Team:EPFL/Poster

Poster: EPFL

Espress'EAU: Test your water
Presented by the EPFL 2020 iGEM team

Andrei Furtuna¹, Harshdeep¹, Laura Iacobucci¹, Yasmine Kouba¹, Ella Miray Rajaonson¹, Julien Salomon¹, Anas Seddiki¹, Sven Basil Spörri¹, Konstantinos Ragios², Amir Shahein², Laura Grasemann², Chun-Jie (Josh) Cai², Shiyu Cheng², Sebastian Maerkl³



¹iGEM Student Team Member, ²iGEM Team Mentor, ³iGEM Team Primary PI, School of Engineering, Swiss Federal Institute of Technology Lausanne (EPFL)


Abstract

Water is an essential resource that we regularly use for drinking, cooking and food processing. Hence, it is important to ensure that it is safe for consumption. Espress'EAU serves as an analysis of general water quality. We aimed to create a safe, low-cost, and easy-to-use system that can enable on-site testing of water at home or in small communities. We hypothesized that genetically engineered yeast could be used as biosentinels by hijacking the yeast stress response pathway using either deletion strains or fluorescent reporter strains that are sensitive to water contaminants. These biosentinels are grown in a DIY system with temperature control, stirring, and two optical detectors for fluorescence and density, allowing us to monitor growth and fluorescence of the yeast, and thus detect the presence of contaminants in the water sample. We hope our project will facilitate frequent water testing of local water sources.

Introduction

Origin of the project

Our project originated from the recent problems encountered in Switzerland regarding the presence of pesticides in groundwater. The Plateau, the most populated region in Switzerland, is the main area affected by this problem.

As we started looking more deeply into the problem, we figured out that the issue is much more widespread than we thought. During the past year, several cases of Chlorothalonil contamination, a recently banned substance in Switzerland, have been reported in the Canton of Vaud. This concerns especially small villages, that do not always have the means to test the quality of the water of their network. Indeed traditional testing methods are rather expensive and since small villages have smaller budgets compared to big cities, their water is only tested once or twice a year.

Goal

This is how Espress’EAU is born: we aimed at creating a low-cost and easy-to-use device that provide users with a fast and generic preliminary analysis of water samples. It would allow small communities as well as individuals to perform on-site testing without any precise expertise needed.

Synthetic Biology

At the core of our water testing device lie genetically modified yeast (S.Cerevisiae) strains that act as bio-sentinels. We studied the literature about the stress response of this organism and identified five target genes (HSP12, YCF1, TRX2, GLR1, GSH1) that are activated under the control of three transcriptional master regulators of stress response (MSN2, MSN4, YAP1). (Martínez-Pastor et al., 1996; Coleman et al., 1999)

When exposed to stress, the transcriptional master regulators enter the nucleus and activate the transcription of target genes. It has been shown that this activation differs qualitatively for different environmental stresses. (Hao & O'Shea, 2011)

Inspired by this mechanism we engineered two types of strains. Firstly, we tested three deletion strains where one of the three stress master regulators was knocked out, impairing the stress response and rendering them more sensitive to environmental stresses. When grown in contaminated water, we expected these strains to have a distinct growth rate when compared with wild type yeast cells. This would be detectable in our device. Secondly, we produced five reporter strains where we integrated one stress response target gene promoter upstream of a reporter gene. When grown in contaminated water, these strains should produce a fluorescent signal which our device is also able to detect.




References

  • Martínez-Pastor, M. T. et al. The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). EMBO J 15, 2227–2235 (1996).
  • Coleman, S. T., Epping, E. A., Steggerda, S. M. & Moye-Rowley, W. S. Yap1p Activates Gene Transcription in an Oxidant-Specific Fashion. Molecular and Cellular Biology 19, 8302–8313 (1999).
  • Hao N, O’Shea EK. Signal-dependent dynamics of transcription factor translocation controls gene expression. Nat Struct Mol Biol. 2011 Dec 18;19(1):31–9.
Deletion Strains
We grew the different deletion strains as well as a wild-type strain in presence of pesticides (solution diluted in methanol) and we analysed their growth behaviour.
  • In presence of Bentazon, all the yeast strains have their growth phases delayed and none of the strains reached the stationary phase at the end of the experiment
  • Deleting the master regulator genes affects the initiation of the cells growth (the knockout strains start approximately 15 hours later than wild type strains). Indeed, knockout strains are less resistant to environmental stresses than the wild type strains, indicating that they are more sensitive.


However, we wanted to know if that effect was due to pesticides or if methanol was having any side effect. We were not able to confirm that the effect we are observing on the growth of our deletion strains is entirely due to the pesticides or the methanol since we do not observe any effect on the growth of the knockout strains when we decreased the methanol concentration to 1%.

In order to asses the difference between pesticides and methanol, for each measure (4 replicates for each measure), a theoretical logistic function has been fitted in order to observe the the growth rate in the log phase of the growth curve. A multiple t-test corrected for the Benjamini-Hochberg procedure was performed to evaluate the significance of the difference of growth rates between the samples containing pesticides and the control sample (methanol solution). Two significant p-values (95% confidence interval) were found when comparing the different pesticides with the control solution of methanol: Diazinon and Metolachlor.




By Anas, Andrei and Laura

Reporter Strains

We have engineered five reporter strains by assembling a cassette that contains the promoter of a stress response gene upstream of a reporter gene (mScarlet-I) and a terminator. We integrated this cassette into the yeast genome using a method based on CRISPR/Cas9. (Shaw, 2019) When exposed to environmental stresses such as pesticides, we expect those yeast cells to activate the transcription of the reporter gene and produce a signal.

We confirmed through fluorescence microscopy that the reporter protein can be expressed and that for the strain carrying the Hsp12 promoter the signal is significantly higher if the cells are incubated with pesticides.






By Andrei, Sven and Yasmine

References

  • Shaw W. Quick and easy CRISPR engineering in Saccharomyces cerevisiae · Benchling [Internet]. [cited 2020 Oct 26]. Available from: https://benchling.com/pub/ellis-crispr-tools#parts-list
Capsule Design

In order to use the yeast strains that were engineered by our bio team, we needed to conceive a container that would allow to detect both optical density and fluorescent signal while mantaining a friendly environment for the yeast to grow and react to the pollutants in water. This gave birth to our capsule: a close environment that allows the rehydration of dry yeast and the monitoring of its growth.

Accessible & Safe

By opting for a ready-to-use type of capsule, we make the handling of modified organisms accessible to anybody. The capsule is made to guarantee the user safety by providing a leak-proof and sealed environment for yeast growth so no contact between the organism and the user is possible.


Low-cost

The capsule prototype we imagined is made from commercially available components for a total price of ~7$/capsule. It can be assembled in less than 5 minutes.


Sustainable

Despite the great benefit of a capsule system in terms of handling, it can rapidly become a source of waste. To limit waste production, we privileged durable and autoclavable materials such as glass so capsules can be re-used several times.


Viability

We demonstrated that our capsules are viable for the growth of freeze-dried yeast by OD600 measurements of commercial dry baking yeast.






By Ella and Laura

Components

3D printed components

Three 3D components were printed using PLA (polylactic acid) to integrate the hardware together:

  1. Vial and sensors (OD, Fluorescence) holder
  2. Magnetic stirrer and temperature control holder
  3. A box to cover the whole device so that sunlight cannot interfere with the readings

Magnetic stirrer

In order to have an homogeneous medium, a stirring mechanism is required. This is done using a DC motor with a variable resistance. A plate is balanced on top of the axle of a DC motor and 2 magnets are placed at 180° to create a magnetic force.


Temperature sensor

Also temperature needs to be controlled and be constant so that the yeast can grow. This is achieved using a low voltage, high precision TMP36 sensor and a thermoelectric peltier element for regulating the temperature around the 30°C mark.






By Harshdeep and Julien

Sensors

Optical density sensor

The growth of the yeast is influenced when in the presence of pesticides. This can be measured by the absorbance of light - which means the more yeast cells there are, the more light will be absorbed.
We used a light to frequency converter and white pin LED both at 180° to each other to measure the light that is absorbed.


Fluorescence sensor

In presence of pesticides, our genetically engineered yeast is expected to express a red fluorescent protein. This red fluorescence is obtained by exciting the mScarlet-i fluorescent protein with yellow light.
To measure the fluorescence phenomena, we use an RGB light sensor and a yellow LED, the light source and the sensor are at 90° of each other.
This is because we want to measure the maximum amount of red light emitted by the excitation of yeast and since the direction changes once it is emitted from the particle, it was more suitable to keep it at 90°.



These two sensors were then placed on the 3D printed piece in order to mantain them at their correct position.




By Harshdeep and Julien

Integrated Hardware

Successful integration

Separate 3D components were constructed for each of the electronic components which were further isolated using a 3D printed box. A C++ script was used to interface between the sensors and an Arduino UNO board was used as the microcontroller in order to collect and store the data on a host machine. This allowed us to connect all the different components both physically and electronically and obtain a working prototype. The complete code, circuit diagrams and 3D schematics are available on Github as the open source project.

Total price

With ~50$ for 3D components, ~40$ for electronic components and ~7$ per reusable capsule, we could propose the Espress’EAU prototype for less than 100$. It is worth mentioning that if it were to be produced, the 3D printing cost could drastically decrease.

Pick-up and delivery service

To implement Espress’EAU in the real world, we imagined a delivery and pick-up system set up by the manufacturers. The users would order batches of capsules containing freeze-dried yeast and medium powder. After use, the capsules are returned to the manufacturer who disposes the biowaste, autoclaves the capsule and prepares it for the next user, thereby creating a sustainable cycle and producing minimal waste.

Results and Future Plans

Synthetic Biology

  • We successfully generated reporter strains for three transcriptional master regulators of stress response in yeast. In the process of doing so we created five new basic parts that are compatible with the Dueber Yeast toolkit.
  • We screened three knock-out strains and wild type yeast against eight pesticides or their metabolites and found significant differences in the growth factor for some strains.
  • Our future work will include the screening of further conditions to answer the question of sensitivity. These experiments will also aim at identifying harmless chemical compounds found in drinking water that trigger our system to understand the potential of false-positive results.

Hardware

  • We designed and built a compact device composed of a 3D-printed case, electronic components, and a reusable capsule.
  • Our hardware is open-source, and can be built for less than 100$. The documentation is freely available on GitHub.
  • The components have been tested. We found a linear relationship between the OD600 measured on a calibrated spectrophotometer and our OD-sensor. The magnetic stirrer was capable of keeping the yeast cells in suspension overnight. The temperature regulation is capable of keeping the temperature around 30° Celsius for optimal growth conditions.

Implementation

  • The components of our device were combined and we showed that the system was stable enough to measure the growth curve of a wild type yeast overnight culture.
  • Our future plans include the testing of the sentinel strains on our device in an attempt to reproduce the effects found on the plate reader.
Integrated Human Practices

Human Practices have been an important aspect of our project, as we wanted to build a system that would suit people’s needs:

  • We interrogated the main people involved in water testing in the area of Lausanne to get more insight on the testing methods and get started with our project
  • The Association des Fontainiers de Suisse Romande (AFSR) helped us shape our project to adapt it to the usage by small communities as well as by the Fontainiers themselves
  • We launched a survey that gave us an overview of what people from around the world think about water quality and how they would envision a water testing device

As a final step of our project, we presented the first prototype of Espress’EAU to two of our stakeholders in order to obtain user feedback:

  • Quentin Morezzi from the AFSR suggested that our device was meeting the Fontainier’s needs in terms of pricing, size and handling ability
  • Endre Horvath, CEO of Swoxid, affirmed that Espress’EAU tackles a very common problem with very few solutions and thus it could serve for various usages






By Andrei, Ella and Julien

Sponsors & Acknowledgement

Acknowledgements

Our team was supervised by the Laboratory of Biological Network Characterization (LBNC) at EPFL and a member of a former EPFL iGEM team. We are grateful for their support throughout the year!

Prof. Sebastian Maerkl

Shiyu Cheng
Amir Shahein
Laura Grasemann
Konstantinos Ragios
Chun-Jie (Josh) Cai

We would like to thank everyone else who helped in one way or the other to realize this project!

Ming Yip
Martine Truan
Luc Patiny
Rachel Aronoff
Josiane Smith-Clerc
Quentin Morezzi
Prof. Urs von Gunten
Dr. Fereidoun Khajerhnouri
Julien Ducry
Endre Horvath
Bernhard Spörri

Sponsors

We would like to thank our sponsors that made the realization of Espress'EAU possible!