According to our human practice and research of bibliography, we find out that the main problems causing traditional biological methods to be inefficient are their disperse concentration, ineffective enzyme and high mortality of bacteria. Our project successfully improve the biological method of treating sewage in all these three aspects.

Our project mainly consist with two parts: CsgA + Spytag and CotA laccase + Spycatcher. These two parts will form a biofilm that provide shelter and for the bacteria and help them share nutrition. At the same time, this design will increase the concentration of laccase. Our experiments sufficiently demonstrate the feasibility of this prosperous project.

The combination of our parts is generally satisfactory in efficiency. We choose B. subtilis to produce laccase, which is efficient multi-copper oxidase. This enzyme has robust properties and is widely applied in industrial fields that required harsh treatment conditions (Tian-Nyu Wang, 2016).

We transfer CotA laccase genome into E.coli. In order to produce laccase extracellularly, our team also fuse signal peptide into the genome. Afterward, we measure the enzyme activity of our designed part, and our engineering turn out to be effective.

We have tried three type of signal peptide, PelB is the most effective one

This signal peptide could improve the enzyme activity by more than 100%

In order to demonstrate that our Spytag could successfully bind with Spycatcher, we add the gene of GFP into the genome and compare the fluorescence of the biofilm between two groups: one contains Spycatcher, while the other group are not. After we separate biofilm from supernatant, we find out that the fluorescence in the liquid of the Spycatcher added group is significantly lower than the Spycatcher absent one. The result shows that most Spytag+CsgA and Spycatcher+GFP are bond onto the biofilm, and this is removed from the liquid, suggesting that the Spycatcher and Spytag could firmly connect with each other, and our bacteria could therefore be suspended on the biofilm.

Also, we do further experiments to prove that the connection of Spycatcher and Spytag could largely improve the enzyme activity. We find that, comparing the mixture of the Spycatcher-CotA + inactive biofilm, Spycatcher-CotA + CsgA-Spytag, CsgA-Spytag + CotA(wild type), and inactive biofilm + CotA(wild type), there is a striking difference of enzyme activity between the mixture our engineered parts and others. This result indicates that our part, Spcatcher-CotA + CsgA-Spytag, could undoubtedly raise the effectiveness of CotA.

To maximize the CotA-biofilm contact area, our design is to apply the biofilm onto tiny beads, as shown in our applied design section. Most of the industrial biofilm carriers are made of ABS or traditional plastic materials; whereas most of them are not biological degradable, and some of them contain tiny toxic substances. Our project use polyhydroxyalkanoates(PHA) as our biofilm carriers. PHA is a eco-friendly material, and it could be produced by crops and kitchen waste. After PHA is discarded into the wild, this material could be degraded by soil within a few month, and the byproducts contain no biohazard substance(Bluepha, 2018). Moreover, an outstanding characteristic of PHA is that it could supply nutrition for the adhering bacterias. In this way, the duration and expectancy of bacterias could be dramatically improved.

To test the adhesivity of PHA beads, we soak them into the bacteria solution

Our team also design a water-filter. This water-filter, as shown in the blueprint, could contain the beads in the central cavity. The inlet and outlet of the filter is connected with the water pump, manometer, which could adjust the flow rate and hydraulic pressure and reach an optimal processing efficiency as we discover in the modeling section.

In summary, we have shown that both of our approaches, using either CotA laccase or biofilm as well as the hardware design, are successful at treating waste water.