UNILausanne - iGEM 2022

Quagga mussel: an invasive species in Switzerland waters

Since 2014, Switzerland has been facing a major problem: the unintentional introduction in our lakes of the quagga mussel Dreissena bugensis. It is an invasive species and has devastating consequences on our biodiversity and water distribution systems because of its rapid growth (Figure 1). Its propagation is currently resulting in a dramatic loss of biodiversity in our Swiss lakes and significant economic problems. At present, this invasive species is found in about ten Swiss lakes (Figure 2). Moreover, quagga mussels are also present in many lakes throughout Europe and several freshwater regions in North America, therefore representing a worrying worldwide problem. In the United States, the costs linked to managing the colonization of quagga mussels amount to 1 billion dollars. Every year in Switzerland, it costs several million francs.
Due to the massive impact on the environment and infrastructure caused by quagga mussels, the 2022 University of Lausanne iGEM team decided to tackle this issue. Our goal is to find a solution to this local, as well as global problem, by working hand in hand with professionals and people affected by this invasive species. We aim to eradicate and stop the spread of quagga mussels in Switzerland and worldwide.

Our team member Filippo on a mussel quest — **Figure 1:** Pipes blocked by quagga mussels investigated by Filippo, one of our team members, in the lake Geneva.

Zebra and Quagga mussel — **Figure 2:** Current distribution of the quagga mussel (*Dreissena bugensis*) in Switzerland since its first observation in the Rhine, near Basel, in 2014. The figure shows the locations of observed cases represented by circles, of unobserved cases by open squares, and the first eDNA (environmental DNA) observation by a star (Haltiner, 2022, p. 160). At the top right of the figure is an image of a quagga mussel.

Quagg’out: The Project

To do this, our project, named “Quagg’out”, is divided into two parts both based on the genetic engineering of the bacteria Escherichia coli and Pseudomonas protegens. First, we set out to kill the mussels using the FitD toxin. Encoded by the fitD gene, this toxin naturally expressed by the bacterium Pseudomonas protegens is known for its insecticidal properties and potency as a molluscicidal agent. Second, we wish to prevent the attachment of mussels to surfaces using zosteric acid, which has anti-adhesive properties. Zosteric acid is an acid naturally produced by the algae Zostera marina, and it possesses anti-insecticidal, anti-fungal, and anti-adhesive properties.

FitD toxin to kill quagga mussels

To this end, we engineered Pseudomonas protegens to over-express the FitD toxin using two different approaches. We introduced in our cells plasmids bearing either (1) an extra copy of the fitD gene or (2) a copy of the fitG gene, which encodes a protein that acts as a positive regulator of FitD expression. Two variant plasmids were built for each of our two strategies, with our genes either expressed constitutively or under the control of the IPTG-inducible lac promoter (Figure 3). We reasoned that testing these two distinct designs would increase our chances of obtaining a stable and substantial increase in toxin production. To test FitD production and its efficacy in killing quagga mussels, we first grew our engineered bacteria in liquid media and induced the production of FitD when appropriate. We then applied live or lysed cells to small water tanks containing the mussels and quantified their molluscicide activities.

Plasmids FitD — **Figure 3:** Different plasmid used for the FitD experiment (FitD constitutive: pSEVA2313_FitD, FitD inducible: pSEVA234_FitD, FitG constitutive: pSeva2313_FitG and FitG inducible: pSEVA234_FitG).

Zosteric acid to prevent attachment of quagga mussels

For this part of the project, we used a strain of Escherichia coli. First, we cloned the TAL and SULTA1 genes in a pET17B backbone creating two version of this plasmid: pET_17b_SULT1A1_Tal and pET_17b_Tal_SULT1A1, making it possible to carry out the catalytic reactions using tyrosine as precursor to generate zosteric acid by passing through an intermediate compound (p-coumaric acid). Second, we cloned cysPUWA and cysDNCQ genes in a first pCola_Duet plasmid and the cysP and cysDNCQ genes in a second plasmid. This strategy gave two versions of the plasmids, allowing for transport and activation of sulfate to react with p-coumeric acid and recycle byproducts in the cell: pCola_Duet_cysPUWA_cysDNCQ and pCola_Duet_cysP_cysDNCQ (Figure 4). All our plasmids, being under the LacI promoter, can be induced using IPTG. Thus, our various plasmids transformed in our bacteria allow the production of zosteric acid, which will then be released into the medium and applied in solution in a small aquarium containing the quagga mussels to test its effectiveness.