Pili⁺

The Mine

The South West of England has a long and complicated mining history that only came to an end within our lifetimes. The remains of the old sites are still dotted around the region today and the entire scope of their legacy is still revealing itself now. Many areas in Cornwall and Devon are beginning to see the negative effects of the metal ions leaching into nearby waters.

The Consolidation Mine forms a part of the Cornwall and West Devon Mining Landscape World Heritage Site. After collapsing in 1992, it has been abandoned and currently contains approximately 220 000 m³ of mine waste. Numerous controlled water risk assessments show that the site is causing pollution of waters by leaching of arsenic, cadmium, copper, chromium, iron, lead, nickel and zinc through the lower lagoon (the site consists of two lagoons separated by three dams) into the St. Day stream. The stream is a tributary of the Carnon River, with potential to carry the harmful metals across Cornwall and even into the English Channel. The mine site is in a popular area for walkers and mountain bikers, whose inhalation, ingestion and dermal absorption pathways are under threat from the toxic contents of the body of water. Although the mine was proven to be a harmful site after an environmental quality inspectionconducted by the Environment Agency in 2007,little progress has been made in decreasing its negative influence on the surrounding areas.

The current methods of purification of the mine’s waters are large scale, but inefficient. They rely on pumps located over 60m below the surface, transporting at least 110 litres a second to special tanks. Contaminated water is then treated with lime in order to neutralise its acidity and to precipitate out the metal salts. The waste products from this process are not recycled or cleaned but are essentially dumped into a lined hole in the ground. The operation costs the government about £1.5m a year to run and the lime itself has to be ferried from Derbyshire, making the whole affair unsustainable economically and ecologically. This could be balanced out by attempts to retrieve and sell the metals, although this is not being attempted.

Where we come in

As members of the community in Devon and the South West we wanted to solve a local problem such as this. That is why we decided to run a synthetic biology project that would target remediation of harmful waters spreading from the Consolidation Mine Site. Half the team made a trip to an old mine site to see first hand the effects of the contamination and to analysethe water composition.

Having previously looked at biofilms and bacterial virulence mechanisms as an area of interest, we discovered the potential power of Type I pili in E. coli and saw that Harvard's 2015 iGEM team could become a solid basis for our research. Our aim from then was to fuse metal binding domains with the terminal proteins of pili and to house the modified bacteria in a robust and responsible filtration unit .

What are pili?

Pili are small hair-like structures found on bacteria. Pili are encoded by the fim operon which contains a number of genes that code for structural proteins, including FimH. The main constituent is FimA *insert reference*, which polymerises to form the main body of the pilus structure. FimH is found at the very end of the complex, and contains the lectin - the mannose binding domain.The other proteins are largely related to the chaperone-usher pathway *insert reference* They have a natural ability to attach to eukaryotic cell surface mannose molecules due to specific binding sites within FimH, the terminal protein. By investigating adhesion mechanisms involved in type 1 pili we aimed to repurpose the structures by introducing new metal-binding domains within the pili of our E.coli.

Implementation and responsible approach

The Applied Design aspect of our project involved building a prototype of a filter that could potentially be used to treat water from the mines. The prototype consists of three components, one of which is a 3D printed hydrocyclone that efficiently separates sediment from water. In order to search for the best methods of inserting E.coli into the system, we visited the Taunton Aquarium and purchased a Fluidised Media Reactor, forming one of the next components of the filter. The use of the experimental design statistical software (JMP), along with numerous experiments we conducted on the Reactor, provided us with required information including the optimal volume of the Reactor or the type of media the bacteria should grow on. The filter is also equipped with components ensuring quality and safety of the whole process, including bactericidal mechanisms preventing the transfer of GM organisms into the natural environment. The design of the filter, at all stages of its development, was carefully discussed with potential stakeholders, including the South West Water company, to insure it would meet the current standards.

In order to optimize the performance of our filter system and to measure its efficiency in various conditions, we constructed a mathematical model. The model, when given information about the metal to be extracted, initial concentration, flow rate, fluidised media reactor volume and target efficiency, calculates the expected efficiency, output concentration of the metal, time required to run the water through the filter and the mass of the metal reclaimed. It determines whether the whole process, assuming the variables given, would be successful and if there is no need for any changes in the initial settings.

We did not want our work in the laboratory to be insular. Over the course of the summer we liaised with a number of researchers, stakeholders and NGOs, all of whom became significant parts of the iterative progression of our project. Their influence and inspiration reminded us that scientists have an interdependent relationship with society and should consider themselves part of a larger community. All of our Human Practices have been executed according to the European Responsible Research and Innovation Framework.

We have released a mobile phone app that can be downloaded from the Google Play Store. The app is a simple visualisation of biological fundamentals underlying the design of our research and it contains trivia related to the world of synthetic biology as an educative function.

References

Andersson, M. et al., Dynamic Force Spectroscopy of E.coli P Pili, Biophysical Journal, Volume 91, 2717-2725 (2006)
Harvard BioDesign, iGEM 2015: https://2015.igem.org/Team:Harvard_BioDesign
Miller, E. et al., The Mechanical Properties of E.coli Type 1 Pili Measured by Atomic Force Microscopy Techniques, Biophysical Journal, Volume 91, 3848-3856 (2006)
Munera, D. et al., Recognitiion of the N-terminal lectin domain of FimH adhesin by the usher FimD is required for type 1 pilus biogenesis, Molecular Biology, 64(2), 333-346 (2007)
Pallesen, L. et al., Chimeric FimH adhesin of type 1 fimbriae: a bacterial surface display system for heterologous sequences, Microbiology, Volume 141, 2839-2848 (1995)

Team:Exeter/Description