Team:Dundee/Project

Algae are photosynthetic organisms which live in aquatic environments. Cyanobacteria, also known as blue-green algae, are prokaryotes that are responsible for the majority of photosynthesis on Earth. During the hot and sunny summer months we experience a phenomenon known as an algal bloom. This is a spectacular increase in the size of the algal population in a water body as the algae take advantage of the seasonal spike in light and warmth. With increased amounts of nutrients leaching into water due to agriculture, blooms are becoming more common. Aside from affecting the environment they occur in, algae can be dangerous for humans as many species of algae produce toxins.

The Perfect Synthetic Biology Project

In the beginning, we thought: what makes a perfect synthetic biology project? First you have to have your idea. Rather than showing this to the public at the end of the project, we thought it would be a much better idea to include them in the project from the very start. In this way, they can bring up any concerns they have and we can try to address those throughout the project through our Human Practices.

With this, our project becomes a lot less about consultation and a lot more about inclusion. For this reason, we feel that our team has become a lot more than the ten of us. These extra team members will be introduced throughout the project.

ToxiMop Project

In order to tackle these toxic algal blooms, our project consisted of 5 subdivisions which come together to reach the best solution;

1. Mopping
2. Detecting
3. Monitoring
4. Informing
5. Mathematical Modelling

1. Mopping

We are to focussing on one hepatotoxin called microcystin, a cyclic non-ribosomal peptide that binds covalently and irreversibly to protein phosphatases in mammalian bodies resulting in an inactivation of the protein.

The study of biochemical processes in human cells is one of the most heavily-researched scientific fields. However, it is not often that scientists exploit the biochemical potential that our bodies offer in order to create new technologies for environmental remediation. We have decided to exploit the human protein-phosphatase 1 (PP1)–microcystin interaction to create a bacterium that will sequester microcystin and prevent its toxic action.

Microcystin cannot freely diffuse through the inner membrane of bacteria but can sufficiently enter the outer membrane. Therefore we engineered the chassis organism Eschericha coli to export human protein-phosphatase 1 (PP1) to its periplasmic compartment. By doing this we have created a biological mop for microcystin that we call the ToxiMop.

2. Detecting

The current procedures for detecting algal toxins involve HPLC (High Performance Laser Chromatography) analysis of contaminated water bodies, and takes approximately 24 hours. Bacterial populations are so dynamic that in the interim between taking a sample for testing and getting a result, the amount of toxin present may have changed dramatically. In addition, this kind of analysis is expensive, so often water bodies will not be tested unless there is already some evidence of algal growth.

Based on the protein – toxin interaction explained above we also constructed a toxin detection system. E. coli’s membrane-bound osmolarity sensor, EnvZ, was modified to include the PP1 protein. The idea was that upon binding to the microcystin, EnvZ is activated and triggers luminescence by upregulating expression of a fluorescent reporter gene. The luminescence can then be recognised by our monitoring device – the Moptopus.

3. Monitoring

Feedback from the biological detector would be recognised by a modular hardware device called the Moptopus. The Moptopus contains a pH meter, dissolved oxygen meter, thermometer, light sensor, humidity meter and a camera. Implementing these sensors allows us to calculate the possibility of an algal bloom occurring and will provide data on demand for potential algal bloom forecasts. The Moptopus is designed to permanently inhabit a water body, providing real-time information on many of the parameters that control algal growth.

4. Informing

With daily monitoring of algal bloom toxicity and data relating to algal bloom growth, the Moptopus can act as an early warning system. This warning system would inform the public in cases where toxicity could be harmful to individuals and pets. Furthermore, by real-time detection of toxicity, the need for the ToxiMop can be determined immediately and the mop deployed when needed.

By monitoring the relationship between environmental conditions and toxicity we can potentially learn more about any environmental links and causes of this toxicity.

5. Mathematical Modelling

Mathematical modelling was the fundamental foundation to the project in understanding and informing the progression of the wet work.

Initially the team was working with two bacterial chassis, E. coli and B. subtilis. We planned to anchor PP1 to the outer surface of B. subtilis or have the protein free flowing in the periplasm of E. coli. If the efficiency of these bacterial chassis as mops depended only upon the number of PP1 they could accommodate, the E. coli chassis has the potential for greater efficiency. This analysis allowed the Wet team to tailor their time and resources accordingly.

Modelling of Tat machinery, which we used to transport the PP1 molecules to the periplasm of E. coli, produced ordinary differential equations able to mathematically explain the processes being carried out in the ToxiMop bacteria. Using this modelling, limiting factors of transport were identified and future improvements to increase the efficiency of the bacterial mop were suggested to the Wet team.

The migration of microcystin into the periplasm of a cell and its binding with PP1 are crucial events to the successful application of the ToxiMop. Visualisation of production and export of PP1 alongside these events was achieved via the creation of a virtual cell environment in Netlogo (a multi-agent programming language). This virtualisation allowed the dynamic alteration of key properties returning immediate feedback to help analyse the corresponding effects.