Team:KUAS Korea/Description

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Project Description

The inspiration for our project started from reading the book “the sixth extinction” by Elizabeth Kolbert. In her amazing book, I(Sungwook) read stories about how human activities were posing a threat towards our wildlife and ecosystems. Among the many episodes she delivers, I was especially astonished by the fact that the amphibian disease chytridomycosis caused by the fungal pathogen B. dendrobatidis(Bd)were pushing amphibian species towards the brink of extinction. I couldn’t believe that amphibian species living in the remotest parts of the globe were being affected. The beautiful anuran Panamian golden frog(A.zeteki) were so heavily affected that they were extinct in the wild. They were only salvaged from complete extinction because of the efforts of conservationist Edgardo Griffith and the EVACC foundation. I thought that we should feel responsibility and should provide a solution. I brought up this issue during our weekly discussion earlier this year and eventually we decided to work on this project.
As we gathered more information, we found a research paper which claims that the origin of this pathogen is the Korean peninsula(1). As a South Korean team, we felt more motivated by the project. We soon became aware that U Florida had previously worked on the same topic in 2017(2). They worked on a project to produce tryptophol which is known to be effective in repressing the growth of the pathogen. However, since their project was simply producing the anti-fungal agent, we saw room for improvement especially in the biosecurity aspect. Also, as a Safety grant recipient, safety consideration largely shaped our project.

Along with tryptophol, violacein is also known to be effective against Bd. Previous iGem projects used violacein as their focus to produce color, but we couldn’t find teams that focused on the antifungal properties of this chemical. Adopting this compound as an antifungal agent seemed like a fresh approach, so we selected this compound. Also, the visible color of the compound would make it easier for us to detect the compound in future wet lab experiments.
We read about experiments which inoculated violacein producing microbes on the skin of frogs to see whether it enhances the survival of the amphibians(3). This approach worked on some species but not in A.zeteki(4). We thought synthetic biology may provide a better solution, since engineered cells, with their versatile properties, might perform better in these probiotic approaches. Our engineered cells will be applied on the skin of the amphibians to confer immunity against the pathogen. More information at Proposed Implementation.

Improving the adhesion of the cells to the mucus layer of amphibians would improve specificity and would reduce the losing cells to the surrounding environment. Thus, we found several mucus binding domains and decided to fuse one of them with surface display protein OmpA. We hope this part would be displayed on the surface of the cells and improve the adhesion of cells. More information on our Design page.

With Improved adhesion, we thought that any cells that detach from the skin should be killed to prevent them from propagating. We designed a quorum sensing Kill switch which only allows cells to survive under conditions of high cell density. With this system, cells would survive on the skin of amphibians, where cells exist in high densities, but would not once they detach from the skin. More on our Design page.

For higher sensitivity, pathogen detection would be necessary. This part was the most difficult to design and our final design has many limitations. We found research shows that tryptophan is emitted by the pathogen but we were unsure whether the pathogen would produce trp in quantities that would be detectable and also trp was too unspecific. As an alternative, we found a plant receptor(CERK1) that dimerizes upon contact with chitin: a fungal cell wall constituent. Chitin is a reasonably specific molecule and the CERK1-ECD(extracellular domain) alone is known to distinguish chitin with peptidoglycan and dimerize. This domain was perfect for detection purposes. We searched igem projects that worked on receptor-based detection and found the project by Uppsala. CadC works as a receptor that dimerizes and acts as a transcription activator of the promoter CadBA. We fused the domains on the end of this receptor as a detection module. More at Design page. But the caveat is that Bd do not have chitinous cell walls. We learned this fact only after we finished the design of the proteins through the interview with Prof. Fisher. But we think our constructs will well adapt to sensing other Bd specific molecules. More at HumanPractices

When we reviewed our designs, we could think of two scenarios that may be potentially harmful. First, the engineered microbes could propagate under soil, disrupting the microbial diversity. And second, predators ingest the microbes along with the amphibians. Soil hosts a whole ecosystem of microbes including beneficial ones such as mycorrhizal fungi. Unregulated violacein production would disrupt this system. Also, if these microbes end up in the digestive organs of predators, it could disrupt the gut microbes. We thought that only allowing violacein production under the presence of light would mitigate these issues since light does not reach under soil or inside digestive organs. Thus, we assembled multiple light sensors(LovTAP, pDawn) using Biobricks. We hope this construct improves the safety of the project. More at Design page.

With our input options(light & chitin) ready, we could combine them to create an AND gate, and improve specificity. For example, we could make the cells to produce violacein only when there is light and when chitin is detected. Violacein is synthesized from tryptophol with 5 enzymes(VioABCDE) catalyzing each reaction. If the enzymes are separated into 2 operons, each with a different promoter, the production of violacein would depend on both inputs, similar to an AND gate. Inputs could be changed by swapping the promoters of each operon. More at Design page.

References

1. O'Hanlon SJ et. al, 'Recent Asian origin of chytrid fungi causing global amphibian declines'. Science. 2018 May 11;360(6389):621-627. doi: 10.1126/science.aar1965. PMID: 29748278; PMCID: PMC6311102.
2. https://2017.igem.org/Team:UFlorida
3. Harris, R., Brucker, R., Walke, J. et al. 'Skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungus'. ISME J 3, 818–824 (2009). https://doi.org/10.1038/ismej.2009.27
4. Becker MH, Harris RN, Minbiole KP, Schwantes CR, Rollins-Smith LA, Reinert LK, Brucker RM, Domangue RJ, Gratwicke B., 'Towards a better understanding of the use of probiotics for preventing chytridiomycosis in Panamanian golden frogs'.
5. Ecohealth. 2011 Dec;8(4):501-6. doi: 10.1007/s10393-012-0743-0. Epub 2012 Feb 11. PMID: 22328095.

KUAS

Korea University

In-Geol Choi

South Korea

Tel: +82 10 3103 7332

Email: kuaskorea@gmail.com

Sponsor and Special thanks for

CSBL at Korea University

Sponsors