Team:Brno Czech Republic/Description

Project description

The goal of our project is to design a mobile device CYANOTRAP that degrades and detoxifies cyanobacterial blooms in lakes and water reservoirs. The key elements of the device are functional cellulosomes to lyse cyanobacteria and degrade their common toxins using genetically modified Bacillus subtilis.

Motivations

When deciding the topic of our iGEM project, we spent many hours discussing various ideas. From synthetic food to saving forests from the bark beetle to terraformation. The final inspiration came when we looked at the dam located near the city of Brno. It is a huge body of water, where the inhabitants of Brno can swim in the heat of summer, try different water sports, ride a ferry boat or ice skate in the winter. However, there is one problem – cyanobacterial overpopulation - which often precludes these activities. The more we thought about it, the more we realised just how many places are affected by an overabundance of these organisms and that this truly is a global issue. As we delved into the research, we have found multiple scientific publications and iGEM projects to take inspiration from while also coming up with our own ideas to tackle this problem.

Cyanobacteria can proliferate easily in water environments, largely due to human activity such as the accumulation of phosphorus [1, 2] which is released into water from wastewater effluents or agricultural areas where it is used in fertilizers [1, 2]. Cyanobacterial blooms not only affect water quality but also produce highly toxic metabolites (Figure 1). These cyanotoxins, often neurotoxins or hepatotoxins, are hazardous for humans and other species. Water organisms affected by cyanotoxins have symptoms such as reduced survival, limited swimming movements leading to paralysis, or reduced fertility. These toxins affect organisms of all taxonomic levels like bacteria, algae, plants, zooplankton or molluscs and fish [3].

Figure 1

Phosphorus accumulation in the water leads to cyanobacterial overpopulation. Cyanobacteria produce harmful toxins.

Today, cyanobacterial blooms are fought in many different ways. These strategies vary depending on the target water quality, geomorphological type of a water body or cyanobacterial overgrowth rate. It can be the management of nutrient levels (nitrogen and especially phosphorus), support of macrophytes colonising the bottom of the lakes, direct intervention in the food web (e.g. fish management) or interventions in hydromorphology. These are mainly long term and complex strategies and their positive effects can not be granted. Other methods, often radical or not eco-friendly, are based on physical or chemical principles, e.g. disinfection of the water, biomass removal or mixing of water levels [4]. 

Our solution instead, offers an effective and innovative biological strategy to fight cyanobacteria, their toxins and their overgrowth itself. Our device can be used constantly to prevent cyanobacterial bloom, or to directly control it when it has occurred. Just read more below!

Design

Our idea is to generate a small self-operating floating system that is powered by solar energy and continuously lyses cyanobacteria and detoxifies water from their toxins. Our device called CYANOTRAP will be remotely operated by a drone. It will fly over the water basin and measure the density of the cyanobacterial bloom at every square meter of the surface. It will then navigate the floating device to the most critical spot in its area of operation. It could also send data to an app that could be utilized by swimmers and fishermen looking for a safe place for their hobbies. This system will provide a faster, more targeted, efficient and safe way to achieve clean water for the sake of the environment, as well as humans.

The core of the device are engineered bacteria that on their surface display enzymes required for the lysis of cyanobacteria cells and for degradation of cyanotoxins. Such a design has several advantages over other solutions: 1) High mobility of the device would allow its targeting to the most infested areas, 2) Number of devices put in operation can easily be scaled according to the acreage of the water surface and degree of infestation, 3) Enzymatic lysis is more environmental friendly than chemical treatments, 4) The device can be used for constant surveillance and prevent formation of water blooms, 5) Use of bacteria as carriers of the enzymes provides longer sustainability as living bacteria can continuously replenish the enzymes. 

The major challenge in such a design is how to keep engineered bacteria within the device, while exposing it to a continuous flow of water from outside. To solve this issue, we propose to immobilize bacteria on the surface of cellulose-coated beads that will be trapped in a chamber with perforated mesh-like walls. This will allow influx and efflux of water while preventing release of the bead-immobilized bacteria. This solution will also increase the surface of bacteria coating and, hence, cleaning capacity of the device. Further mesh barriers placed in front of the water inlets will protect the system from larger clumps and clogging. (Figure 2).

As a cellular chassis, we chose Bacillus subtilis as it is a Gram-positive bacterium able to live in lower temperatures. We will also use its ability to export synthesized proteins to its outer surface [5]. We believe this will distinguish our project from those using modified E. coli against cyanobacteria. We will engineer Bacillus subtilis to trigger lysis of a common cyanobacteria Microcystis aeruginosa. This will be accomplished by using lysozyme and microvirin, which binds to a cyanobacterial cell wall, bringing it closer to lysozyme. Secondly, we plan to degrade the most common cyanobacterial toxin microcystin. Microcystin can be degraded via the MC pathway consisting of three Mlr enzymes with less toxic, non-proteinogenic amino acid Adda as a final product [6]. All these enzymes will be displayed on the cell surface using cellulosomes which are described in detail below.

Our project centers around the cellulosome. It is a protein complex which can be naturally found on the surface of anaerobic cellulose-degrading bacteria. The cellulosome is composed of catalytic and noncatalytic subunits. The non-catalytic subunit is called scaffoldine[7]. We decided to take advantage of this natural complex and redesign it to serve our purpose.

Because we want our device to carry out two separate functions – to lyse cyanobacteria and to degrade microcystin – we decided to design two different cellulosomes which can be seen in Figure 3. Both cellulosomes are produced by a bacterial cell, immobilized on a cellulose bead via a cellulose-binding module (CBM) connected by a linker to the LysM domain, which interacts with the cell wall of this bacterium. This immobilization is crucial for continuous production of recombinant proteins and for preventing the release of genetically modified bacteria into nature.

The rest of the cellulosome differs depending on its function. Design for the lysis of cyanobacteria is pictured in Figure 3. Synthetic scaffoldin L is attached to the bacterial cell wall by LysM domain. LysM is then connected to cohesin by a linker. Cohesin interacts with dockerin – domain fused with an effector enzyme. In this case, the effector enzyme is Lysozyme. The successful production of lysozyme from Bacillus licheniformis in Bacillus subtilis has been demonstrated without any impact on its viability [8]. The potential of chicken lysozyme C to lyse Microcystis aeruginosa was proved by team Peking iGEM 2014 [9]. However, we still need to determine which of these two lysozymes will be the best for our project in terms of Bacillus subtilis viability, environmental conditions and enzymatic activity. Microvirin is attached at the end of the scaffoldin structure. Microvirin binds to Microcystis aeruginosa [9], and thus holds cyanobacterial cells in close proximity to lysozyme, increasing the effectiveness of its lysis.

The second cellulosome is designed to degrade microcystin. Three enzymes MlrA, MlrB and MlrC of the MCpathway are attached to the scaffoldin D by corresponding dockerins (Figure 3).

In total, we've designed seven synthetic genes, transcription of which is controlled by a constitutive promoter. They contain three different RBSs - when we combine our genes into a synthetic operon, the strongest RBSs should compensate for the lower transcription rate of the gene located at the 3' end of the operon.

With all these ideas and designs, we believe CYANOTRAP to be a useful and innovative application of synthetic biology.

Summary

To sum it up, we plan to fulfil our goals by constructing two different cellulosomes. After confirming the expression and assembly of the cellulosome on the surface of Bacillus subtilis, we will conduct measurements testing its functionality. Sadly, because of the global pandemic, our time for working in the wet lab was reduced. Instead, we focused on detailed planning and designing CYANOTRAP. 

As we planned our project, we came up with many ideas to improve our device and make it more effective at tackling the cyanobacterial overpopulation while adhering to safety guidelines. Therefore, we plan to implement other features in the future. For example, we would like to modify Bacillus subtilis to accumulate phosphates from the water. This extracted phosphorus can be then reused, and it will no longer support cyanobacterial growth. This means that our device will not only reduce cyanobacterial numbers but also further prevent their growth.

Impact of the pandemic

Man proposes, Covid disposes. Our team was also affected by the global SARS-CoV-2 pandemic. Unlike many other teams, we were allowed to spend our summer in the wet lab, but our time there was reduced. Therefore, we didn’t have time to repeat some crucial experiments and we had to postpone assembly of whole cellulosomes as well as the implementation of phosphorus accumulation until next year.

Instead, we focused on educational activities to entertain people at home during the quarantine. We came up with online courses on various biological topics from room plants and extinct animals to synthetic biology or microbiology. We also prepared a talk about GMOs to educate the general public about this issue. You can find out more about these activities in the Education chapter.

After all, we survived the pandemic healthy and motivated as we proved that we can work as a team even in these challenging times. Stronger and determined, we are looking forward to another year in iGEM.

References

1. Parrish, J., 2014. The Role of Nitrogen and Phosphorus in the Growth, Toxicity, and Distribution of the Toxic Cyanobacteria, Microcystis aeruginosa. Master's Projects and Capstones. 8.

2. Hart,  M. R., Quin, B. F., Nguyen, M. L., 2004. Phosphorus runoff from agricultural land and direct fertilizer effects: a review. J. Environ. Qual. 33 (6), 1954-72.        https://doi.org/10.2134/jeq2004.1954 

3. Zanchett, G., Oliveira-Filho, E. C., 2013. Cyanobacteria and cyanotoxins: from impacts on aquatic ecosystems and human health to anticarcinogenic effects. Toxins, 5 (10), 1896–1917. https://doi.org/10.3390/toxins5101896 

4. Stroom, J. M., Kardinaal, W. E. A, 2016. How to combat cyanobacterial blooms: strategy toward preventive lake restoration and reactive control measures. Aquat Ecol 50, 541–576. https://doi.org/10.1007/s10452-016-9593-0

5. Fu, L. L., Xu, Z. R., Li, W. F., Shuai, J. B., Lu, P., Hu., C. X.,, 2007. Protein secretion pathways in Bacillus subtilis: Implication for optimization of heterologous protein secretion. Biotechnol. Adv. 25, 1–12. https://doi.org/10.1016/j.biotechadv.2006.08.002

6. Dziga, D., Wasylewski, M., Wladyka, B., Nybom, S., Meriluoto, J., 2013. Microbial Degradation of Microcystins. Chem. Res. Toxicol. 26, 841–852. https://doi.org/10.1021/tx4000045

7. Hu, B., Zhu, M., 2019. Reconstitution of cellulosome: Research progress and its application in biorefinery. Biotechnol. Appl. Bioc. 66, 720–730. https://doi.org/10.1002/bab.1804

8. Zhang H, Fu G, Zhang D., 2014. Cloning, characterization, and production of a novel lysozyme by different expression hosts. J. Microbiol. Biotechnol. 24 (10), 1405–1412. https://doi.org/10.4014/jmb.1404.04039   

9. https://2014.igem.org/Team:Peking

Figure 2.
Our device floats on the water surface and contains cellulolytic beads with bound Bacillus subtilis.

Figure 3.
Strain L produces cellulosome containing Lysozyme and microvirin. Its function is to lyse cyanobacterial cells. Strain D produces cellulosome which contains Mlr enzymes to degrade toxin microcystin. All enzymes are connected to the scaffoldin via dockerins and cohesins.

Figure 4.
Members of our team during the SARS-CoV-2 pandemic.