The development of a system that contributes to the detoxification of pharmaceutical waste requires a well thought out plan. Follow along to learn more about our project all the way from the initial project ideation to future project extension possibilities!
How did we choose our topic?
Our team is located in the coastal city of Turku, right by the Baltic Sea. The Baltic Sea currently suffers from many types of polluting agents one of which is pharmaceutical waste. We wanted to tackle this problem by producing knowledge on the detoxification of these compounds especially targeting diclofenac (2-[2-(2,6-dichloroanilino)phenyl]acetic acid), a non-steroidal anti-inflammatory drug that is commonly found in pain relieving gels. You can read more about this on our project description and pharmaceutical waste page.
How can diclofenac and other pharmaceuticals be detoxified?
Once we had the problem in mind, we set out to search for biological processes that we could harness for our biotechnological application with the tools of synthetic biology. These turned out to be enzymes called laccases. Laccases are multi-copper oxidases (MCOs) that can catalyze reactions of many substrates, for example organic compounds, dyes and pharmaceuticals. They can be found in fungi, bacteria, insects and plants and they are involved in many different functions, for example in lignin degradation, pigmentation, pathogenesis of fungi and wound healing in plants. Due to their low substrate specificity and their “green nature” (i.e. require only oxygen molecules and produce water as a by-product), possible applications of laccases have been extensively researched. However, low efficacy has limited their wider use in applications and, for example, there is not yet any commercially available laccase-based solution for water purification. (Arregui et al., 2019.)
Since laccases constitute a large group of enzymes from a variety of source organisms, we needed to decide which specific laccases we wanted to use in our project. We knew that we would be working with prokaryotes, so we excluded all eukaryotic options. This way we could avoid lacking essential post-translational modifications in our produced enzymes and thus increase the likelihood of them being active. Out of bacterial laccase options we attempted to find laccases that the three previous iGEM teams that we were inspired by didn’t get any experimental results from, and/or were shown to work well at pH 7 with diclofenac as substrate. These turned out to be CotA from Bacillus subtilis, CueO from Escherichia coli, and Yak from Yersinia enterocolitica subsp. palearctica strain 7.
CotA is a multicopper laccase enzyme from Bacillus subtilis in which it has been proven to act as a spore coat (Enguita et al., 2003). Previously, it has been shown to catalyse, for example, the degradation of dyes, polycyclic aromatic hydrocarbons (PAHs) and diclofenac (Zhang et al., 2018; Zeng et al., 2016; Chen et al., 2020). The suggested reaction mechanism of CotA catalyzed degradation of diclofenac is shown in Figure 1. CueO, in turn, is a multicopper oxidase from E. coli in which it regulates the copper resistance (Grass and Rensing, 2001). To our knowledge, it has not been previously studied in degrading diclofenac. You can read more about CueO and its reaction mechanism on our contribution page. The third laccase, Yak, is a multicopper oxidase and it has been shown to be thermostable as well as tolerant to alkalines (Singh et al., 2016). Yak has been studied much less than the other two laccases that we used, but it has been proven to be able to degrade diclofenac and aspirin (Arregui et al., 2019).
Our partnership with team UChicago also helped us make this choice as they provided valuable insights on the enzymatic structures and their opinion on which ones they would be most interested in utilizing for their project.
Where should the laccases be produced and how should their production be regulated?
The chosen laccases needed a production host for our project and proposed implementation plans. Our lab plans would later on include performing measurements to compare the functions of the three chosen laccases, and thus, we knew we needed an efficient overexpression method for the laccase genes to obtain a large enough enzyme yield. This for us meant the use of the E. coli BL21(DE3) strain, which is one of the most used strains for recombinant protein production. It is an E. coli B strain derivative that lacks the ompT and Ion proteases, and harbors a prophage DE3 from the bacteriophage λ (Jeong et al., 2015). The absence of two proteases results in the cell’s decreased ability to degrade produced proteins and thus increases the yield for later-stage experiments. The DE3 prophage, on the other hand, carries a T7 RNA polymerase gene regulated by the lacUV5 promoter (Jeong et al., 2015). These traits coupled with a suitable vector, a so-called pET vector, forms what is known as the pET system. The gene of interest in a pET vector is under the strong control of the T7 bacteriophage’s transcriptional and translational regulatory systems (Fig. 2).
The pET system is induced by IPTG, short for isopropyl β-d-1-thiogalactopyranoside, which is an analog for allolactose. In nature, allolactose regulates the function of the lac-operon through the removal of the lac repressor that inhibits transcription. This induces gene expression of the operon. In IPTG induction, the lac repressor that inhibits the function of the E. coli BL21(DE3) RNA polymerase, is removed from the lacUV5 promoter thus activating the transcription of the T7 RNA polymerase gene. IPTG also removes the lac repressor from the T7 promoter of the pET series vector. This enables the T7 RNA polymerase produced by the cell to bind to the T7 promoter of the expression vector, which starts transcription of the gene of interest.
When induced, the pET system allows for the redirection of nearly all of the cell’s resources towards the production of the desired protein. Within a matter of hours after induction the protein of interest can constitute over half of the cell’s total protein content (Mierendorf et al. 1998). This is why we decided to rely on the E. coli BL21(DE3) strain in combination with a pET series expression vector. The strain is, however, not suitable for long term maintenance of expression plasmids (ThermoFisher). Thus, we planned to use the E. coli DH5α strain instead for subcloning steps and storage.
How should the cells be lysed and the laccases purified?
Before the produced laccases can be purified from the rest of the cell’s proteins, we needed a method for lysing our engineered E. coli cells from the induced cultures. We decided to do this by sonication because our department has the necessary equipment for it and it is a reliable method for which we already had a protocol. Sonication is a physical cell lysis technique, meaning that it relies on external forces to break the cells instead of chemical means. In sonication, sound energy, often at ultrasonic frequencies, is applied to the cells of a culture to disrupt their cell membranes. However, also disadvantages of methods need to be evaluated during project design to minimize their effects if possible. An obvious one with sonication is the unwanted release of proteases as cells are lysed. To slow down the degradation process of our produced laccases, we made sure to remember to keep the cells cold when time for this would come.
Once the cells are lysed, we would be able to continue to protein purification steps. This offers multiple methodological alternatives. We wanted to choose a straightforward option that would likely not have detrimental effects on the activity of our enzymes. The addition of a His-tag seemed worthy of a try, as it often meets these criteria and could be found to have been used for this purpose in literature about laccases (Li et al., 2017). Furthermore, it could be found in many expression vectors belonging to the pET series, i.e. the vector type we wished to utilize. It needs to be noted that a His-tag can be added to either end of the protein, the C-terminal or N-terminal end. Based on previous work on one of our enzymes, CotA, we concluded to opt for a C-terminal His-tag for increased enzymatic activity compared to an N-terminal His-tag (Li et al., 2017).
Enzymes with a His-tag can easily be purified through Ni2+ affinity chromatography. In brief, our His-tagged laccases would bind to the Ni2+ -ions of the Ni2+ affinity resin while all other proteins would pass through the column. The bound laccases could then be released by the addition of a buffer containing imidazole and collected. The usage of imidazole is based on its competitive binding to the Ni2+ -ions acting as ligands. You can find the exact procedure on our methods and experiments page.
After the production and purification steps, the obtained proteins would be run on an SDS-PAGE gel to verify their correct size before proceeding to measurements.
Which expression vector did we choose and what design considerations did it require?
Our choices regarding enzyme production and purification methods led us to have to select our expression vector from a very limited array of options, if we wanted to make things as easy as possible for us. First, we asked around our department if any group already had an empty pET series expression vector with a C-terminal His-tag. We got valuable recommendations of which plasmids should be suitable for our project and ended up using the pET36b(+) plasmid. The plasmid also harboured a kanamycin resistance gene enabling antibiotic selection.
Knowing the expression vector enabled the design of our final laccase harbouring constructs. We decided to order our genes through gene synthesis, which also allowed the addition of appropriate restriction sites in the beginning and end of our fragments to facilitate subcloning. Since the pET36b(+) plasmid also contains tags that we do not need nor want to use within our project, we designed the restriction sites to get them removed. Using the NdeI and XhoI restriction sites all unnecessary tags could be cut out leaving our laccase genes with only a C-terminal His-tag.
As our work was to involve two laccases from the iGEM Darmstadt 2020 team’s plans, CotA and CueO, we decided to use the sequences that they had uploaded to the registry. If we were to obtain useful results by using these sequences, we could refer to their registry pages and add our data there as experiences. This could help future teams. Unfortunately, when checking through their sequences we found that CueO had two NdeI restriction sites that needed to be changed in order for our plans to work. This modified sequence was then uploaded to the registry as a new Part (BBa_K3872001). The sequence for Yak could not be found from the registry, so we designed our construct based on the one reported in literature (Singh et al. 2014). This sequence was also uploaded to the registry as a new Part (BBa_K3872000). Once the constructs have been assembled and transformed into the E. coli BL21(DE3) cells, they are verified through test digestions and sequencing.
How do you measure the activity of laccases and determine the most promising one for our proposed implementation?
There are many options available for determining the activity of laccases, as they are enzymes that are not specific to only one substrate. One of the most commonly applied approaches is the ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay. It is a spectrophotometric technique based on a color reaction caused by the oxidation of ABTS by laccase. In the reaction, ABTS is oxidized to a more preferred state of its cation radical, which results in a color change from colorless to green (More et al. 2011). Enzyme activity can be determined by measuring the change in absorbance at 420 nm (More et al. 2011).
We had to, however, return to the design of this part of the project later on when we faced problems performing the ABTS assay in the lab (you can read more about it here). The revised plan utilized syringaldazine instead of ABTS as a substrate for the produced laccases (Sigma-Aldrich). The procedure is otherwise similar, but the absorbance is measured at 530 nm.
We designed positive and negative controls for our assay to ensure the validity of our results. As a positive control we would use a commercially available laccase, and as negative controls we would have tests without substrate and without enzyme. We also recognized at an early stage that measuring the activities of our laccases solely by the means of ABTS or syringaldazine would not be enough for us. This is because these methods would possibly not tell us anything about the functions of our laccases in regards to diclofenac, the target of our project. Using ABTS or syringaldazine would nonetheless be easier, so the plan was to use them for an initial screening before transferring to diclofenac. We had trouble finding an easy kinetic technique suitable for activity measurements for diclofenac and thus, a plan involving liquid chromatography–mass spectrometry (LC–MS) use was developed.
During the design of what measurements to conduct and how, we also realized the importance of getting information on our laccases at conditions simulating our local wastewater. This was taken into account by designing our measurements to include pH 7 as a condition by altering our reaction buffers, and trying to figure out a way to control the temperature with the equipment we had at hand. Our partnership team also asked us to perform experiments at pH 5 as a comparison. The question on pH and temperature values was raised multiple times during our partnership meetings, which greatly affected our design phase. The most promising laccase for us would be the laccase that was found to be most active especially in regards to diclofenac in wastewater conditions.
Furthermore, our plans included receiving wastewater samples from our local WWTP for the determination of its current diclofenac concentration at the aeration phase with LC-MS. This sample could then be treated with our purified laccases after which the concentration would be measured again. Knowing the diclofenac concentration at the specific time of our project would also allow preliminary estimations to be made on the necessary laccase quantity for our proposed implementation to be functional.
How about the development of the proposed implementation system?
Our proposed implementation is a photoautotrophic system based on the usage of cyanobacteria for laccase production. Therefore, after evaluation to determine which of the three laccases could be the most potential for our biotechnological application we would want to express it in cyanobacteria. Cyanobacteria are photosynthetic, meaning that they get their energy from light, carbon dioxide and water. This trait poses some clear advantages compared to heterotrophic systems that rely on, for example, organic energy sources, in terms of setting up sustainable production platforms. This is also why we were attracted to having them as our second chassis for our project.
The cyanobacterial species we were interested in using was the common model organism Synechocystis sp. PCC 6803 (Synechocystis from here on). This is due to the extensive knowledge that we have on it both as a species and from an engineering standpoint. With a change in production host for our laccases, also for example the expression plasmid had to be changed to accommodate the other organism. This meant that we also designed additional expression constructs besides the pET36b(+) based ones. The construct assembly for the cyanobacterial expression plasmids would follow a system based on a modular cloning strategy described by Zelcbuch at al. (Zelcbuch et al., 2013).
First, the laccase genes delivered in pUCIDT-Kan plasmids would be subcloned downstream of the S3 RBS sequence of the pNiv plasmid (NsiI-XhoI). However, as the pUCIDT-Kan plasmid also contains additional NsiI restriction sites resulting in bands of similar size to the laccase genes, a third restriction enzyme would need to be added to maximize successful ligation. For the genes encoding CotA and CueO this was to be done using BspHI and for the yak gene with PvuII.
Next, the RBS and gene combination would be transferred to the pDF-lac2 expression vector (SpeI-SalI). The pDF-lac2 plasmid, modified from pDF-lac, is a shuttle vector between E. coli and Synechocystis which has the IPTG inducible promoter PA1lacO-1. (Thiel et al., 2018). This allows molecular biology steps to be performed in E. coli DH5α cells before transformation of the final expression constructs into Synechocystis. Utilization of this system for construct assembly would enable the change of RBS later for production optimization, as these translational control elements have been found to have a significant effect on translational efficiency. S3 was chosen as the RBS for our project as it had been shown to result in high translational efficiency with both genes involved in a previous study. (Thiel et al., 2018.)
We planned to order the genes for all laccases with the cyanobacterial modifications through gene synthesis simultaneously with the laccase genes to the E. coli expression constructs to save time. We also planned to begin the construct assembly steps before we had results from our laccase experiments to maximise time available for cyanobacterial work.
The pDF-lac2 expression vector harbouring the gene coding for the most suitable laccase would be introduced into Synechocystis through natural transformation (for more information see the Phototroph Handbook chapter on cyanobacterial transformations). This transformation technique was chosen for its efficiency and ease-of-use. After a successful transformation the engineered strain would be characterised.
How would we want to extend our project?
Many additional modules and elements were thought about already during the initial project design phase. Unfortunately we quickly realized that with the very limited time and resources we had available for the project, these ideas would probably never take shape in our lab this year. Still, we found them important to present and hope that they could be of use for someone in the future. With a better support system we believe that they would have had potential to make interesting follow-up engineering cycles for us.
Addition of a signal peptide and its characterization
Our proposed implementation plan discusses the production of laccases in engineered cyanobacteria grown in a closed photobioreactor system. From there the produced laccases would travel through a filter to join the wastewater where they would detoxify diclofenac. For this to be possible, a signal peptide should be added to the expression constructs for the secretion of laccases into the culture media. We would have wanted to test at least one signal peptide and perform tests to verify its secretion efficiency. This would have taken us one step closer to the development of our proposed implementation system.
Introduction of a killswitch
Our proposed implementation plan discusses the need for a killswitch incorporated in our engineered cyanobacteria. You can read more about it on our proposed implementation page. The addition and evaluation of the killswitch’s functionality in the lab would therefore be of interest to us.
Production optimization efforts for improving efficiency
It should be remembered that research on synthetic biology with cyanobacteria is still a relatively new field and far less developed than heterotrophic systems. Consequently, current production efficiencies that have been obtained in Synechocystis sp. PCC 6803 often fails to meet the requirements for an industrial scale setup. Research on optimization strategies is therefore ongoing and comprises attempts from many different perspectives. These strategies include for example expression improvements on both transcriptional and translational levels, which would have formed interesting additions to a project like ours.
Laccases are enzymes that can be used to target many types of compounds ranging from different types of pharmaceutically active compounds to for example dyes. However, since the focal point of our project is diclofenac, it would be natural to explore site-directed mutagenesis for improved enzyme specificity. Another enzyme engineering approach could be, for example, to aim at an increased enzyme activity at the conditions prevalent in wastewater, which currently are suboptimal for laccase function. These plans would require proper simulations and later on preferably also experimental structural analysis of the mutated laccases.
Exploration of filter options for the proposed implementation
Our proposed implementation mentions the reliance on an effective filter that only allows for the release of laccases to wastewater, thus preventing release of engineered cyanobacterial cells. Comparing the performance of possible filters could provide useful insight for further development of the system.
Shift from laboratory batch-culture measurements to a continuous-culture setup
Since the proposed implementation system is intended to be a continuous-culture system, it would be of relevance to conduct measurements accordingly also in the lab once the strains are otherwise completed. This could be made possible with the availability of a suitable photobioreactor setup.
How did we succeed with these design plans?
Our methods and experiments -page contains the precise protocols for how we built and tested our design plans. Whereas our results page goes in depth on how we succeeded at every step of our plan, and what could be learnt from it all. Check out our engineering success page for a summarized version of the above.
- Arregui, L., Ayala, M., Gómez-Gil, X., Gutiérrez-Soto, G., Hernández-Luna, C. E., de los Santos, M. H., Levin, L., Rojo-Domínguez, A., Romero-Martínez, D., Saparrat, M. C. N., Trujillo-Roldán, M. A., Valdez-Cruz, N. A. (2019) Laccases: structure, function, and potential application in water bioremediation. Microbial Cell Factories, 18(1), 200, https://doi.org/10.1186/s12934-019-1248-0.
- Chen, L., Li, Y., Lin, L., Tian, X., Cui, H., Zhao, F. (2020). Degradation of diclofenac by B. subtilis through a cytochrome P450-dependent pathway. Environmental Technology & Innovation, 20, 101160, https://doi.org/10.1016/j.eti.2020.101160.
- Enguita, F. J., Martins, L. O., Henriques, A. O., Carrondo, M. A. (2003). Crystal structure of a bacterial endospore coat component: A laccase with enhanced thermostability properties. The Journal of Biological Chemistry, 278(21), 19416-19425, https://doi.org/10.1074/jbc.M301251200".
- Grass, G., Rensing, C. (2001). CueO is a multi-copper oxidase that confers copper tolerance in Escherichia coli. Biochemical and biophysical research communications, 286(5), 902-908, https://doi.org/10.1006/bbrc.2001.5474.
- Jeong, H., Kim, H. J., & Lee, S. J. (2015). Complete Genome Sequence of Escherichia coli Strain BL21. Genome announcements, 3(2), e00134-15, https://doi.org/10.1128/genomeA.00134-15.
- Kataoka, K., Komori, H., Ueki, Y., Konno, Y., Kamitaka, Y., Kurose, S., Tsujimura, S., Higuchi, Y., Kano, K., Seo, D., Sakurai, T. (2007). Structure and function of the engineered multicopper oxidase CueO from Escherichia coli -- deletion of the methionine-rich helical region covering the substrate-binding site. Journal of Molecular Biology, 373(1), 141-152, https://doi.org/10.1016/j.jmb.2007.07.041.
- Li, L., Xie, T., Liu, Z., Feng, H., & Wang, G. (2017). Activity enhancement of CotA laccase by hydrophilic engineering, histidine tag optimization and static culture. Protein Engineering, Design and Selection, 31(1), 1–5, https://doi.org/10.1093/protein/gzx064
- Mierendorf, R., Yeager, K., & Novy, R. (1994, May). The pET System: Your Choice for Expression. inNovations, 1(1).
- Mierendorf, R. C., Morris, B. B., Hammer, B., & Novy, R. E. (1998). Expression and Purification of Recombinant Proteins Using the pET System. Methods in molecular medicine, 13, 257–292, https://doi.org/10.1385/0-89603-485-2:257.
- More, S. S., P S, R., K, P., M, S., Malini, S., & S M, V. (2011). Isolation, Purification, and Characterization of Fungal Laccase from Pleurotus sp. Enzyme research, 2011, 248735, https://doi.org/10.4061/2011/248735.
- Sigma-Aldrich. Enzymatic Assay on laccase (EC 184.108.40.206). Retrieved Oct 18, 2021 from https://www.sigmaaldrich.com/FI/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-laccase.
- Singh, D., Rawat, S., Waseem, M., Gupta, S., Lynn, A., Nitin, M., Ramchiary, N., Sharma, K. K. (2016). Molecular modeling and simulation studies of recombinant laccase from Yersinia enterocolitica suggests significant role in the biotransformation of non-steroidal anti-inflammatory drugs. Biochemical and biophysical research communications, 469(2), 306-312, https://doi.org/10.1016/j.bbrc.2015.11.096.
- Singh, D., Sharma, K. K., Dhar, M. S., & Virdi, J. S. (2014). Molecular modeling and docking of novel laccase from multiple serotype of Yersinia enterocolitica suggests differential and multiple substrate binding. Biochemical and biophysical research communications, 449(1), 157–162, https://doi.org/10.1016/j.bbrc.2014.05.003
- Thiel, K., Mulaku, E., Dandapani, H., Nagy, C., Aro, E. M., & Kallio, P. (2018). Translation efficiency of heterologous proteins is significantly affected by the genetic context of RBS sequences in engineered cyanobacterium Synechocystis sp. PCC 6803. Microbial cell factories, 17(1), 34, https://doi.org/10.1186/s12934-018-0882-2.
- ThermoFisher: BL21(DE3) Competent Cells. Retrieved Oct 9, 2021 from: https://www.thermofisher.com/order/catalog/product/EC0114#/EC0114.
- Zelcbuch, L., Antonovsky, N., Bar-Even, A., Levin-Karp, A., Barenholz, U., Dayagi, M., Liebermeister, W., Flamholz, A., Noor, E., Amram, S., Brandis, A., Bareia, T., Yofe, I., Jubran, H., & Milo, R. (2013). Spanning high-dimensional expression space using ribosome-binding site combinatorics. Nucleic acids research, 41(9), e98, https://doi.org/10.1093/nar/gkt151
- Zeng, J., Zhu, Q., Wu, Y., Lin, X. (2016). Oxidation of polycyclic aromatic hydrocarbons using Bacillus subtilis CotA with high laccase activity and copper independence. Chemosphere, 148, 1-7, https://doi.org/10.1016/j.chemosphere.2016.01.019.
- Zhang, Y., Dong, W., Lv, Z., Liu, J., Zhang, W., Zhou, J., Xin, F., Ma, J., Jiang, M. (2018). Surface Display of Bacterial Laccase CotA on Escherichia coli Cells and its Application in Industrial Dye Decolorization. Molecular Biotechnology, 60(9), 681-689. https://doi.org/10.1007/s12033-018-0103-6.