Sub Divisions

The project is divided into three conceptual parts:

Part A: Since DCM metabolism is toxic to cells, we will test the previously reported tolerance of ~20 mM DCM in M. extorquens. In order to pursue the possibility of using other bacteria for this system, we will also establish the toxicity of DCM metabolism in E. coli and Pseudomonas strains. Finally, we will attempt to evolve tolerance of the various bacteria to DCM.

Part B: The gene responsible for DCM metabolism, dcmA, is regulated by an adjacent gene, dcmR. As this system is currently poorly characterised, we will establish the role of dcmR, and whether it acts as a repressor or activator. The binding site of the regulatory gene, dcmR, has not yet been characterised. We will therefore truncate upstream regions of dcmA to find the binding site, and thus further characterise the dcmR/A regulatory network. In order to test the above, we are constructing a DCM reporter consisting of a translational fusion between DcmR to mCherry.

Part C: In order to increase the efficiency of the DCMation system, we will use directed evolution to attempt to improve the catalytic turnover of the dcmA gene product by using random mutagenesis. Furthermore, we will use microcompartments to contain DCM metabolism within the cell, and thus avoid associated problems with toxicity.