Team:UZurich/Description
Description
The Challenge
Microbial pollution in water is a major issue across the globe [1]. Though it is more prevalent in developing nations, first world countries also suffer from it. Even Switzerland, a nation famed for its drinking water has issues with microbial pollution, such as legionnaires disease. Legionnaires disease is caused by the bacteria Legionella pneumophila and causes 500 cases yearly in Switzerland of which up to 10% end fatally [2].
Legionella and other bacteria are particularly difficult to combat in water distribution systems, due to their ability to form biofilms, allowing them to better cope with biotic and abiotic stressors. As such, many tests across the distribution system are necessary to insure safe drinking water. Currently, many testing methods exist, but they all have drawbacks: either they are labour intensive, expensive, have low sensitivity or are not quantifiable.
The Goal
To assess the bacterial contamination in a water sample many approaches exist. Cheap and rapid options for detecting bacteria are either unspecific, labour intensive or not quantifiable. For a more in-depth comparison of the different approaches please visit our implementation page.
Our goal is to improve on these methods, by creating a bioreporter system that would be equally cheap and rapid but allow for greater quantification and simplicity than currently available systems.
Bioreporters are a subcategory of biosensors, that utilize a whole organism as a base for a biochemical detection system.
We believe a bioreporter approach would fulfill our needs perfectly, as a bioreporter system can reproduce itself and is thus extremely cheap to mass produce [3] and can be designed to possess a rapid and simple output. Additionally, through a well characterized output biosensing systems can be made highly quantifiable.
The Tool: Plant Pattern Recognition Receptors
The most important part of a biosensing system is the sensor itself, the detector module. We chose to work with plant immune system receptors as they have been honed over millions of years of evolution to be extremely good at sensing pathogens [4] and are thus predestined for biosensing applications.
FLS2-BAK1 complex [12]
Pattern Recognition Receptors (PRRs)
PRRs form a part of plants' innate, non-adaptive immune system which recognize conserved microbial epitopes (MAMPs, Microbe associated molecular patterns) with high affinity and specificity. We focused on a subsection of PRRs that are membrane-localized and consist of a receptor and co-receptor. Upon presence of a bacterial epitope these receptors dimerize and activate a signalling cascade that initiates the plants immune response. If you want to learn more about PRRs, we wrote a guide to introduce you to the topic.
Using PRRs in a biosensing system is a completely novel approach that has never been attempted before. To even express PRRs in microorganisms has never been tried before. As such we wanted to lay the groundwork and introduce this system to synthetic biology in a modular way, to allow for future advancements of PRRs in biosensing.
PRR based Biosensing
The detector module
We chose one co-receptor (BAK1) and three PRRs: EFR, FLS2 & CORE, which recognize the bacterial elongation factor-tu, flagellin & cold shock protein respectively [5]. All of these receptors are well studied and recognize an extremely broad spectrum of bacteria, with the elongation factor-Tu being highly conserved in all bacterial groups we have analysed [6]. The three main receptors only dimerize with their co-receptor BAK1 in the presence of a bacterial epitope. These properties allow us to construct a PRR-based biosensor that detects the total bacterial load of a sample.
The reporter module
To test for dimerization between BAK1 and EFR/FLS2/CORE after exposure to a contaminated sample, we decided to use a split protein system by attaching one half of a fluorescent protein to each the receptor and co-receptor. The system’s two halves don’t exhibit any fluorescence on their own, but when brought into close proximity to each other, they reconstitute the complete, functional fluorescent protein. The dimerization of the receptors in the presence of bacteria therefore initiates fluorescence of the system.
We have designed our system by attaching a split luciferase [7] to the intracellular domains of the receptors and co-receptors. In the presence of microbial contamination these receptors should dimerize and create a luminescent output. By integrating these receptors into S. cerevisiae, we can create a simple bioreporter that can be dry frozen [8], stored and transported to remote locations with ease. By directly fusing our output to the receptor, we aim to obtain a luminescence signal that correlates directly with the amount of bacteria present in a sample. Thus a sample's bacterial load could be quickly and easily quantified using a luminometer or other related devices.
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Implementation
Our testing kit is designed to work in an incredibly simple way: The kit is delivered in a test tube - all one has to do to apply our system is add a water sample to the tube together with the luciferase substrate and seal it. The quantification can be done by placing the tube directly into a luminescence meter and reading the luminescence output. The luminescence is expected to be proportional to the total bacterial cell count. This can be done with a single sample in a handheld luminescence meter or in the lab, where a microplate format could be used to enable high-throughput testing of many samples. In this approach neither our detector organism nor the contaminated sample can pose a risk or escape, since the tube only needs to be unsealed once, when the sample is inserted, and can be left sealed until disposal, by autoclaving or disinfection.
Created with BioRender.com
From our initial planned implementation, much has changed throughout the development cycle. Due to feedback from professionals, we have adapted our goal, our target audience and our system itself, to better match the needs. We have learned many important limitations of our system and have taken them into account during our design-build-test cycles. You can learn more about how the Integrated Human Practices helped us to reshape and improve our project on our Human Practices page.
Our detector organisms need to be packaged in a responsible way, not only to ensure they can properly function but also due to any potential mishandling. Safety is very dear to us, and while developing a concept for possible implementation of detector organisms into a device, we considered this heavily. Our imagined primary application market has the benefit that professionals will be handling the organisms. In such an environment, autoclaving ensures proper disposal. For more on the precautions we’ve taken during the development of our project please read our Safety page.
Future Prospects
One of our main goals of this project was not just to create an implementation, but to lay the groundwork for PRR based biosensing. PRRs are being heavily researched in the context of transgenic immunity in GMO plants [9] for agriculture. In biosensing they are currently hardly explored. By developing a modular system for PRR biosensing we want to ensure that future advances of PRRs in agriculture can be integrated directly into a biosensing approach.
This works because most plant PRRs are highly similar in biochemical structure [10] and our system is designed in a way where our output works identically, no matter what PRR is inserted.
PRR biosensing has a lot of potential in its future, as PRR research is being driven in an agricultural context, from discovering new receptors with new epitopes to engineering and designing new PRRs with improved or expanded epitope affinities [11].
Additionally, since the receptors are similar, any output modifications done in one PRR biosensor likely maintain functionality with another receptor, again making the system highly modular. Important examples of desirable output modifications would be: A colorimetric, visible to the naked eye output for pathogen specific systems or a signalling cascade-based threshold output.
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Improving the world, one sample at a time
We are incredibly privileged in Switzerland, our water standards are extremely high and it often gets overlooked, just how much is put into this safety. We hope to introduce a new way of approaching water testing with our plant immunity based receptors, and thereby contributing to the continuously improving movements to combat water pollution. Its unique application purpose in supplementary pathogen detection offers valuable information. To be contributing to global security is an honor and also a responsibility. With accurate quantification, safe methods and unfazed dedication - we hope to make a change. One receptor dimerization, one water sample, one biobrick at a time.
[1] https://www.who.int/water_sanitation_health/water-quality/en/
[2] (German) Swiss Health Department: Legionella, https://www.bag.admin.ch/bag/de/home/krankheiten/krankheiten-im-ueberblick/legionellose.html
[3] van der Meer, J., Belkin, S. Where microbiology meets microengineering: design and applications of reporter bacteria. Nat Rev Microbiol 8, 511–522 (2010). https://doi.org/10.1038/nrmicro2392
[4] Alberto P. Macho, Cyril Zipfel, Plant PRRs and the Activation of Innate Immune Signaling, Molecular Cell, Volume 54, Issue 2, 2014, Pages 263-272, ISSN 1097-2765, https://doi.org/10.1016/j.molcel.2014.03.028.
[5] Kunze, G., Zipfel, C. et al. The N Terminus of Bacterial Elongation Factor Tu Elicits InnateImmunity in Arabidopsis Plants, The Plant Cell, Vol. 16, 3496–3507, December 2004, DOI: https://doi.org/10.1105/tpc.104.026765
[6] See our Design page.
[7] Wang F.Z. et al., Split nano luciferase complementation for probing protein-protein interactions in plant cells, J. Integr. Plant Biol. 2019; (Published online November 22, 2019. https://doi.org/10.1111/jipb.12891)
[8] J.-F. Berny, and G. L. Hennebert. "Viability and Stability of Yeast Cells and Filamentous Fungus Spores during Freeze-Drying: Effects of Protectants and Cooling Rates." Mycologia 83, no. 6 (1991): 805-15. Accessed October 26, 2020. doi:10.2307/3760439.
[9] Ranf, Stefanie. (2018). Pattern Recognition Receptors—Versatile Genetic Tools for Engineering Broad-Spectrum Disease Resistance in Crops. Agronomy. 8. 134. 10.3390/agronomy8080134.
[10] Ulrich Hohmann, Kelvin Lau, Michael Hothorn, The Structural Basis of Ligand Perception and Signal Activation by Receptor Kinases, Annual Review of Plant Biology 2017 68:1, 109-137, https://doi.org/10.1146/annurev-arplant-042916-040957
[11] Freddy Boutrot and Cyril Zipfel, Function, Discovery, and Exploitation of Plant Pattern Recognition Receptors for Broad-Spectrum Disease Resistance, Annual Review of Phytopathology 2017 55:1, 257-286, https://doi.org/10.1146/annurev-phyto-080614-120106
[12] Yadong Sun, Lei Li, Alberto P. Macho, Zhifu Han1, Zehan Hu, Cyril Zipfel, Jian-Min Zhou, Jijie Chai, Structural Basis for flg22-Induced Activation of the Arabidopsis FLS2-BAK1 Immune Complex, Science 01 Nov 2013, Vol. 342, Issue 6158, pp. 624-628, DOI: 10.1126/science.1243825