In order to verify the function of our project, we first need to construct the expression vector. In this module, we designed a whole set of workflows to construct the plasmid and verify our engineering success. Firstly, we synthesized and purchased the genes of interest and vector from the company. After amplifying the vector and sequence, homologous recombination or restriction enzyme digestion colony was used to connect our target gene sequences and plasmids backbone, and then the constructed vector was transferred to the chassis. Finally, colony PCR was used to verify the successful construction and transfer of the vector.

These constructed parts make up the modules of our project. In the degradation module, we planned to use co-incubation to verify the effect of PEBP on enhancing the microplastic binding ability of bacteria. Microplastic degradation is the main goal of the project, so weighing, Fourier transform infrared spectroscopy and scanning electron microscopy were used to verify the degradation effect of engineered bacteria microplastics to ensure the accuracy of our verification. The engineered bacteria need to transfer electrons and carbon dioxide to translate the carbon dioxide to cellulose. In order to transfer electrons, we added pilA and nqrf parts to engineering bacteria and verified them using MFC methods. For individual pilA parts, we verified their ability to increase flagellar length using silver dyeing method. And for nqrf, we verified its ability to reduce NADH content using NADH kit. In the carbon dioxide transfer module, we designed the carbonic anhydrase parts of Pseudomonas.aeruginosa and Rhodopseudomonas.palustris respectively, and planned to use bicarbonate ion indicators to test the ability of engineered bacteria to transfer carbon dioxide. Ultimately, we hypothesized that our engineered R.palustris could fix carbon dioxide into cellulose, so we used Fourier infrared spectroscopy, Congo red staining, and other methods to verify cellulose production. The safety module is designed to enable the bacteria to initiate suicide under certain conditions while preserving the plasmid, and the hok/sok system is used in this module. Antitoxin Sok is activated with a citrate promoter. Therefore, the citrate promoter followed by a GFP protein is used to verify the induction effect of citrate. And the anti-plasmid loss efficacy is verified with antibiotics. Ultimately, we wanted this system to be a co-culture system, so the co-culture growth curve was tested to verify the feasibility of co-culture.

Eventually our engineering products need to be put into practical use. Since the wet experiment has not been able to carry out this step, we have used many modeling methods to verify the practical application feasibility of the project, including Functional three-electrode system, Tidal dynamic simulation Mangrove soil sampling and so on. In practical applications, being able to monitor the amount of PE microplastics in the soil sample on the spot crucial, so we constructed hardware to try to quickly detect the amount of microplastics. Construction of the hardware using fluorescence microscopy and detection of contact angles.