Team:UZurich/Implementation

iGEM UZurich 2020

Proposed Implementation

Introduction

Our system has two main advantages over other comparably cheap systems. It is more rapid, giving an output in less than 20 minutes and quantifiable using only a luminescence meter. This makes it ideal to use as a test kit for multiple samples, to monitor changes in the total bacterial cell count, for example across a distribution system. The total bacterial cell count, though not sufficient to diagnose issues or determine safety, gives an overview of changes in water quality and allows experts to make decisions on how to carry on and track the origin of issues, filling a supplementary role to other systems. Though our system has a current, useful, implementation, this not the limit of the potential of PRR based biosensing. Its future potential and implementations are just as important.

How it works

Our product would be shipped in a sealed tube or a sealed microplate, with the yeast in a dry frozen state, able to be kept at room temperature for at least a year [1]. When needing to take a sample, one simply unseals the tube, places the water sample in the tube together with the luciferase substrate and reseals it to prevent any chance of GMO escape into the environment. The water then reactivates the yeast and starts the detection process. In a few minutes the yeast is fully active. The receptors in the organism’s membrane begin dimerizing, if there is bacterial presence in the sample. Once the receptors dimerize, the split luciferase reconstitutes and gives a luminescent output. Within 20 minutes the output should be readable by a luminescence meter. Then all one needs to do is read the luminescence output and compare it to a reference chart to obtain the expected bacterial load.

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This can be done on-site and the tube can be inserted directly into a portable luminescence metre. Alternatively in a laboratory context many samples could be inserted into a microplate and read simultaneously to allow for higher throughput. After usage, the tube can be autoclaved or disinfected to easily & correctly dispose of both the pathogens in the sample and our GMO organism.

Comparison with existing systems

Current testing methods for bacterial testing in water samples are extremely diverse, but all systems have drawbacks. Below you see a comparison of different systems of measurement that are used in a water quality context, in comparison to ours:

Test Price Sensitivity Specificity Speed Quantifiable?
Lab methods (PCR, Flow Cytometry, Sequencing)[2] High High High System dependant Extremely
Dipsticks litmus test [3] Approx 5$ per sample Medium Coliforms Rapid (less than 30 min) Limited, colour change
Culture based methods [4] Approx. 1$ per sample [5] Medium High [6] Slow (1-2 days) Indirectly
ATP Systems [7] 5$ per sample High None Rapid (less than 20 min) Yes, luminescence
PRR based Approx. 1$ per sample Medium Bacteria (Receptor dependant) Rapid (less than 20 min) Yes, luminescence
PRR based (Future prospects) Approx. 1$ per sample Medium Specific to epitope Rapid (less than 20 min) Yes, e.g luminescence

As you can see, our system is expected to be either cheaper or more quantifiable than comparable methods and at least equally rapid and low labour intensity. The total bacterial cell count (TBC) is important to measure in a distribution system since fluctuations in the TB indicate issues with disinfection steps and regrowth. As such quantification is a relevant tool to measure precisely.
With information about changes in the TBC, experts can observe large distribution systems and track issues with regrowth and biofilm formation to its source. For example one can detect and identify issues with disinfection steps in a water plant.

Our system is aimed for expert use, because the proper disposal of our GMO system, though easy to perform, needs to be ensured every time. Additionally, since a luminescence meter is required for use, laboratory settings which usually already possess a luminometer are most suited for its use. We believe it can function as an early detection test, alongside other more complex methods to determine water quality and help ensure water safety.

Future Potential

Though our current system serves a purpose, it is far from the full potential of PRR based biosensing. Both the PRRs themselves and the surrounding system have further development potential:

First the PRRs themselves, there is a huge variety known today and many more to still be discovered. Additionally current research into chimeric and engineered receptors suggests that we will be able to design PRRs with affinities to specific epitopes of our interest in the future. This is a huge advantage, as current research further suggests that PRRs can recognize viruses [8].
Detecting specific viruses would be a revolution in microbial sensing as very costly and complex techniques are necessary for their identification. Viruses are extremely important for water safety, due to their direct impact on human and animal health and as indicator species.

As it stands, one should be able to directly implement such a novel PRR into our current system, simply by replacing the receptor sequence in the plasmid. But why stop there, when the rest of the system has many more options?
Additionally current research into chimeric and engineered [9] receptors suggests that we may be able to design PRRs with improved or expanded ligand affinity.
Another benefit of our approach is that by proving the functionality of one PRR in a detector organism, we believe that many others should be functional as well, due to their biochemical similarity. This means that our system has great potential to be modular, as not only the PRR can be swapped with relative ease, but the output system can be adapted to a specific use as well.
We identified output modifications that could expand the utility of our system:

  • A colorimetric system based on beta galactosidase or another colorimetric output, for on site equipmentless testing
  • A downstream cascade, allowing for signal amplification or threshold based approaches
  • Transcription factor systems, for amplified or reusable systems
  • Alternative outputs to further reduce cost, as currently the Luciferase substrate makes up a major component of the price

On a macroscopic scale there are many additional possible improvements as well, such as adding a neutralizer directly to the sample tube, or even integrating the system directly into a handheld device with luminescence measurement potential.

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[1] 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.
[2] Deshmukh, Rehan A et al. “Recent developments in detection and enumeration of waterborne bacteria: a retrospective minireview.” MicrobiologyOpen vol. 5,6 (2016): 901-922. doi:10.1002/mbo3.383
[3] Gunda NSK, Dasgupta S, Mitra SK (2017) DipTest: A litmus test for E. coli detection in water. PLoS ONE 12(9): e0183234. https://doi.org/10.1371/journal.pone.0183234
[4] https://www.who.int/water_sanitation_health/publications/hpc/en/
[5] Price can be reduced by ordering components individually, but this increases manual labour significantly
[6] Species identifiable, but few are culturable
[7] Vang, Ó. K. (2013). ATP measurements for monitoring microbial drinking water quality. DTU Environment
[8] Gouveia Bianca C., Calil Iara P., Machado João Paulo B., Santos Anésia A., Fontes Elizabeth P. B., Immune Receptors and Co-receptors in Antiviral Innate Immunity in Plants, Frontiers in Microbiology, 7, DOI: 10.3389/fmicb.2016.02139, ISSN=1664-302X
[9] 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