Team:Stockholm/Poster
Poster: Team Stockholm
S-POP: A modular biosensor for the detection of POPs in water
After centuries spent using oceans for waste management, we have finally realized the impact of water pollution. Persistent organic pollutants (POPs), including PFOS (perfluorooctanesulfonic acid) and PCBs (polychlorinated biphenyls), have been of great concern, due to their toxicity in low concentrations and bioaccumulating properties that lead to alarming concentrations in the ecosystem. Current detection methods, which are performed by analyzing massive amounts of water samples in the lab, cannot properly measure the low levels of POPs nor differentiate them. Our project, Sensing-Persistent Organic Pollutants (S-POP), aims to solve this issue by creating a monitor containing two major parts, a modular E. coli biosensor coupled with a microbial fuel cell (MFC). When E. coli is activated by the pollutant a quorum sensing (QS) molecule is produced. Upregulated by the QS-molecule, engineered Shewanella oneidensis produces an oscillating electrical signal that corresponds to the type and quantity of pollutants in the sample.
Introduction
Water is essential for life on our planet. Without clean water sources, the entire ecosystem suffers and there is no valuable solution in sight yet. To reach the Sustainable Development Goals (SDGs), the methods for monitoring aquatic pollutant concentrations need to be cheaper, more accessible and more accurate (1).The Problem
Persistent organic pollutants (POPs) like PCBs and PFOS are a group of problematic pollutants that are not readily degraded in the environment and bioaccumulate in aquatic organisms (2, 3). Exposure to POPs can lead to reproductive disorders, neurobehavioral impairment, increased risk of cancer, genotoxicity and increased birth defects (4). The Baltic sea, which is landlocked and less resilient than other ecosystems due to lower biodiversity, is in great danger because of POP pollution (5, 6, 7).Our Solution
Shewanella oneidensis produces an electrical output in anaerobic conditions through an electricity-producing gene which, when knocked out, can be reintroduced and controlled by an engineered plasmid. Induction of this gene using quorum sensing molecules produced by an engineered E. coli can couple electricity production to pollutant detection. For better detection, the signal-to-noise ratio can be improved by using an oscillatory circuit to control the current. The fully developed S-POP system can be used to continuously monitor pollutant concentrations and improve efforts for pollution prevention.References
(1) Practices of Science: Scientific Error. Exploring Our Fluid Earth. (n.d.). Retrieved on October 26, 2020. from https://manoa.hawaii.edu/exploringourfluidearth/physical/world-ocean/map-distortion/practices-science-scientific-error
(2) Jones, K. C., & de Voogt, P. (1999). Persistent organic pollutants (POPs): state of the science.Environmental Pollution 100(1-3), 209-221.
(3) Schmidt, C. W. (1999). No POPs. Environmental Health Perspectives 107(1), A24.
(4) Persistent organic pollutants (POPs). World Health Organization. (n.d.). Retrieved on October 26, 2020. from https://www.who.int/foodsafety/areas_work/chemical-risks/pops/en/
(5) Wiberg, K., Assefa, A. T., Sundqvist, K. L., Cousins, I. T., Johansson, J., McLachlan, M. S., . . . Cato, I. (2013). Managing the dioxin problem in the Baltic region with focus on sources to air and fish. Naturvårdsverket.
(6) Bergknut, M., Laudon, H., Jansson, S., Larsson, A., Gocht, T., & Wiberg, K. (2011). Atmospheric deposition, retention, and stream export of dioxins and PCBs in a pristine boreal catchment. Environmental Pollution 159(6), 1592-1598.
(7) Magnusson, K., & Norén, K. (October, 2012). The sensitivity of the Baltic Sea ecosystems to hazardous compounds. Swedish Chemicals Agency - www.kemi.se. Retrieved on October 25, 2020. from https://www.kemi.se/download/18.6df1d3df171c243fb23960c4/1591097407345/pm-9-12-baltsens.pdf
Engineering
Figure 1: Division of engineering tasks
We designed S-POP as a modular system made of an E. coli detection module and a Shewanella oneidensis oscillatory electrical output module, linked to each other by quorum sensing signalling molecules. We created genetic circuits using SnapGene and optimised them until simulations confirmed we could get a unique oscillatory electrical output upon detection of a specific pollutant. Genetic circuits for detection were characterised using fluorescence measurements and Western blotting, while electricity production was measured by building a microbial fuel cell. The division of tasks is shown in Figure 1 above. For each experiment and simulation, we identified problems and solved them methodically so that scientifically correct results could be produced (Figure 2). This included the use of negative and positive controls as well as repetitions of measurements. A troubleshooting guide to Western Blotting was also created to help future teams.
Figure 2: Engineering cycle for our project
Model
Figure 3: Animated descriptive diagram of the oscillatory system
A system of delayed differential equations was used to predict whether our genetic circuit could create pollutant-specific electrical oscillations. Oscillations are established in a Shewanella oneidensis mtrB knockout strain thanks to the co-existence of positive and negative feedback loops. These loops are controlled by the production and degradation of quorum sensing molecules emitted by E. coli upon binding of pollutants. Our first system contained the feedback loops and electricity reporter parts under the same promoter. This led to time-dependent oscillation phases, which could be a problem since cells are spatially distributed and QS-signalling is diffusion dependent. To avoid destructive interferences, the genetic circuit was adjusted so that oscillations without time-dependent phases could be obtained. Higher levels of mtrB expression (i.e. higher current) were observed in silico for induced cells than non-induced cells.
Figure 4: Simulation of oscillatory circuit
MFC
The wildtype strain of S. oneidensis MR-1 (WT) and the mtrB knockout (KO) strain were cultivated in the microbial fuel cell (MFC) we built and electricity production was monitored for 450 h.
Figure 5: Measurement of current over time in red, with the different phases in cyan
Initial addition of Lysogeny Broth (LB) growth medium resulted in a steady current increase to 20 μA over 260 h, suggesting that either a biofilm generated on the electrode or cells in suspension produced electricity. Addition of fresh LB resulted in a rapid current increase to 130 µA followed by a plateau. This confirmed successful biofilm growth and stabilization due to biofouling of the electrode and/or nutrient limitation in the chamber. Change to M9 medium with lactic acid as the only carbon source resulted in, after an initial current drop, a steady increase upon addition of more lactic acid to the media, suggesting growth limitation by nutrient availability. The KO strain was grown in similar conditions and no electricity generation was observed, as hypothesized. The introduction of a circuit containing mtrB can be expected to re-establish controlled electricity production in the MFC.
Characterization
Successful gene insertion was confirmed through sequencing after cloning. Western Blots, fluorescence measurements, and Chromobacterium reporter strain assays were used for functionality analysis and protein expression determination.
Fluorescence
The QS-construct, pConst-LuxR-pLux-GFP (Q) showed an increase in fluorescence intensity upon induction with the AHL synthase-producing construct and synthetic AHL (3O6C-HSL) respectively, in comparison with non-induced conditions. Similar results were observed for the homologue QS construct, pConst-RhlR-pRhl-GFP (O14, O19) which displayed increased fluorescence intensity upon induction with the AHL synthase-producing construct and synthetic AHL (C4-HSL). No significant difference was observed for the pollutant sensing promoters prmA (B7, B8) and bphR1 (C) in induced and non-induced conditions with associated pollutants PFOS and 1,1-biphenyl respectively.Figure 6: Net fluorescence intensity of constructs expressing (a) mCherry and (b) GFP. The values shown represent averages of 3 technical replicates, and have been adjusted for cell density. Error bars represent standard deviation
Figure 7: GFP visualisation of constructs O14, O19 and Q under UV light
Western Blot
In accordance with fluorescence results, positive bands for LuxI (A), RhlI (G) and LuxR (Q) were observed on the WB. No positive bands were noted for remaining constructs in non-induced as well as induced conditions.Figure 8: WB results of samples with positive bands for positive control, LuxI (A), RhlI (G) and LuxR (Q) in that order. M + biphenyl, F + PFOS and P3.2 + biphenyl were induced by pollutants
Chromobacterium Assay
Chromobacterium violaceum CV026, an AHL reporter strain, was confirmed to produce violet pigment upon induction of 10 mM AHL. However, no color change was detected upon co-culturing of C. violaceum with AHL producing constructs (LuxI and RhlI), perhaps due to the concentrations of AHL produced being below C. violaceum sensitivity levels.Viability Assay
Viability assays showed no notable impact in the survival and growth of our pollutant-sensing host organisms as well as Top 10 E. coli BL21 up to pollutant threshold values of 10-3 M of 1,1-biphenyl and 125 µM PFOS.
Conclusion
The results from the MFC experiment suggest that the WT S. oneidensis strain could form a biofilm on the MFC anode rendering electricity production as opposed to the mtrB knock-out strain. Hence, reintroduction of mtrB containing circuits under the control of an inducible promoter would presumably generate electricity in a controlled manner upon promoter induction. Successful functionality of the QS signaling constructs was observed through fluorescence results and protein expression of the QS proteins; LuxI, RhlI and LuxR was observed through positive WB bands, suggesting viable QS transmission between the cellular modules. The viability assay suggests survival and growth of our host cells at pollutant concentrations well above levels found in nature and industrial environments. The pollutant sensing genes did not operate as hypothesized, and require further optimization.
Implementation
Figure 9: Schematic of the two potential implementation locations of S-POP microbial fuel cells into the sewerage system
S-POP has been designed as a modular biosensor to address the need for a rapid, reliable and economical system to detect and distinguish between the aquatic pollutants threatening our water bodies. The modularity of S-POP supports flexible implementation options. Future modifications could allow detection of various additional aquatic pollutants, whilst implementation of parallel systems enables multiple pollutant monitoring within the same aquatic environment.Bioethical safety measures are key in implementation, with focus on bacterial strain containment through filtration and sterilization as well as system maintenance and quality control through regular check-ups by experienced technicians. Quick source identification and continuous monitoring are crucial in ensuring early action for pollutant control and S-POP offers an on-the-spot, almost real-time detection system for this need. When implemented, S-POP could be a valuable tool for environmental agencies and industries to monitor pollutant levels both downstream and upstream of industries, water treatment plants, and households.
Contribution
S-POP offers a modular biosensor platform for future iGEM teams to adapt and modify for the detection and differentiation of multiple aquatic pollutants, with a standardized signalling output in the form of electricity production. The Shewanella oneidensis module was designed to be adequate for all types of pollutants, while the E. coli detection module can easily be adapted to other pollutants with appropriate promoters.
Additional characterization of BioBricks such as pollutant-sensing promoters prmA and bphR1 was conducted, including improvement through addition of fluorescence markers and Myc-tags for WB and fluorescence characterization. 
A combination of literature research, valuable feedback from experts in the field and numerous experimental trials and optimizations resulted in a Western Blot guide that will hopefully prove helpful to future iGEM teams and researchers when troubleshooting WBs.
We created a YouTube series aiming to make science accessible and fun through a series of DIY-experiments. The goal was to provide a creative platform for future iGEM teams to build upon and to interest audiences in science and synthetic biology.
Attributions
We would like to thank all the people that helped us during the journey either by providing valuable advice or by providing material.
Supervisors
Prof. Johan Rockberg. Primary iGEM PI and the head of a research group at the Division of Protein Technology at KTH Royal Institute of Technology. Prof. Ute Römling. Secondary iGEM PI and the head of a research group at the Department of Microbiology, Tumor and Cell Biology at Karolinska Institutet.Instructors
Maximilian Karlander. Team member of iGEM Stockholm 2015 and Ph.D. student at the Department of Protein Technology at KTH Royal Institute of Technology. Aman Mebrahtu. Team member of iGEM Stockholm 2016 and a Ph.D. student at the Department of Protein Technology at KTH Royal Institute of TechnologyAdvisors
Emma Vincent, researcher at the Institute of Environmental Medicine, Karolinska Institutet. Amirreza Khataee, postdoc researcher at the Division of Applied Electrochemistry at KTH Royal Institute of Technology. Nerea Capon Lamelas, team member of iGEM Stockholm 2019 and a Field Application Specialist at Promega Corporation. Rasmus Bengtsson, team member of iGEM Stockholm 2017 and a Graphic Designer and Marketing Communicator at TCO Development.Lab Support
Johannes Gescher, Melanie Knoll, Jonatan Martin Rodriguez, Martin Gustafsson, Rakel Wreland Lindström, Steffen Georg, João Pereira, Sudhanshu Pawar, Gunaratna Kuttuva Rajarao, and research staff on floor 3, Department of Protein Science, Albanova, KTH Royal Institute of Technology.iGEM Teams
iGEM Aalto-Helsinki, iGEM Uppsala, iGEM Bielefeld, iGEM BITS Goa, and iGEM UCopenhagen.Human Practices Support
Kristina Stark Fujii, Sara Villner, Åsa Andersson, Karin Norström, Sándor Bereczky, Vilma Lagebro, Stella Axelsson, Josephine Berg, and Iida Tynkkynen.Financial Advisors
Stamatina Rentouli and Puck Norell.Acknowledgments
SGEM (Stockholm Genetically Engineered Machine) and Ester Lisnati Jayadi.Sponsors
KTH Royal Institute of Technology, KTH Opportunities Fund, KI Innovations AB, PN Biomedicine Grant (Medicinska Föreningen union, KI), Benchling, Integrated DNA Technologies, New England Biolabs, Red Bull, and SnapGene.