To guarantee that the PET surface display system can degrade enzymes, we must first ensure a successful construction. On the C terminal, an anchor protein plays a vital role in determining the feasibility of the system. A prediction model was used to select the best anchor - out of the genome of Candida Tropicalis- for testing. In addition, a yeast-enhanced green fluorescent protein (yeGFP) was adopted as the reporter to screen effective display structures. On the N terminal, a signal peptide helps guide the system out towards the cell membrane. The successful surface display of PETase and MHETase demonstrated the practical value of the system. In the future, it can also expand its applications by displaying other proteins.
In addition, we also conducted interviews with a number of plastic production companies to inquire about the possibility of our project implementation and aspects that can be improved. We hope that our projects can be put into practical application in the future to benefit mankind.
Prediction of anchor proteins
Firstly, we constructed a network prediction model to help us screen for GPI-anchored protein candidates from the genetic data of Candida Tropicalis.
According to the characteristics of anchor proteins, we successfully obtained 129 candidates with the prediction model. The principle was as follows:
Design of screening methods
To measure the GPI-anchor protein candidates' effect on the display, we chose the yeast enhanced green fluorescent protein (yeGFP: BBa_K3829002) which was codon-optimized to be the reporter. Furthermore, reduced glyceraldehyde-phosphate dehydrogenase promoter (P-GAPDH: BBa_K3829001) and reduced glyceraldehyde-phosphate dehydrogenase terminator (T-GAPDH: BBa_K3829000) were used to initiate and terminate transcription. In addition, GFP fluorescence fused with protein candidates at the C-terminal can be microscopely observed to check for the success of the system.
Composition of the genetic circuits. a) Gene circuit was constructed by P-GAPDH: BBa_K3829001, yeGFP: BBa_K3829002 and T-GAPDH: BBa_K3829000, which was to express the fused GFP-anchored proteins in the cytoplasm. b) Gene circuit was constructed by P-GAPDH: BBa_K3829001, yeGFP: BBa_K3829002 and T-GAPDH: BBa_K3829000, which was to express the fused signal peptide-GFI-anchored proteins on the cell membrane surface.
Results
Negative control was the uracil-deficient Candida tropicalis (URA3-KO). Positive control introduced the gene of GFP-anchored protein fusion protein compared to NC. The green fluorescence was able to be observed with laser confocal microscopy, to indicate that the three parts of the system could be effectively translated. On the other hand, since the signal peptide was absent, the fluorescence was diffusely distributed in the cytoplasm.
In the experimental group, fluorescence was distributed on the surface of the cell membrane. The signal peptides on the N terminal of GFP-anchor protein fusion protein helped guide the protein to the cell membrane. Furthermore. the C-terminal GPI site of the anchor protein can be modified to anchor it to the cell membrane, thereby presenting the effect of displaying the protein on the surface. By observing the strains containing different anchor proteins one by one, we successfully screened 25 anchor proteins that could display GFP around the cells, among which the results of 4609 and 5105 were shown.
Representative images of other strains that can successfully display anchor proteins around cells.
Validation of surface display systems
To further confirm that the protein was successfully displayed on the cell surface, we used an indirect immunofluorescence method. The schematic diagram of indirect immunofluorescence method was as follows:
Project Feasibility Evaluation by Plastic Production Companies
DuPont is a global enterprise based on scientific research. It provides scientific solutions that can improve the quality of human life in the fields of food and nutrition, health care, clothing, home and construction, electronics and transportation. The engineering plastics produced by the company occupy an important position in the field. After our interview with Dr. Wang of DuPont, he also put forward some constructive opinions and expectations for our project, and felt that the development prospect of our project was good.
The physical method has been operated with low recovery cost, but many plastic materials can not be recycled, and the performance of plastic recycled for many times will be reduced, and it is not suitable for plastic products with high safety requirements; Although the chemical method can recover the plastics that cannot be treated by the physical method, the operating conditions are harsh and there is wastewater pollution. For the biodegradable PET plastic project developed by us, the other party has put forward biosafety problems and equipment construction requirements. If these problems can be solved and the current degradation efficiency can be improved, it will be a very promising project.
Similarly, in our exchange interview with Lanzhou JIN TU DI plastic products Co., Ltd. (Jin Tu DI), the other party also raised questions about the safety and equipment requirements of our project. Although our project is still in the lab stage, we have made full assumptions for its practical application.
With regard to biosafety, we have described in the safety section and Wiki implementation section of "wiki result". The uracil deficient Candida tropicalis used in the project is safe and controllable.
As for the equipment construction requirements, we have also made corresponding explanations in "wiki implementation", and designed a sketch of equipment construction of large waste recycling station, hoping to be applied in our projects in the future.
Results
Green fluorescence was detected in 9 of the anchored proteins with confocal laser microscopy.
References
Chen Z, Wang Y, Cheng Y, Wang X, Tong S, Yang H, Wang Z. “Efficient Biodegradation of Highly Crystallized Polyethylene Terephthalate through Cell Surface Display of Bacterial PETase.” Sci Total Environ, 20 Mar 2020, 709:136138.
Andreu C, Del Olmo ML. “Yeast Arming Systems: pros and cons of different protein anchors and other elements required for display.” Appl Microbiol Biotechnol, volume 102, issue 6, Mar 2018, pp. 2543-2561.
Eisenhaber, Birgit, et al. "A sensitive predictor for potential GPI lipid modification sites in fungal protein sequences and its application to genome-wide studies for Aspergillus nidulans, Candida albicans Neurospora crassa, Saccharomyces cerevisiae and Schizosaccharomyces pombe." Journal of molecular biology 337.2 (2004): 243-253.
Möller, Steffen, Michael DR Croning, and Rolf Apweiler. "Evaluation of methods for the prediction of membrane spanning regions." Bioinformatics 17.7 (2001): 646-653.
