppose the following hypotheses for DCM activating the transcription of dcmR: Either double repression or double activation.
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| - | [[File:oxfordigem_repact.png|400px|centre]]<BR>
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| - | Looking at the basal rate of sfGFP production (in the absence of any dcmR) we would expect a higher level of expression for the repression construct as this would be presumed to have a constitutive promoter that is repressed by dcmR. In the case of activation we would expect a low basal rate of expression since dcmR is required for transcription activation. This will allow us to determine whether dcmR acts as a repressor or activator of downstream genes. <BR><BR>
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| - | Once either of these networks is confirmed we will then add a second plasmid that will contain dcmR - the heart of our biosensor:
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| - | [[File:oxfordigem_dcmrmcherry.png|1000px|centre]]
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Latest revision as of 01:06, 18 October 2014
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