Through our proposed implementation plans we hope to contribute to the development of a new diclofenac-detoxifying solution which could be integrated at wastewater treatment plants (WWTP). The current removal efficiency of diclofenac at our local WWTP is only 27%, which is a lot lower than for many other compounds. To tackle that local problem with synthetic biology, our solution is to create a closed photobioreactor system in which the laccase enzymes would be produced by genetically modified cyanobacteria. Cyanobacteria provide a sustainable production alternative for heterotrophic systems as they are photoautotrophic organisms. This means that they get their energy from light, carbon dioxide and water, as opposed to for example organic compounds that are often produced in ethically questionable ways. The produced enzymes from the cyanobacteria would flow from the closed system through a filter first to a so-called “kill tank” and after that through a second filter to the wastewater. This is done to prevent any genetically modified organisms (GMOs) from being released from the system.
Our system would make use of all recent advancements regarding photobioreactor design. This includes, for example, the measures taken to improve light conditions to make them as uniform within the culture as possible. Preventing major light fluctuations caused by shadowing is important for optimal cyanobacteria cultivation, if we would want them to produce laccases at their highest capacity.
We have consulted our local WWTP (Turun Seudun Puhdistamo Oy) about the proposed implementation of our system. According to them, our solution could be added either right before or after the process called aeration. In that phase microbes remove organic material and nitrogen from wastewater. The beginning of the aeration phase is technically the first reasonable point to add our laccases because before that, conditions and processing is so harsh that the laccases would get denatured. After the aeration phase there are only two phases left in the purification process; secondary clarification phase and sand filtration phase. That means that our laccases would still have time to detoxify diclofenac before the water leaves the wastewater treatment process, but would not be denatured in the process.
All systems containing genetically modified organisms should contain features that minimize biocontainment related hazards. For this reason, also our plans include multiple design elements specifically targeting biosafety issues. This is done even though the engineered cyanobacteria could only accidentally be released from our closed photobioreactor system.
One non-biological component is the filter that was previously described to separate the photobioreactor first from the kill tank and then the kill tank from wastewater. The purpose of the filter is to prevent the release of GMOs by only allowing the produced laccase enzymes to pass through. Relying on solely a filter is, however, not enough to ensure the operational safety of our system in regards to prevention of unintentional release of modified cyanobacteria. This is why the kill tank has been set in place. The tank situated between the photobioreactor and wastewater forms the site for killswitch activation. It has a detection mechanism, mounted near the filter isolating the tank from the photobioreactor, for the detection of cells. If cyanobacterial cells were to be detected in the tank it would trigger the release of zinc ions to activate a metal-inducible killswitch. This way the system would not cause further harm to the Baltic Sea by dispersing extra zinc ions continuously.
A killswitch is a genetic element that can cause controlled cell death when induced. There are many types of kill switches available with distinct killing mechanisms. The killswitch that we would want to have in our engineered cells uses a strategy where conditional lethality depends on intracellular degradation of DNA (Čelešnik et al. 2019). This would thus, in addition to killing the GMOs, also destroy the genetic material in the cells, which would minimize the risk of horizontal gene transfer from occurring (Čelešnik et al. 2019). Minimizing the risk of transfer of characteristics from our engineered strain to other microbes is especially important as they carry antibiotic resistance genes. The killswitch with these features is the KSPcopM195-BCD-nucA, which comprises of the non-specific DNA/RNA nuclease NucA in combination with its inhibitor NuiA, and is induced by the PcopM195-BCD promoter (Čelešnik et al. 2019). This killswitch was chosen as it has shown to be capable of complete autodestruction when induced in experiments with our chassis Synechocystis sp. PCC 6803 (Synechocystis from here on) as opposed to only marked or weak autotoxicity (Čelešnik et al. 2019). Another option we considered was a toxin-antitoxin system with the MazF toxin from E. coli, which has also been studied in cyanobacteria (Cheah et al. 2013).
Another genetic element within our constructs that can be viewed to assist in biosafety concerns is our chosen promoter for laccase production. The pDF-lac2 expression vector that would be used for our engineered Synechocystis strain carries the PA1lacO-1 promoter. The promoter is induced by IPTG, a chemical that is not found in nature at concentrations that are high enough for gene expression. This does not solve biocontainment issues, but prevents the production of laccases if the engineered cyanobacteria are outside the bioreactor system.
Lastly, the usage of specifically Synechocystis as the cyanobacterial species should decrease the likelihood of potential ecological hazards. This is due to it being a freshwater cyanobacterium that has been isolated from a Californian lake and, hence, it would most probably not do well in the conditions prevalent in the Baltic Sea. This hypothesis should however be confirmed through extensive tests with the wild-type strain currently used in the lab.
Additionally, we have familiarized ourselves very closely with the legislation concerning the use of genetically engineered organisms in Finland. These laws and directives include for example (The Board of Gene Technology):
- The Gene Technology Act (377/1995)
- The Government Decree on Gene Technology (928/2004)
- The Decree of the Ministry of Social Affairs and Health principles of risk assessment of the contained use of genetically modified microorganisms, on classification of the contained use, and on containment and other protective measures (1053/2005)
- The Decree of the Ministry of Social Affairs and Health on notifications and applications relating to the contained use of genetically modified organisms, on keeping a record of the contained use and on an emergency plan (272/2006)
- The Decree of the Ministry of Social Affairs and Health on Inspection Procedures under the Gene Technology Act (198/2007)
- The Government Decree on Chargeable Performances under the Gene Technology Act (1255/2018)
Furthermore, we took a look at the legislation concerning the deliberate release of genetically engineered organisms into the environment, but that doesn't really concern us since we are not releasing GMOs into the environment purposefully.
We also looked briefly at the GMO legislation of the other countries around the world - in case that our solution could someday be in use globally. You can read more about this on our partnership page.
Our project falls under all of these laws and we would carefully obey the legislation when it comes to our proposed implementation. Even though all of these legislations don’t concern us as long as we are only working in a lab, we wanted to take all aspects into account and be truly conscious about legislation-related things as one of our core values is to obey the law.
It needs to be noted that research on harnessing the potential of cyanobacteria for biotechnological use is still in the very early stages compared to heterotrophic systems. As a result, current production efficiencies that have been obtained in Synechocystis rarely meet the requirements for an industrial scale setup. Thus, our proposed implementation would most likely not be suitable yet as is, but we hope that future advancements within the field will make our plans viable.
In addition to production improvements, also the laccases we are using might need alterations to make them more suitable for a system like this. The enzymes we have chosen for our project should be working at the pH of wastewater, which is 6.5-7.5, but the low temperature conditions, 12-25 degrees Celsius, are suboptimal for them. Our proposed implementation system could be particularly inefficient during the winter months when wastewater temperatures are the lowest, and consequently prove to be unreliable as a year-round detoxification method.
Another important aspect to consider when designing a new biotechnological application is the possibility of potential misuse no matter how good the developer’s intentions are. With our system, one of the most relevant questions raised is whether our system could be used to alter diclofenac, and other biologically active compounds, before their human consumption. This could result in not only decreased therapeutic response but also be potentially hazardous for the people involved.
- Čelešnik, H., Tanšek, A., Tahirović, A., Vižintin, A., Mustar, J., Vidmar, V., & Dolinar, M. (2019). Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium Synechocystis sp. PCC 6803. Biology Open, 5(4), 519-528.
- Cheah, Y. E., Albers, S., & Peebles, C. (2013). A novel counter-selection method for markerless genetic modification in Synechocystis sp. PCC 6803. Biotechnol Prog., 29(1), 23-30.
- The Board of Gene Technology. Legislation. Read 8.10.21, available at: https://geenitekniikanlautakunta.fi/en/legislation
- Turun Seudun Puhdistamo Oy. Read 22.7.2021, retrieved from www.turunseudunpuhdistamo.fi