Design

In order to decompose nonylphenol and Bisphenol-A which is a kind of EDC, a type of chemical that can disturb our reproductive system's normal function, the use of protein is definitely one of the best choices. But among these many kinds of proteins, which one do we need to solve the problem? After looking through some paper, laccases, which can be found in many different species of plants and fungi is what we have been looking for.

Laccase 1326

But there is an issue we have to settle first before we can make our plan work. As you know, proteins can only work at their full efficiency when they are placed in a straightly regulated environment. However, the polluted water in the sewage treatment plant or polluted water in general where EDC is usually located is somewhat unsuitable for the majority of them to work. So, we decided to pick one of the variants of laccases (Laccase 1326^[1]) discovered in 2018 which can endure high temperatures (the enzymes display more than 50% of its maximal activity at 20–70°C, with the max enzymatic activity at 60°C), salty environment and work just fine in some more moderate environments as our main focus in this project.

To improve the downstream processing

E.coli is a commonly used host for synthetic biology, plus most of its pathways are fully understood, therefore, the chance of having accidents is low. We decided to used it to kickstart our project.

In this case, we are using a pET vector to carry our gene into E.coli. This is because it has a strong promoter(T7) and a lot of common restriction sites which sometimes can be very convenient to work with. Furthermore, this vector has a medium copy number, so it will not give too much stress to the cell and negatively affect it.

It is clearly expensive and unsustainable to kill the bacteria and extract the laccase, therefore, we would like to apply a secretion system to E.coli. Secreting the protein has been a method used by experts to let the protein works more efficiently for many years. As EDC degradation is limited to the rate of bacteria EDC uptake, the efficiency of laccase may decrease because of it. Through a recommendation from the team of the University of Macau, we learned about NSP4, which is a secretion peptide that fits our design. Although it is a newly discovered single peptide, researchers have been using it for their research for a while, so we believe that using this secretion peptide can lower our risk of failure drastically.

Maximizing productivity in outdoor environment

On the other hand, the selection of the host of our genes has also been carefully determined. Instead of using E. Coli, we think that Synechocystis sp PCC 6803, which is a kind of cyanobacteria, would be a better option. This decision was to maximize productivity in an outdoor environment, targeting EDC issue in nature.

Cyanobacteria use photosynthesis in order to produce energy for itself, in addition to that, extra energy can be harvested for other purposes. Therefore, as a renewable and sustainable microbial "cell factory", cyanobacteria can be used to produce a variety of chemicals. In the past few years, the industry and researchers have made great efforts to adjust metabolic pathways and fluxes to optimize target production.

Why we chose Synechocystis sp PCC 6803

When purifying sewage, we need to know whether the sewage will have substances that can affect our host, for example, zinc pollution. According to previous studies, some cyanobacteria can be affected by organic chemicals or heavy metal contamination, delaying their growth or causing death. In addition, parts of the phytochemicals involved in photosynthesis of biological or chemical substances also bear the possibility of being affected by these pollutants.^[2]

As it is an autotroph, PCC 6803 can support itself and produce a fair amount of protein that we want through just photosynthesis, which can save us a lot of effort otherwise needed to keep them alive. Moreover, this species of cyanobacteria have an advantage that others do not. Zinc, which is rich in polluted waters, is poisonous to cyanobacteria when it is in high concentration. But in the case of PCC 6803, however, giving it enough light will prevent it from taking in too much zinc, protecting it from being poisoned.^[3]

Substances like insecticidal are commonly found in the sewage in different areas. Previous studies indicated that high concentrations of insecticidal acetamiprid can reduce photosynthesis efficiency of Synechocystis FACHB898 by inhibiting electron transportation in their reactions, but the effects are limited.

Laccase Secretion in Cyanobacteria

Secreting the protein has been a method used by experts to let the protein works more efficiently for many years. As the EDC degradation is limited by the rate of the bacteria's EDC uptake, the efficiency of the laccase may be limited because of it. So in order to let laccase 1326 work at its full efficiency, we decided to add pilA1^[4]—a more understood signal peptide found in cyanobacteria—into our vector to act as a signal to send the protein out of our cell.

pPMQAK1, which is widely used for research in cyanobacteria, is a good candidate for our project. However, a better vector called—pSCB-YFP—has been made in 2018. Not only does it have YFP on it, but it can also coexist with pPMQAK1. Therefore, we have decided to use pSCB-YFP instead of pPMQAK1 as our vector.^[5]

Machine

If we only have a bunch of engineered cells in our hands is obviously not enough to effectively get rid of all the EDCs in the water. So in order to utilize our engineered system, we need a property designed machine in a Sewage treatment plant. For more information, please visit Hardware

Team:PuiChing Macau/Design