Contents

• 1) Biosensing
  • 1.1) Introduction
  • 1.2) Usage
  • 1.3) Considerations
  • 1.4) Future challenges
• 2) Bioremediation
  • 2.1) Introduction
  • 2.2) Usage
  • 2.3) Considerations
  • 2.4) Future challenges
• 3) Bioconversion
  • 3.1) Introduction
  • 3.2) Usage
  • 3.3) Considerations
  • 3.4) Future challenges
• References

Biosensing

Introduction

When we were creating our heavy metal biosensors, the goal we had in mind was a simple test that anyone could use, even if they had no knowledge of or experience in synthetic biology. To do this, we would need to have a test which gave an easy to interpret output, such as colour or fluorescence. We propose that our biosensor could be freeze dried on cellulose discs, so that when the discs are rehydrated in heavy metal contaminated water, they fluoresce under blue light. Our proposed implementation of these is to use a cassette where the discs can be swapped out and reused for multiple simple tests of various heavy metals (see figure 1).

Figure 1. Proposed designs for our biosensor. Biosensor constructs are immobilised on cellulose discs, and when in contact with heavy metals they will fluoresce green. The casette is reusable, and the immobilised biosensors can easily be swapped out.

End users

From our human practices research, we found that people living in rural areas would benefit from a simple diagnostic test for heavy metal contamination. However, these are not the only possible end users. Environmental agencies around the world could also use an accurate field test for heavy metal contamination; our interactions with the Scottish Environmental Protection Agency confirmed their interest in this. Other end users include industry and people in laboratory settings, if they need to test easily whether a sample heavy metal contamination. However, since they would not be in the field, it would be possible for them to use our biosensor as a chemical test instead.

Safety considerations and dual use

Generally speaking, our biosensor is safe and not adverse to human health. The only components are lysate, T7 polymerase, NTPs, our linear DNA construct and DFHBI, none of which are toxic to human health. However, our proposed implementation involved being able to switch out the cellulose discs after use, which would be contaminated with heavy metals if they showed a positive result. Recycling of these may become an issue that would have to be addressed if they were used in practice. Likely, it would simply follow the correct procedures for disposal of toxic materials in the area they are used in. Our biosensor could also have some dual-use, as people could modify the designs of our biosensor to detect physically harmful resources such as uranium, or socially harmful resources such as gold or other valuable metals, which could lead to mining and environmental damage. However, these designs already exist and are public knowledge, and access to materials such as DFHBI or DFHO is generally limited.

Future challenges

If our biosensor were to be properly implemented and distributed, there would be many other factors we would need to consider for its usage. One example is temperature, as T7 polymerase may not work with sufficient processivity at low temperatures to produce an output. On the other side, too high temperatures may also denature T7 polymerase. Another possible variable we have yet to account for is pH, as many water samples contaminated with heavy metals also tend to be quite acidic due to the mining effluxes. Furthermore, long term storage of our biosensor is another challenge that would need to be investigated if we were to implement it. Finally, the scalability and economic feasibility have also not been addressed; DFHBI is quite expensive, and getting purified enzymes and NTPs in bulk would also not be trivial. We acknowledge that these challenges would need to be addressed if our biosensor were to be implemented in a real world scenario.

Bioremediation

Introduction

While going through the many iterations of the design cycle while making our bioremediation device, we also had ease of use for people who did not know any synthetic biology in mind. Our vision was to create a hydrogel, which could be put into water and then swell, and then manually removed. And to some extent, we achieved this. Our hydrogel keeps its consistency after swelling, and also functions in chelating heavy metals out of the water. Before we could implement our device in real life though, there are many variables we would have to consider.

End users

The main end user of our bioremediation device would be people living in rural areas, whose main source of water is contaminated with heavy metals. We would want to implement our device in conjugation with the biosensor, so people in rural areas can test their water for heavy metal contamination, then if it is contaminated remove the heavy metals with our hydrogel. This would work by putting the hydrogel into a reservoir of water, leaving it for a certain amount of time, and then removing the hydrogel manually, yielding a reservoir of heavy metal free water. This is the individual household implementation model we have been envisioning all project. This would work both if a household had central plumbing, and no central plumbing. Another implementation model we envisioned was municipal water treatment, as the hydrogel would require very little setup compared to current techniques such as chemical precipitation or fractionation. However, the issue of scalability of our hydrogel arises when dealing with this, and would have to be considered before it was used.

Safety considerations and dual use

The biggest safety consideration is that there are currently no guidelines to follow for the handling and disposal of a hydrogel that has chelated heavy metals. Since the hydrogel would have quite a high concentration of heavy metals, it is likely that manual removal without protective equipment could result in absorption of heavy metals into the skin. Also, it would need special guidelines for disposal and recycling, which like the biosensor, would likely follow local guidelines for the handling of such materials. Furthermore, having a simple means to generate a high concentration of heavy metals could be used for malicious intent, for example the enrichment or extraction of toxic heavy metals. However, simple means like electrophoresis already exist to achieve this, and thus our hydrogel likely would not cause any social impact in this regard. The design we have developed could also be modified so that malicious proteins are fused to the hydrogel, as we have shown that proteins still function when immobilized to a cellulose hydrogel using our protocol and design. However, generating such a construct would require a well equipped laboratory anyway, so would not allow for dual-use by the general public.

Future challenges

If our design were to be implemented, there would need to exist safety guidelines for handling and disposing of used hydrogels. Since we are proposing implementing our design in rural areas, the infrastructure likely doesn’t exist to dispose of such contaminated materials. Before our design could actually be implemented, the safe and environmentally considerate handling and disposal of our hydrogels would need to be fully addressed.

PET Bioconversion

Introduction

Considering that there are currently no biological Parts capable of collecting microplastics dispersed in the aqueous environment, we decided to take a different perspective to mitigate the problem of plastic pollution in water bodies. Microplastics are created and move through nature because plastics exposed to the natural environment gradually degrade over time into pieces less than 5mm in diameter (1). We therefore wanted to design a PET biodegradation device for current PET plastic recycling industry with the capacity to degrade recycled PET plastic into chemical monomers, thus reducing PET plastic waste exposure and newly formed microplastics. By demonstrating that PET degradation products can be transformed into high-value products, the economic returns to the communities concerned can be increased, creating a positive incentive cycle for plastic recycling.

How would it be used?

After designing the silica beads decorated with PETase and MHETase enzymes we wanted to create a device with these immobilised enzymes with enzymes such that they could be used easily by a recycling facility (Figure 2). Silica beads have completed their reaction with the PET, the solution is passed through a large pore size filter membrane to separate (enzymes and reaction products) from (large impurities, undegraded PET). The initially filtered solution is then passed through a smaller pore size membrane to separate the working enzyme from the reaction substrate (TPA & EG), this step also allows the working enzyme to be recovered for reuse. The isolated TPA and EG substrates can be chemically purified or specifically uptake by engineered organisms for downstream use.

Considerations

Due to the low solubility of the PET plastic and silica beads, the soluble end products (TPA and EG) can be separated from the insoluble substances relatively easily after the reaction. In other words, the insoluble substances in the device can settle at the bottom of the device while the end products remain soluble in the reaction buffer. Ideally, the technician could gently pour the liquid out, leaving the immobilised protein in the device. However, there may be PET plastics or impurities that cannot be degraded into monomers. And reusability is an important advantage of immobilised proteins, as our experiments have demonstrated, with the FAST-PET enzyme retaining more than 65% of its activity at the second use. Therefore, enzyme recovery after the reaction is an important issue. For this case we have designed a double filtration system that allows the separation of product and impurities and the recovery of the enzyme is made easy (Figure 3). Since the size of silica beads (Celite 545) ranges from 20-100 µm, the upper filter with pore size >100 µm can let the silica beads and soluble products pass. The impurity will leave on it. The second filter need to have a pore size smaller than 20 µm in order to separate the immobilized enzymes and soluble product. If the silica beads stuck the second filter in real practice, we can replace it with an impenetrable layer to let immobilized enzymes precipitate and collect the liquid containing final product.

We have taken many considerations into account already when designing our device, and thus it could be ready for implementation without many additional changes. These include:

1. The cell-free system avoids issues with toxicity to host cells, and long-term storage of cells
2. Cell-free systems are more predictable for industrial applications
3. There is an extremely small chance of genetic material leaving the industry, since there are no living cells or genetic material in the working device
4. The immobilised enzyme can easily be reused with filtration or centrifugation
5. Silica beads are cheap and eco-friendly, and can help industry reach their sustainable development goals or carbon neutralisation goals
6. Silica immobilisation is cheap and easy
7. Diatom biosilica exists as a renewable material: its production is driven entirely by photosynthesis under mild conditions (2).

Future challenges

To see if our proof of concept could actually be used in industry in real life, we would need to conduct more tests on the efficacy of our enzymes, develop our downstream TPA bioconversion pathway, and characterise usage in various conditions better.