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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 [1]. 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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| - | Using models that Ollie has made we will hold tetracycline constant at a specific concentration that allows a robust response such that variation in concentration between the individual colonies will not affect the overall output. Using, then, the only input as DCM concentration we will characterise the system shown above. <BR><BR>
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| - | Ideally we are looking for a quick 'ON' response such that as the user pours their waste DCM into the container the bacteria rapidly switch on the reporter output (green fluorescence). We also want to design a quick 'OFF' switch such that when the bacteria from part C have fully degraded DCM the reporter output if switched OFF and the user can easily interpret this as a signal to pour the bioremediated contents away. This will be achieved by close communication between the engineering model and the biochemistry data. We will model the affects of manipulating GFP degradation rates (using degradation tags), expression levels, and plasmid copy numbers in an attempt to design the best response profile that will serve as a functional biosensor for our bioremediation kit.
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| - | <h2> References </h2>
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| - | [1] J. La Roche, S. D., and T. H. O. M. A. S. Leisinger. "Identification of dcmR, the regulatory gene governing expression of dichloromethane dehalogenase in Methylobacterium sp. strain DM4." Journal of bacteriology 173.21 (1991): 6714-6721.
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