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.
Presented by Aalto-Helsinki 2020
Pharmaceuticals pose multiple issues in the environment: persistence, possible biological activity, and synergistic toxicity . 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).
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 . Therefore, SINISENS has potential use in wastewater treatment plants to monitor the removal performance of micropollutants.
“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 .
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.
Conclusion: E. coli cells are viable in wastewater.
MphR is a transcription factor highly specific for macrolide antibiotics . 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.
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).
Conclusion: We can correlate macrolide concentration with fluorescence intensity.
“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).
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 . 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).
Conclusion: We increased the sensitivity of our biosensor with hyperporinated GKCW104 cells.
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  (Fig. 7).
Conclusion: We would need to improve our biosensor’s sensitivity so it could be used for WWTPs.
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.
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.
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 . 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).
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.
Biggest thank you to our sponsors
And a special thank you to Helsinki Region Environmental Services Authority HSY:
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