This year, we managed to successfully integrate the synthetic gene coding the Immobilization module into the chromosome of Bacillus subtilis. However, there is still a lot of work to be done in order to convert Bacillus into an effective killer of cyanobacteria.
Next year, we plan to assemble the cellulosomes, clone the rest of our genes into vectors, transform them into Bacillus subtilis and thus enabling our bacteria to display the scaffoldins on its surface. Synthetic scaffoldin is attached to the bacterial cell by its LysM domain. The first scaffoldin displays lysozyme and microvirin to attach and lyse cyanobacteria and the second scaffoldin binds enzymes of the MC pathway to degrade cyanobacterial toxin microcystin.
To make CYANOTRAP even more efficient, we want it to accumulate phosphorus from the water. This could enable phosphorus recycling and it will also lead to the loss of optimal conditions for cyanobacteria, as the phosphorus intake is required for their growth . Phosphates can be incorporated into larger polyphosphates by the enzyme polyphosphate kinase. By expressing this enzyme inside a bacterial microcompartments (BMC), newly synthesized polyphosphates can be encapsulated in this BMC and thus protected from enzymes which degrade them .
We also have a couple of other ideas to improve CYANOTRAP. The realisation of these however is much more complicated as they require further research. Our first idea is to add a biosensor monitoring the concentration of microcystin and other cyanobacterial toxins in the water. This will allow us to not only measure the effectiveness of the device but also to send CYANOTRAP into the most polluted areas. This biosensor is inspired by the iGEM team Cornell 2019 . They used aptamers - short DNA molecules which specifically bind to the target molecule. Although we love this idea, we had to postpone its realisation, as designing aptamers is expensive and complicated.
And last but not least there is our idea for a quorum sensing-based autolysis system, which would prevent genetically modified Bacillus subtilis from escaping into the environment. If a bacterial cell leaves the device, it will no longer receive quorum-sensing molecules. As these molecules are blocking the synthetic autolysis pathway, their absence will trigger the destruction of the escaped GM bacteria. We really wanted to include this addition to our project but after complex research, we decided to postpone it as well, as quorum sensing mechanisms of Gram-positive bacteria have currently not been explored in great detail and we would have to deal with other problems like diffusion of the quorum-sensing molecules in the water stream.
Step by step, we hope to gradually complete our ideas into a beautiful and complex device. We hope to introduce our next steps in iGEM 2021. See you there!
Parrish, J., 2014.. The Role of Nitrogen and Phosphorus in the Growth, Toxicity, and Distribution of the Toxic Cyanobacteria, Microcystis aeruginosa. Master's Projects and Capstones. 8.
Liang, M., Frank, S., Lünsdorf, H., Warren, M.J., Prentice, M.B., 2017. Bacterial microcompartment-directed polyphosphate kinase promotes stable polyphosphate accumulation in E. coli. Biotechnol. J. 12, 1600415. https://doi.org/10.1002/biot.201600415
Scheme of the phosphorus accumulation into the BMC
Scheme of quorum sensing-based autolysis system. Bacterium only prospers when being among other bacteria producing quorum-sensing molecules.