Proposed Scenario

Harmful Algal Blooms

The most direct implementation of our microcystin degrading bacteria is in treating harmful algal blooms (HABs), during which large quantities of the toxins are released. According to previous case studies (Backer et al., 2008 & 2010), the concentration of microcystin in HABs can be as high as 1mg/L. Previous research on natural degraders indicated low efficiency, and a large volume of bacteria culture may be required. We believe the utilization of genetically engineered bacteria can solve this problem. Moreover, chassis E. coli bacteria are more tolerant to harsh conditions that cyanobacteria may dwell in, and the system can be more stable in long-term implementations.

In lab experiments, we tried to replicate the environmental conditions by controlling the temperature at 25 – 30 °C, at which HABs are likely triggered by the high water temperature; the pH at around 9.5, as previously case studies (Backer et al., 2010) suggested. Our engineered bacteria demonstrated excellent degradation ability and efficiency – thus, we believe they can be used to treat an “emergency” HAB outbreak and significantly remediate the situation within hours.

Regular Water Treatment Plants

Microcystin-LR, the most toxic of all microcystins, has been categorized as a Class 2B carcinogen (IARC, 2012) and a concentration requirement has been set at 1 μg/L (WHO, 2004). Considering the severe health impact of microcystins, we believe that it is important to regularly monitor microcystin levels, and that microcystin degradation should be part of routine water treatment.

Experiments and modeling have suggested that our bacteria is capable of degrading microcystin at low levels. The by-product, linearized microcystin, is essentially non-toxic at environmentally relevant concentrations. This eliminates the common concern for chemical degradation that products or reagents may cause secondary pollution.

Moreover, previous works (Han et al., 2018) have found that the extracellular enzyme display system is capable of displaying different proteins on the same bacteria, which provides wider applicability of these degraders. In the future, we hope to explore this technique and construct more “versatile” bacteria that can greatly simplify the water treatment process.

Novel Algae Technology

Many new biotechnology build upon an algae chassis. They utilize algae metabolism to produce biodiesels (Rosenberg et al., 2008), take advantage of its photosynthesis, or use it as a food source. (Varshney et al., 2015) However, the toxins produced by cyanobacteria as secondary metabolites makes handling algae more risky and challenging for users.

In experiments of co-culturing engineered E. coli and microcystis aeruginosa (microcystin producer), we found no influence on the survival or metabolism of algae, which indicates excellent compatibility. (More details below in “Safety”.) Therefore, we believe our bacteria can improve the safety of a greater system built upon cyanobacteria.

Hardware

As shown in FIG. 3, We preliminarily designed a bioreactor to degrade microcystin in water bodies. It mainly consists of a large container filled with alginate beads that immobilize the engineered E. coli to prevent them from unintended release. Degradation is carried out in the cylindrical container. (Dziga et al., 2013)

In FIG. 3, the cylinder is shown to be transparent for clarity. We hope to make it non-transparent and employ a light-induced kill switch in the bacteria to address unwanted leaks into the environment.

Microcystin contaminated water is pumped into the bioreactor to be treated, and the water is released after a time interval. Then another portion of water will be taken in, and the cycles go on. A standard will be created to determine how much time is needed to degrade a given concentration and volume of microcystin contaminated water. This way, we can create a composite system that first measures the microcystin concentration (or allows direct data input), then automatically determines the time needed for the interval between each water intake, and executes the cycles.

Due to time and equipment restraints, we were unable to fully construct this bioreactor and test it within this iGEM season. In the future, we wish to optimize these aspects: (Dziga et al., 2014)

1)Size of the bioreactor (aspect ratio);

2)Size of the alginate beads and immobilization ability;

3)Amount of bacteria needed;

4)Efficiency at different microcystin concentrations;

5)Long-term stability and reusability of the system.

Safety

Lab Safety

The organisms used in our experiments are:

1)1)E. coli BL21 (DE3) (engineered & control)

2)Microcystis aeruginosa (microcystin producer)

3)Sphingopyxis sp. m6 (natural microcystin degrader)

These organisms fall under the category of Biosafety Level 1. However, microcystis aeruginosa reproduces particularly quickly, and produces toxins; we also used synthesized toxins in the experiments. Considering these, we decide to perform experiments according to Biosafety Level 2 protocols.

We used the labs of Southeast University School of Public Health, so luckily there were many experts to instruct us on safety precautions, and all experiments were supervised by lab assistants. Prior to experiments, all wet lab members took 2 online seminars and 1 in-lab workshop on safety procedures led by lab assistants. The rigor of the safety procedures was challenging, but we all learned a lot!

Bacteria-Algae Interactions

By learning stakeholders’ attitudes in using GMO for bioremediation (Sayler and Ripp, 2000), we found people very concerned on the artificial bacteria’s influence on the environment. We decided to co-culture the bacteria with microcystis aeruginosa (a strain of microcystin-producing algae) and analyze their interactions in the following 3 aspects:

1.The surviving population of algae. This would be measured by directly counting the algae cells in a small sample from the culture.

2.The metabolic activity of algae. This would be measured by performing quantitative PCR on gene sequences psaB, psbD, rbcL, fbp.

3.The microcystin production of algae. The concentration of microcystin produced by algae at experimental scope is rather low, and is unsuitable for HPLC detection. The production would also be measured by performing quantitative PCR on genes mcyA, mcyB, mcyD, mcyG that are responsible for microcystin synthesis. (Nishizawa et al., 1999)

After monitoring these indicators for 7 days, we discovered that our bacteria has no influence on any of these aspects. Moreover, many novel algae technologies are constructed based on their metabolism, and our bacteria can degrade toxins without interfering. They have the potential of becoming a component of a larger system of algae technology.

Overall, our bacteria’s compatibility with environmental algae has been verified, and they should be safe for primary implementations.

Future Improvements

Lab Safety

Our engineered system still has a long way to go before practical implementation. The realistic situation in water bodies will be far more complicated in environments. Here, we summarize a few key points that we wish to improve on in the future:

1)Construct and optimize the designed hardware and the auto-control timer system

2)Test the system using water samples retrieved from the environment

3)Adapt a kill switch that matches the hardware design, preferably green light induced

4)Explore simultaneous display of a greater variety of chemical degrading proteins at the surface of E. coli

5)Analyze the compatibility between the bacteria and more water-dwelling organisms