Team:Oxford/Results

Wetlab Results:

For the bioremediation aspect of DCMation, we have managed to:

1. purify the pUNI-ABTUNJK and pSB1C3-ABTUNJK plasmids, which encode the microcompartments
2. verify expression of pUNI-ABTUNJK plasmids in E. coli using Western blotting
3. insert the ABTUNJK vector into pSRKGm using Gibson assembly, for expression in P. putida
4. insert the dcmA gene into the pCM66 backbone using Gibson assembly, since pCM66 is suitable for expression in Methylobacterium extorquens DM4
5. insert dcmA into the pRSFDuet, and transformed this into DH5α cells for hypermutagenic PCR on the dcmA gene
6. fuse dcmA and sfGFP using PCR, followed by restriction and ligation to insert the dcmA-sfGFP transcriptional fusion into pRSFDuet. This was then transformed into DH5α cells and imaged using fluorescence microscopy
7. insert microcompartment-tagged dcmA into pRSFDuet
8. insert the microcompartment-tagged sfGFP into pME6010, and used fluorescence microscopy to image this

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')

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.