Our main goal has been to construct a biosensor to detect and quantify macrolide antibiotics meant for use in wastewater treatment plants. Engineering success has been demonstrated in the overall project by following the engineering design cycle. As an overview, we gathered information about environmental issues and wastewater treatment plant needs. We thoroughly discussed which kind of device could be beneficial. We constructed a basic part (see Building and Testing a Basic Part - ermC below) and the full biosensor (see Optical Device Construction below). Additionally, after observing unsatisfactory performance, we improved and adapted our design (see Optical Device Troubleshooting below).
Project Design - General Overview
We gathered information about the main environmental issues facing Europe. Our attention was caught by issues related to water pollution. We wanted the device developed during our project to be of use in real life, so it was crucial to maintain communication with wastewater treatment plants and make sure all their needs are met.
We were considering several ways to use synthetic biology to tackle problems currently being faced by wastewater treatment plants. Some of them included designing bacteria capable of degrading or recycling microplastics, as well as microbial systems detecting certain pollutants, such as pharmaceuticals or perfluoroalkyl chemicals, more efficiently.
In order to evaluate these ideas, we asked experts from several fields, including, among others, wastewater treatment plant employees, microplastics experts and environmental institutions. The key questions we had for the interviewees concerned feasibility and applicability of our device: we wanted to be able to develop a promising tool within the 3 months that we had access to the lab, and we wanted it to be useful for wastewater treatment plants.
Based on our research and experts' advice, we decided to design a biosensor to detect and quantify macrolide antibiotics.This seemed to be the most appropriate choice since (i) these compounds have been in the ‘watch list’ of pharmaceuticals for EU-wide monitoring in aquatic environments for several years , (ii) wastewater treatment plants confirmed that a device like ours would be useful to monitor the pharmaceutical removal process on-site as current detection methods are quite expensive  and (iii) clarithromycin is one of the marker compounds used for monitoring the removal efficiency in Switzerland, which is currently leading the research in this area .
MAIN GOALS - TESTING KEY ELEMENTS
We set three main goals for our project: (i) assure the cells used for the biosensor survived in wastewater, (ii) construct the optical device for macrolide detection and quantification, (iii) concentrate macrolides in the cells or in the sample if necessary, since macrolides are present in wastewater in very low amounts .
To achieve our first goal, we performed cell viability tests, where cells were grown with various media, including wastewater. More detailed description can be found here. These experiments were initially conducted using a plate-reader and later additionally confirmed with a flow cytometer. The results confirmed that cells can survive in wastewater.
Optical Device Construction
While constructing the biosensor, our main concern was the low concentration of macrolides in wastewater . Therefore, we decided to model the expected range of the biosensor. The results indicated that the biosensor may need to be 10-100 times more sensitive to detect the low concentrations of macrolides found in wastewaters (Fig. 1). To solve this issue, we decided to use Rosetta to predict mutations in the binding site of MphR that would increase the binding affinity of MphR to macrolide antibiotics. More about mathematical and protein modelling can be found here. In addition, to further increase the detection of macrolide antibiotics, we decided on studying ways of concentrating them inside our biosensor cells.
Our genetic circuit (Fig. 2) was constructed, so that when there are no macrolides present in the sample, MphR, a repressor protein , binds to pMphR (naturally a promoter of macrolide resistance genes)  preventing the production of enhanced green fluorescent protein [eGFP]. In case there are macrolides, these compounds bind to MphR, preventing the protein from DNA binding. This allows egfp to be expressed. Although for the proof-of-concept studies we are using a fluorescent output, the final product used in wastewater treatment plants may be an electrochemical biosensor, since in many cases there are thought to be more sensitive  and the output is easier to be interpreted (see our Implementation page). In our lab experiments we also included ermC, which is regulated by the same promoter as mphR. This gene ensures the biosensor survives even in high concentrations of macrolides . The constitutive promoter we selected is a medium strength promoter (BBa_J23106) to avoid production of too many repressor proteins and egfp being silenced regardless of macrolide concentrations. All proteins expressed in our genetic circuit contain the same ribosomal binding site, with medium strength 0,764 (BBa_B0029) to decrease the effects of metabolic burden.
In order to build a biosensor, we decided to use Modular Cloning protocol , since it is modular, it allows for addition of more parts than RFC10 at the same time and is compatible with iGEM standards. Our final optical device has been divided into two level 1 assemblies: called a repressor cassette and output cassette, so it is easier to spot which part has malfunctioned in case we face any issues (Fig. 3).
We conducted a plate-reader experiment, where we grew cells transformed with the optical device in various erythromycin concentrations for 20 h. Non-transformed E. coli cells grown in LB with 100 µl/ml erythromycin were a positive control. A negative control comprised non-transformed cells grown in LB with no antibiotic. More details about the experiment, as well as the protocol can be found on our Experiments page. Although there seems to be a correlation between erythromycin correlation and the fluorescent output, the differences are too insignificant for the biosensor to be practical (Fig. 4). It was apparent our current design requires an improvement. See the Optical Design Troubleshooting section below.
Building and Testing a Basic Part - ermC
Since our biosensor aims to detect macrolide antibiotics, it is fundamental the cells survive in their presence. However, many products of antibiotic resistance genes act by pumping the compound out from the cell or by modifying its chemical structure. This would greatly affect the detection and reliability of the results. That is why we decided ermC is a good candidate, since it encodes a protein that methylates selected residues of 23S rRNA, thus making the cells immune to the effect of macrolides and licosamines .
For our proof of concept studies, we placed ermC under a constitutive promoter (BBa_J23106), so we can test the biosensor in a wide range of macrolide concentrations. In our genetic circuit it would be expressed together with mphr (Fig. 2). However, in our final biosensor may not be necessary, since the concentrations of macrolide antibiotics in wastewater are rather low . This would make the biosensor safer to use, since there would be no risk of releasing an antibiotic resistance gene into the environment.
We have constructed our repressor cassette using Modular Cloning protocol (see our Experiments page). We sequenced the assembly to make sure it is correct before assembling the full optical device.
We have grown cells transformed with repressor cassette (Fig. 5) in 1, 10, 50 and 100 µg/ml of erythromycin, clarithromycin and, as a control, spectinomycin. The results showed that ermC is expressed and provides resistance to macrolide antibiotics. The part can be found in iGEM’s registry. More about the test can be read on our Experiments page.
OPTICAL DEVICE TROUBLESHOOTING
We have sequenced the optical device to see whether, despite seeing a correct length band, there has been a mistake that results with little egfp being expressed. The results show no differences between what we assembled and what we expected.
Our initial hypothesis was that the codon optimization of natural egfp sequences may have affected the secondary structure of the mRNA and decrease or block the translation of the protein. We examined the probability of this happening with an online tool (https://rna.urmc.rochester.edu/RNAstructure.html)  and decided this issue is unlikely to be a source of the issue. Another problem we considered was a suboptimal strength of the promoter: MphR may be overexpressed, thus always being bound to pMphR with erythromycin having little effect, causing such low EGFP production.
To investigate what is the issue, we performed several tests. First, we constructed a new level 1 output cassette, but with egfp replaced by sfgfp. Then we performed a plate reader experiment, where we compared the intensity of their fluorescent signal. For more details see our Experiments page. sfGFP turned out to produce much stronger fluorescence. This may potentially solve our problem and decrease the time needed for the measurement.
To see the effect of MphR on egfp expression, we performed a plate-reader experiment, where we measured the fluorescence of level 1 output cassette, which does not express MphR gene so there is no repressor present. We also measured fluorescence of the optical device in various erythromycin concentrations for comparison. For more details see our Experiments page. We did observe that MphR represses the egfp, but the difference between the output of cells expressing and not expressing the repressor is not significant (Fig. 7). Worth mentioning, we were already expecting such results since in our mathematical model it can be seen that the increase in macrolide concentrations result only in slight changes of the fluorescence (Fig. 8). Nonetheless, we wanted to test our system in the laboratory to confirm the model and the experiment were in accordance.
IMAGINE & IMPROVE
For the future, we have five potential ways to improve the range and sensitivity of our biosensor:
- Constructing a new optical device, where the constitutive promoter was replaced by an inducible pBAD promoter (BBa_I0500) . This would allow us to test various induction strengths and select the most optimal one.
- Assembling two more optical devices, both containing sfgfp instead of egfp. One of them comprised MphR under the original constitutive promoter, the other had an inducible pBAD promoter. This would show whether using a protein that produces a stronger signal will help to differentiate between output of various macrolide concentrations.
- Constructing alternative inducible optical devices with MphR mutants with changes to the ligand binding sites. We expect these changes to improve the binding affinity of MphR to erythromycin (to know more, see our Modelling page).
- Concentrating macrolides inside the biosensor cells by using a modified strain with removed element of several multidrug resistance pumps and altered FhuA pore with a constantly open conformation .
- Concentrating macrolides in the sample using isoelectric focusing on a gel  as a proof of concept for concentrating using paper-based microfluidics.
1. Agency for Healthcare Research and Quality. (2018). 2017 national healthcare quality and disparities report (Report No. 18-0033-EF). U.S. Department of Health and Human Services. https:// www.ahrq.gov/ research/ findings/ nhqrdr/ nhqdr17/ index.html
2. 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
3. Verordnung des UVEK zur Überprüfung des Reinigungseffekts von Massnahmen zur Elimination von organischen Spurenstoffen bei Abwasserreinigungsanlagen, 814.201.231 § 2 (2016). https://www.admin.ch/opc/de/classified-compilation/20160123/index.html#a2
4. Kanoh, S., & Rubin, B. K. (2010). Mechanisms of action and clinical application of macrolides as immunomodulatory medications. Clinical Microbiology Reviews, 23(3), 590-615. doi:10.1128/cmr.00078-09
5. 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. https://doi.org/10.1021/acssynbio.7b00287
6. 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
7. Gardner, L., Zou, Y., Mara, A., Cropp, T., & Deiters, A. (2011). Photochemical control of bacterial signal processing using a light-activated erythromycin. Molecular Biosystems, 7(9), 2554. doi: 10.1039/c1mb05166k
8. Yi, H., Li, M., Huo, X., Zeng, G., Lai, C., & Huang, D. et al. (2019). Recent development of advanced biotechnology for wastewater treatment. Critical Reviews In Biotechnology, 40(1), 99-118. doi: 10.1080/07388551.2019.1682964
9. UniProt: a worldwide hub of protein knowledge. (2018). Nucleic Acids Research, 47(D1), D506-D515. doi: 10.1093/nar/gky1049
10. Haddock, Traci & Densmore, Douglas & Appleton, Evan & Carr, Swati & Iverson, Sonya & Freitas, Monique & Jin, S. & Awtry, Jake & Desai, Devina & Lozanoski, Thomas & Shah, Pooja & Agarwal, Yash & Lewis, Kathleen & Pacheco, Alan. (2015). BBF RFC 94: Type IIS Assembly for Bacterial Transcriptional Units: A Standardized Assembly Method for Building Bacterial Transcriptional Units Using the Type IIS Restriction Enzymes BsaI and BbsI.
11. Reuter, J. S., & Watson, R. M. (2017). Welcome to the Predict a Secondary Structure Web Server. Predict a Secondary Structure Web Server. https://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predict1/Predict1.html
12. Lee, N., Gielow, W., & Wallace, R. (1981). Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the L-arabinose regulatory region of Escherichia coli. Proceedings Of The National Academy Of Sciences, 78(2), 752-756. doi: 10.1073/pnas.78.2.752
13. 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, AAC.01882-16. doi: 10.1128/aac.01882-16
14. Zhao, C., Ge, Z., & Yang, C. (2017). Microfluidic Techniques for Analytes Concentration. Micromachines, 8(1), 28. https://doi.org/10.3390/mi8010028
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