Team:EPFL

Solving copper pollution with synthetic biology.

The problem

Mildew and copper fungicides

Mildew is a fungal disease that attacks plants including grape vines and greatly reduces the yield from affected crops.

In order to treat plants attacked by mildew, copper-based fungicides have been in use for over a hundred years thanks to their effective action and compatibility with organic farming practices. Although copper has little effect on the taste and safety of produce from treated plants, it can accumulate to toxic levels in the surrounding soil, inhibiting new cultivation and reducing biodiversity.

More on the problem
Copper concentration in topsoil across Europe.1 White: < 9 mg/kg, dark blue: > 65 mg/kg. The legal “investigation threshold” for topsoil copper is 40 mg/kg in Switzerland.2
24–30%

of the 2021 French wine production is expected to be lost due to mildew.3

4 kg/ha

of copper fungicides can be used at most each year in the EU.4

355 mg/kg

of copper can be found in certain regions of Switzerland, more than 8 times the investigation threshold.5

Our solution

Cuopper Re
cuperation
trieval
cycling
newal
generation
cuperation

Our solution aims to employ bioremediation to remove copper from fungicide contaminated rainwater. Our strategy is to generate transgenic yeast strains that express the endogenous copper-binding protein CUP1 on the surface to bind to and allow the removal of copper from water. To complement these yeast strains, we designed a bioreactor to allow yeast to interact with rainwater and remove pollutants such as copper.

3D structure of CUP1 obtained with NMR.6

Meet CUP1

CUP1 is a copper methallothionein, a small protein with a high affinity for copper ions. CUP1 is naturally present endogenously in the yeast Saccharomyces cerevisiae, where it is responsible for protecting against copper toxicity and acts as a source for copper for proteins with utilize this metal.

Thanks to its cysteine residues, a single copy of CUP1 can bind up to 8 copper ions, making it an ideal protein for biology-based copper binding and removal.

More on the design

Using and improving CUP1

Adding CUP1 to the surface

In order to harness the copper binding ability of CUP1 for bioadsorption, we have introduced CUP1 into a protein expression system which allows CUP1 to be expressed on the surface of yeast. We confirmed successful expression and localization of CUP1 on the surface of yeast.

Enhancing CUP1 copper binding

To further improve the potential for copper adsorption by CUP1, we designed of novel construct which featured two copies of CUP1 in tandem, theoretically doubling its binding capability.We have designed, expressed and tested seven different dimers, each with linker sequence of varying protein flexibility separating the two CUP1 sequences.

Explore our experiments

Discover more about our measurements of copper from vineyard soils, our cloning and expression of CUP1, our protein and immunofluorescence measurements that confirmed production and surface localization in yeast, our design of novel dimers of CUP1 with protein linkers of varying flexibility and our copper measurement chemistry assays.

NotebookResultsDiscussion
Immunostaining of yeast cells expressing our protein.

Implementation

To make full utility of our yeast strains, we designed a bioreactor to enable yeast suspended in beads to interact with rainwater and remove pollutants such as copper. We designed, manufactured and built the device. To make our device easy to use, we also implemented a graphical user interface on an associated tablet that controls of the bioreactor activities.

More on Implementation

Human Practices

In addition to working with experts for biological and hardware components, we engaged with stakeholders impacted by the effects of copper pollution including winegrowers, phytoremediation specialists and water purification authorities.

More on Human Practices

Education & Communication

In outreach activities designed to promote the understanding of and opportunities presented by synthetic biology, in addition to the specific problem we aimed to address, we created a childrens book and a series of podcasts, visited high schools, hosted an event to educate fellow students about iGEM and published a description of our project in a magazine for the general public.

More on Education & Communication

Sponsors

References
  1. Ballabio, Panagos, Lugato, Huang, Orgiazzi, Jones, Fernández-Ugalde, Borrelli & Montanarella (2018)
    Copper distribution in European topsoils: An assessment based on LUCAS soil survey
    Science of The Total Environment, vol. 636, pp. 282-298
    CrossrefGoogle Scholar
  2. Confédération suisse (2016)
    814.12 Ordonnance sur les atteintes portées aux sols
    Fedlex
    Full text
  3. Ministère de l'Agriculture et de l'Alimentation (2021)
    Viticulture n°2021-103, Infos rapides
    Agreste : la statistique agricole
    Full text
  4. European Commission (2018)
    Regulation (EU) 2018/1981
    EUR-Lex
    Full text
  5. Federal Office for the Environment FOEN (2021)
    Map of Heavy Metal Soil Contamination
    Full text
  6. Peterson, Narula & Armitage (1997)
    CU-METALLOTHIONEIN FROM SACCHAROMYCES CEREVISIAE, NMR, MINIMIZED AVERAGE STRUCTURE
    CrossrefGoogle Scholar