We chose Pseudomonas vancouverensis DSM8368 as a promising organism for bioremediation [1], [2], [3], even though we were, to our knowledge, the first scientists to utilize it for synthetic biology. We knew that using a non-model organism for metabolic engineering would be difficult, especially considering we found only one documented, successful attempt of transforming P. vancouverensis [4]. But we believe in the potential of this organism.
We think P. vancouverensis can be used in bioremediation of many pollutants. It is known for its ability to grow on numerous unconventional carbon sources, for example, resin acids [3] or 2-butoxyethanol [5] next to naphthalene and phenanthrene [1], [2], [6]. Its resilience to toxins and other hostile conditions results in many possible areas of application. Therefore, next to cleaning rivers P. vancouverensis could be used for bioremediation in ecosystems like forests or grasslands.
P. vancouverensis could also be useful in agriculture for its biocontrol activity [7]. It has shown antifungal activities against crop fungal pathogens (like Colletotrichum fragariae) [8], [9], and antibacterial effects against fire blight (Erwinia amylovora) [10]. Other studies show that P. vancouverensis promotes plant growth because it acts as a biological fertilizer by solubilizing phosphate [11] and potassium [12], [13], [14] . Co-cultivation can help crops as well as aquatic plants to withstand high stress conditions [4], [15], [16]. Way more research needs to be done in this field, but with bacteria supporting our efforts to efficiently grow food, we might get a step closer to ending world hunger.
We hope to encourage and inspire future iGEM teams to use P. vancouverensis, because we had a great time working with it and in our opinion the species deserves more attention.With the documented protocol for transformation and our insight into cultivation, we hopefully paved the path for other iGEMers using this chassis.

Read transformation protocol

To make a river cleanup possible without needing maintenance, P. vancouverensis needs to be immobilized in a stationary device. There are just two research groups, that looked into the immobilization of P. vancouverensis before. The group around Alptekin Aksan tested the immobilization in a silica gel matrix [17], [18], [19], [20]. Another group (around Geelsu Hwang) observed how the bacteria sticked to sand that is polluted by naphthalene way better, than to just sand [21]. In further research it was discovered, that P. vancouverensis has a very hydrophobic surface and therefore sticks to hydrophobic substances. The hydrophobicity of its cell surface seems to increase with naphthalene intake, making it stick to the substrate even more [22]. So, the current theory is, that when the bacteria take up PAHs, they morph their cell surface to have even more PAHs stick to them [23]. Therefore, we made the bacteria immobilize themselves on silica surface as well. Further than that, we couldn't rely on existing protocols for this organism. Developing a completely new approach to immobilize an organism that is not sufficiently researched would have needed way more time than we have in one iGEM year. Therefore, we tried three methods that have been proven to work with Pseudomonas putida [28]. All three methods attempt to immobilize bacteria in beads that are permeable to water and chemicals but not bacteria (see our results page to learn more).
We hope that there will be a lot of further research in the area. That's why it was important to us to use the limited time we had the create a base, future iGEM Teams can build on. We would also like to encourage young scientists to work with non-model organisms even if there is almost no existing research. We hope that our project showing that approved methods from related model organisms can be transferred, helps others to be more confident in researching non-model organisms.

We added all necessary pyrene degradation enzymes to the part registry, to help future teams work with these proteins. See our parts page.
To get insights on the function and usage of at least some of those parts, we decided to characterize one enzyme complex. This complex is a hetero-tetramer consisting of two dimers with different functions. The first dimer consisting of phtAc and phtAd is an electron transporter, which uses NADH during the degradation of phenanthrene-4-carboxylate. The other dimer consisting of pdoA2 and pdoB2 contains the active site of the enzyme used to degrade phenanthrene-4-carboxylate to cis-3,4-dihydroxy-phenanthrene-4-carboxylate [24].
We planned multiple different activity assays to characterize these proteins as complex and monomers. Thereby we could prove their function and derive kinetic parameters for phtAc and phtAd’s catalytic activity. This electron transportation-system is used by variety of ring-hydroxylating-oxygenases, making it relevant in multiple PAH-degradation pathways [24–26]. Therefor we hope, our characterization will show useful to future iGEM teams, that might also tackle different pollutants.

To apply our modified P. vancouverensis in a real-world setting, we needed to embed the organism into a suitable container that could be placed in rivers, providing optimal flow conditions for breaking down pollutants in the water. Our goal was to determine the ideal internal structure that would facilitate the required flow properties while being easy to manufacture. We explored three designs: a maze, cylinders, and spheres.
To identify the most effective design, we simulated the flow dynamics of each structure using the industry-standard software Ansys [27]. This approach is relatively novel within the iGEM competition, as few teams have explored flow analysis in this context. Setting up the software and obtaining reliable results was challenging and time-consuming.
We documented our efforts on the model page to help coming iGEM generations implementing flow analysis into their project more quickly and obtain reliable results.

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