SINISENS - A step towards cleaner waters

Presented by Aalto-Helsinki 2020

Tytti Jämsä*, Carla Coll Costa**, Daria Pająk**, Emilia Barannik*, Gustav Åberg*, Natalia Lindholm*, Amanda Sandelin**, Artur Gynter*, Julia Manninen**, Maria Rajakenttä**

*Aalto University iGEM team member
**University of Helsinki iGEM team member

The presence of macrolide antibiotics in nature is a growing concern as they have been on the 'watch-list' of pharmaceuticals for EU-wide monitoring in aquatic environments for several years. They can be harmful for the environment and human health because they are persistent and can remain biologically active. Additionally, they may promote the development of antimicrobial resistance. According to various experts, there will likely be regulations regarding the monitoring of macrolide antibiotics in the near future. However, current methods for measuring them are time-consuming, expensive and require expertise. Our solution, SINISENS, is designed to aid wastewater treatment plants to monitor the concentrations of macrolide antibiotics and could be used to optimize the removal process. SINISENS is an optical on-site biosensor based on a genetic circuit that utilises a transcription factor called MphR to detect macrolide antibiotics. In the presence of these compounds, SINISENS produces green fluorescence as an output signal.

Problem and Solution

Pharmaceuticals pose multiple issues in the environment: persistence, possible biological activity, and synergistic toxicity [1][2]. Moreover, antibiotics can lead to the development of antibiotic resistance in environmental bacteria. It is expected that laws regarding pharmaceutical removal will likely be implemented in the future for wastewater treatment plants (WWTPs).

“The additional purification step, ozonation, is highly energy consuming and the production of activated carbon has high environmental footprint. Implementation of a biosensor before and after the purification step could be used for the optimization of the process.” -Paula Lindell, Group Manager, Viikinmäki wastewater treatment plant, HSY

To optimize the energy-consuming removal of pharmaceuticals, a quick, easy-to-use and affordable sensor would be needed. There is no such device in the market, which is why we created SINISENS, a biosensor to detect and quantify macrolide antibiotics. Our sensor would be used on-site in WWTPs before and after the micropollutant removal step to help optimize it (Fig 1).

Figure 1. Wastewater treatment plant process flow including implementation of our biosensor.

In Swiss WWTPs, micropollutant removal is measured on the basis of selected indicator substances. Clarithromycin, belonging to the group of macrolide antibiotics, is used as one of these indicator substances [3]. Therefore, SINISENS has potential use in wastewater treatment plants to monitor the removal performance of micropollutants.

Project Goals

“Pharmaceuticals in wastewater currently pose a challenge due to their potential risks already in low concentrations, unclear regulations regarding their monitoring and removal requirements, and lack of cost-effective detection methods.” -Anna Mikola, Professor of Practice in Department of Built Environment, Aalto University

To assure our biosensor would be useful for optimization of micropollutant removal in wastewater treatment plants (WWTPs), we set three goals:

1. VIABILITY: Our biosensor, engineered from E. coli, should survive in wastewater.

2. DETECTION: To build a genetic circuit that is able to detect and quantify macrolides.

3. SENSITIVITY: The detection limit of our biosensor should be 100 ng/L to meet the needs of WWTPs [4].

1. Viability

To assess viability of E. coli in wastewater, we transformed a plasmid which produces sfGFP under IPTG induction into E. coli. We grew the cells in Luria-Bretani broth (LB), wastewater (WW), milli-Q and mixture of WW and LB (Fig. 2). Increase in fluorescence indicates viability of the cells as they are able to produce GFP. We also validated this data with flow cytometry, where we used viability dye for cells that were grown in the same set-ups for three hours.

Figure 2. E. coli cells producing fluorescence in different experimental set-ups containing Luria-Bretani broth (LB). The graph includes data from three repeats (each comprising 3 technical repeats).

Conclusion: E. coli cells are viable in wastewater.

2. Detection

Genetic Circuit
MphR is a transcription factor highly specific for macrolide antibiotics [5]. In our circuit, mphr is transcribed under pBAD promoter (Fig. 3). This genetic circuit was an outcome of fine-tuning with the help of modeling and experiments. MphR will bind to pMphR to repress production of sfGFP when no macrolides are present. When macrolides are present, they will bind to MphR and release it from the promoter, resulting in production of sfGFP. The circuit was constructed into a plasmid and is called an inducible optical device from here on.

Figure 3. Genetic circuit for detection of macrolides. In absence of macrolides, MphR repressor protein binds pMphR preventing expression of sfgfp. When two macrolide molecules bind to MphR, it releases the DNA, allowing transcription.

Results from Detection
E. coli transformed with our optical device plasmid were grown in different erythromycin concentrations (0, 1, 10 and 100 mg/L) and 0.1 % arabinose. Fluorescence was measured for 15 hours. We obtained higher fluorescence when erythromycin concentration increases (Fig. 4).

Figure 4. Optical Density (OD) normalized fluorescence produced by E. coli transformed with an inducible optical device grown in different erythromycin concentrations and 0.1% arabinose. The graph includes data from two repeats (each comprising 3 technical repeats).

Conclusion: We can correlate macrolide concentration with fluorescence intensity.

3. Sensitivity

“Since the macrolide antibiotics are typically present in very small concentrations in wastewater, it will be important to ensure that the MphR regulatory system can be used to detect them.” -Ville Paavilainen, University Researcher in Institute of Biotechnology, University of Helsinki

We predicted modifications to the active site of MphR to increase its binding affinity to erythromycin and clarithromycin in Rosetta. After testing 5 best outputs experimentally, one of them gave promising results (Fig. 5). The fluorescence signals are more easily distinguishable from each other compared to the genetic circuit with wildtype MphR (Fig. 4).

Figure 5. Optical density (OD) normalized fluorescence produced by E. coli transformed with an inducible optical device with modified MphR grown in different erythromycin concentrations and 0.1% arabinose. The graph includes data from two repeats (each comprising 3 technical repeats).

Conclusion: We increased the sensitivity of our biosensor with Rosetta modelling by mutating transcription factor MphR.

Hyperporinated Outer Membrane
We obtained an E. coli strain, GKCW104, from Krishnamoorthy and colleagues from Oklahoma university, which has a hyperporinated outer membrane produced upon arabinose induction [6]. This was achieved by modifying outer membrane protein FhuA. We transformed our genetic circuit, this time mphr expressed under constitutive promoter, to the GKCW104 strain and were able to differentiate the fluorescence outputs already after 2 hours of growth (Fig. 6).

Figure 6. Optical density (OD) normalized fluorescence produced by GKCW104 cells transformed with a constitutive optical device grown in different erythromycin concentrations and 0.1% arabinose. The graph includes data from two repeats (each comprising 3 technical repeats).

Conclusion: We increased the sensitivity of our biosensor with hyperporinated GKCW104 cells.


Assessing Sensitivity
In order to get insights on how useful our biosensor would be for WWTPs we used MatLab to plot the percentage of macrolide bound to MphR depending on the macrolide concentration. This is, we plot the Hill Equation for a wildtype MphR and a MphR with a modified RBS [7] (Fig. 7).

Figure 7. Hill Equation of a wildtype MphR and a MphR with a modified RBS. In the left-part of the plot we can see the concentrations of macrolides in WWTPs.

Conclusion: We would need to improve our biosensor’s sensitivity so it could be used for WWTPs.

Improving Sensitivity
In order to improve the sensitivity of our biosensor we used Rosetta to predict mutations in nine amino acids in the ligand binding site of MphR that would increase the binding affinity of this transcription factor (TF) for erythromycin and clarithromycin, the two most common macrolide antibiotics (Fig. 8). We chose the five best outputs from the modelling to test them in the lab.

Figure 8. Amino acids of MphR in which we predicted the modifications to increase the binding affinity to erythromycin and clarithromycin.

Fix the Flow
Most people are not aware how the wastewater treatment process actually looks, which can lead to many issues, such as pharmaceuticals being disposed improperly. We thought it might be helpful to transform this rather unattractive topic into something more exciting and approachable.

As our main educational effort, we developed a mobile game targeted for younger teens to familiarize them with synthetic biology, wastewater treatment and antibiotic resistance. The aim of the game is to build a wastewater treatment plant (Fig. 9). The game had two test rounds with the general public and is translated to 13 different languages. The game can be played directly on our wiki page.

Figure 9. Fix the Flow is a mobile game where you can clean the wastewater flow with the help of bacteria and different machines. Bacteria can be upgraded with the help of plasmids.

Antibiotic Resistance Campaign

“Residues of antibiotics are one of the important classes of harmful compounds and the development of antibiotic resistance is a great global concern.” -Jari Männynsalo, Environmental Specialist, The Water Protection Association of the River Vantaa and Helsinki Region

One of the causes of antibiotic resistance is the improper use of antibiotics [8]. To tackle this, we created a campaign to raise awareness on how to prevent antibiotic resistance spreading. We created a website, flyers and an informative video which we spread in social media and at a local pharmacy (Fig. 10).

Figure 10. Flyer from our antibiotic resistance campaign.

Our biosensor has potential for being used in wastewater treatment plants (WWTPs) to optimize micropollutant removal. However, to achieve this, we would need to address three key points:

Creating a multiple use biosensor, which would be more affordable for WWTPs.

Developing a biosensor with an electrochemical output instead of an optical one, since electrochemical biosensors can be more sensitive.

Further increasing the sensitivity of our biosensor to meet the low detection limit of 100 ng/L of macrolides in WWTPs.

“If regulations come that require the removal of pharmaceuticals, a device will be needed to measure whether this removal process succeeds. In that sense, your innovation would be useful.”* -Ari Kangas, Ministerial Adviser, Ministry of the Environment, Finland

*Note: The following is the opinion of an official individual and does not represent the Ministry of the Environment's general position.

References and Sponsors

Biggest thank you to our sponsors

And a special thank you to Helsinki Region Environmental Services Authority HSY:

1. "Science for Environment Policy": European Commission DG Environment News Alert Service, edited by SCU, The University of the West of England, Bristol.
2. Arnold, K. E., Boxall, A. B. A., Brown, A. R., Cuthbert, R. J., Gaw, S., Hutchinson, T. H., et al. (2013). Assessing the exposure risk and impacts of pharmaceuticals in the environment on individuals and ecosystems. Biology Letters (2005), 9(4), 20130492. doi:10.1098/rsbl.2013.0492
3. Verordnung des UVEK zur Überprüfung des Reinigungseffekts von Massnahmen zur Elimination von organischen Spurenstoffen bei Abwasserreinigungsanlagen, 814.201.231 § 2 (2016).
4. Schafhauser, B. H., Kristofco, L. A., de Oliveira, Cíntia Mara Ribas, & Brooks, B. W. (2018). Global review and analysis of erythromycin in the environment: Occurrence, bioaccumulation and antibiotic resistance hazards. Environmental Pollution, 238, 440-451. doi:10.1016/j.envpol.2018.03.052
5. Zheng, J., Sagar, V., Smolinsky, A., Bourke, C., LaRonde-LeBlanc, N., & Cropp, T. A. (2009). Structure and function of the macrolide biosensor protein, MphR(A), with and without erythromycin. Journal of Molecular Biology, 387(5), 1250-1260. doi:10.1016/j.jmb.2009.02.058
6. Krishnamoorthy, G., Wolloscheck, D., Weeks, J., Croft, C., Rybenkov, V., & Zgurskaya, H. (2016). Breaking the permeability barrier of Escherichia coli by controlled hyperporination of the outer membrane. Antimicrobial Agents And Chemotherapy, 60(12), 7372-7381. doi: 10.1128/aac.01882-16
7. Kasey, C. M., Zerrad, M., Li, Y., Cropp, T. A., & Williams, G. J. (2018). Development of Transcription Factor-Based Designer Macrolide Biosensors for Metabolic Engineering and Synthetic Biology. ACS Synthetic Biology, 7(1), 227–239.
8. "Tackling Drug-Resistant Infections Globally: Final Report And Recommendations". 2016. Amr-Review.Org.