Team:Oxford/Results
From 2014.igem.org
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This page summarises our achievements during the project. Essentially our project can be divided into three sections: biosensing, bioremediation and realisation.
![](https://static.igem.org/mediawiki/2014/4/4d/Oxford_plus-sign-clip-art.png)
Introduction
In the native DCM-degrading bacterium the following genetic construct exists:![](https://static.igem.org/mediawiki/2014/7/7f/Oxford_charac3.png)
We have observed and characterised the effect of DcmR regulating both promoters; in both directions under the influence of DcmR.
This is a new tool for molecular biologists wishing to manipulate stoichiometries of genetic products in the same construct.
We have also characterised PdcmA repression as a double repression system with DCM modulating DcmR activity resulting in derepression of genes downstream of PdcmA. This DCM sensitivity is not observed in the PdcmR direction. Wetlab Results:Is DcmR a repressor or an activator at PdcmA/PdcmR promoters?
![](https://static.igem.org/mediawiki/2014/b/b7/Oxford_CharacGlen1.png)
DcmR at the bidirectional promoter - A new tool for molecular biologists
![](https://static.igem.org/mediawiki/2014/1/18/Oxford_CharacGlen2.png)
Through analysing this new bidirectional promoter we have discovered a new tool for molecular biologists. Our new intergenic region is able to modulate different stoichiometries between genes inserted either side of it in response to the presence or absence of DcmR (available in our BioBrick BBa_K1446003). The relative stoichiometry (PdcmA/PdcmR activation) of ~0.9 being reduced to ~0.3 upon DcmR addition.
DCM interacting with DcmR - the basis for our biosensor
For the PdcmA promoter both with and without DcmR:
![](https://static.igem.org/mediawiki/2014/e/ec/Oxford_CharacGlen3.png)
For the PdcmR promoter both with and without DcmR:
![](https://static.igem.org/mediawiki/2014/2/2c/Oxford_CharacGlen4.png)
In the context of our biosensor this means that DCM is capable of modulating expression of sfGFP through interaction through the PdcmA repressor DcmR.
Ultimately we can conclude that DCM acts to encourage expression of sfGFP through derepression: consistent with our double repression model.
Discussion:
The results displayed here demonstrate the repression nature of DcmR on PdcmA and PdcmR bidirectional promoter. The modulation by DCM on the PdcmA promoter we have observed offers a constructive route towards a Dichloromethane biosensor. In this way we are able to use a reporter gene downstream of PdcmA to respond to the concentration of DCM.
Additionally the nature of the bidirectional promoter offers a uniquely useful tool to molecular biologists. The relative stoichiometry of two genes inserted either side of the bidirectional promoter can be can be modulated through the addition of DcmR (BBa_K1446003 - Oxford iGEM). This can be utilised easily for investigations into the effect of protein stoichiometries between two interacting proteins.
![](https://static.igem.org/mediawiki/2014/4/4d/Oxford_plus-sign-clip-art.png)
For the bioremediation aspect of DCMation, we managed to achieve the following:
In the wet lab:
1. Express ABTUNKJ in E. Coli
2. Expression of dcmA-sfGFP in ''E. Coli''. This was verified by:
- Microscopy
- Western Blot
- Visual observation
3. Proof of successful mechanism for targeting into microcompartments by fluorescence microscopy
4. Assay design - successful assay set-up
We also developed the following models:
1. Microcompartment Shape Model (see 'Predicting the microcompartment structure')
2. Prediction of number of enzymes per microcompartment (see 'Modelling the number of enzymes in a microcompartment')
3. Effect of microcompartments on collision rates (see 'Microcompartment rate of collision model')
4. Stochastic diffusion of formaldehyde within microcompartments (see 'Modelling the diffusion of formaldehyde inside the microcompartment')
5. Star-peptide model (see 'The Star-Peptide Model')
![](https://static.igem.org/mediawiki/2014/e/e5/DCMAsfGFP.jpg)
1. Express ABTUNKJ in E. Coli
2. Expression of dcmA-sfGFP in ''E. Coli''. This was verified by:
- Microscopy
- Western Blot
- Visual observation
3. Proof of successful mechanism for targeting into microcompartments by fluorescence microscopy
4. Assay design - successful assay set-up
We also developed the following models:
1. Microcompartment Shape Model (see 'Predicting the microcompartment structure')
2. Prediction of number of enzymes per microcompartment (see 'Modelling the number of enzymes in a microcompartment')
3. Effect of microcompartments on collision rates (see 'Microcompartment rate of collision model')
4. Stochastic diffusion of formaldehyde within microcompartments (see 'Modelling the diffusion of formaldehyde inside the microcompartment')
5. Star-peptide model (see 'The Star-Peptide Model')
![](https://static.igem.org/mediawiki/2014/4/4d/Oxford_plus-sign-clip-art.png)
Realisation Results:
For the containment of our bacteria, we have managed to:
1. synthesise novel agarose beads that have a polymeric coating which limits DCM diffusion into the beads. This allows optimum degradation by the bioremediation bacteria, while physically containing the bacteria for safety reasons
2. verify the functioning of the biopolymeric beads by measuring diffusion using indigo dye 3. use computer-aided modelling to design a prototype of the DCMation system, and physically constructed this container
4. 3D print a cartridge to hold our biosensor bacteria, which can easily be replaced by the user
5. construct a prototype circuit that lights up when the photodiodes detect light emission from our biosensing bacteria that are contained in the cartridge. This lets the user have a simple yes/no response to whether the contents of the container are safe for disposal.
For the containment of our bacteria, we have managed to:
1. synthesise novel agarose beads that have a polymeric coating which limits DCM diffusion into the beads. This allows optimum degradation by the bioremediation bacteria, while physically containing the bacteria for safety reasons
2. verify the functioning of the biopolymeric beads by measuring diffusion using indigo dye 3. use computer-aided modelling to design a prototype of the DCMation system, and physically constructed this container
4. 3D print a cartridge to hold our biosensor bacteria, which can easily be replaced by the user
5. construct a prototype circuit that lights up when the photodiodes detect light emission from our biosensing bacteria that are contained in the cartridge. This lets the user have a simple yes/no response to whether the contents of the container are safe for disposal.
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