Contribution of parts
- We made improvements on existed part reporter GFP（BBa_K3402050) so that it will be more suitable to express in our host Candida Tropicalis, which becomes our new part yeGFP:BBa_K3829002.
- We also filtered new promoter （P-GAPDH: BBa_K3829001）and terminator （T-GAPDH: BBa_K3829000）to express downstream gene effectively, and have added the data of them to the corresponding BioBricks.
- Figure 1: Composition of basic Bio-bricks.
- 1. yeGFP（BBa_K3829002）
- In our research, yeGFP was used as a reporter gene. In addition to showing whether the promoter can successfully start the transcription of downstream genes, it is also used to observe whether the anchor protein can display the fused genes on the cell surface. It is a useful tool for us to efficiently screen the best anchor protein.
- 2. P-GAPDH (BBa_K3829001)
- In our research, P-GAPDH was used to start the transcription of downstream genes. The experimental results showed that the selected P-GAPDH could effectively start the transcription of downstream genes.
- 3. T-GAPDH (BBa_K3829000)
- In our research, T-GAPDH was used to terminate the transcription of the target gene. The results showed that T-GAPDH could effectively terminate gene transcription in Candida tropicalis.
- 4. Surface Display System
- By screening ankyrin, we successfully constructed a cell surface display system platform to display the target protein, which is convenient to display other functional proteins in the later stage.
- Figure 2: Sketch of surface display system.
- 5. Through observation using scanning electron microscopy (SEM), the anchor protein expressed in the periplasm of yeGFP was successfully screened.
- Figure 3: Representative images of screened anchor proteins
- 6. Based on the above experiments, it was further determined that ankyrin could successfully display the protein on the outside of the cell by immunofluorescence test. The surface display system of Candida tropicalis was successfully constructed.
- Figure 4: Representative image of 4609 (further screened anchor protein).
Contribution of experimental operation
Contribution of manual of different medias for live broadcasting (with ASTWS-China)
- We have initiated and attended several online broadcasting events using different medias. Since most members are familiar with tencent and zoom, together with ASTWS-China, we would presented user guidance we found for other two live broadcasting platforms BILIBILI & 小鹅通. And we are open to questions if you would like some help from us.
- How to broadcast using bilibili:
- PC
- BILIBILI 1
- BILIBILI 2
- Mobile
- BILIBILI
- How to broadcast using 小鹅通:
- BILIBILI
- For more, could follow the wechat official account of 小鹅直播助手 （wechat account: gh_6b47eeda13f5)
Contribution of list of previous iGEM projects using PETase and MHETase (with ASTWS-China)
- In order to save the time of searching and sorting information for the new iGEMers. we reviewed the related projects over the years and summarized the successful projects results as follows. We hope this list can be supplemented continuously in the future by other iGEMers if they want. In summary, the previous iGEM teams are mainly committed to solving the problems related to PET degradation: 1) improving the degradation efficiency and 2) improving the thermal stability of PETase (2019 Exeter). 3) Improving the secretion efficiency of PETase if necessary (2018 Yale).
- 2021
- 2021 ASTWS-China: https://2021.igem.org/Team:ASTWS-China
- 2021 IvyMaker-China: https://2021.igem.org/Team:IvyMaker-China
- 2020
- 2020 ASTWS-China: https://2020.igem.org/Team:ASTWS-China
- 2020 KEYSTONE: https://2020.igem.org/Team:KEYSTONE
- 2020 TJUSLS_China: https://2020.igem.org/Team:TJUSLS_China
- 2019
- 2019 TU_Kaiserslautern: https://2019.igem.org/Team:TU_Kaiserslautern
- 2019 Exeter: https://2019.igem.org/Team:Exeter
- 2019 Toronto: https://2019.igem.org/Team:Toronto
- 2018
- 2018 UMaryland: https://2018.igem.org/Team:UMaryland
- 2018 Yale: https://2018.igem.org/Team:Yale
- 2017-2014
- 2017 Baltimore_Bio-Crew: https://2017.igem.org/Team:Baltimore_Bio-Crew
- 2016 Harvard_BioDesign: https://2016.igem.org/Team:Harvard_BioDesign
- 2016 TJUSLS China: https://2016.igem.org/Team:TJUSLS_China
- 2016 Tianjin: https://2016.igem.org/Team:Tianjin
- 2016 UESTC-China: https://2016.igem.org/Team:UESTC-China
- 2015 Berlin: https://2015.igem.org/Team:Berlin
- 2014 METU_Turkey: https://2014.igem.org/Team:METU_Turkey
Contribution of list of reference papers about PET degradation (with ASTWS-China)
- 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.
- 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.
- Cui Y, Chen Y, Liu X, Dong S, Tian YE, Qiao Y, Mitra R, Han J, Li C, Han X, Liu W. “Computational Redesign of A PETase for Plastic Biodegradation Under Ambient Condition by The GRAPE Strategy.” ACS Catalysis, Volume 11, issue 3, 13 Jan 2021, pp.1340-1350.
- Harry P Austin. et al. “Characterization and Engineering of A Plastic- degrading Aromatic Polyesterase.” Proc Natl Acad Sci USA, volume 115, issue 19, 2018, pp. E4350-E4357.
- Ikuo Taniguchi. et al. “Biodegradation of PET: Current Status and Application Aspects.” ACS Catal, volume 9, 2019, pp. 4089−4105.
- Joo S. et al. “Structural Insight into Molecular Mechanism of Poly (Ethylene Terephthalate) Degradation.”Nature News, volume 9, issue 1, 2018, pp. 382.
- Kawai F, Kawabata T, Oda M. “Current Knowledge on Enzymatic PET Degradation and Its Possible Application to Waste Stream Management and Other Fields.” Appl Microbiol Biotechnol, volume 103. issue 11, Jun 2019, pp. 4253-4268.
- Smith MR, Khera E, Wen F. “ Engineering Novel and Improved Biocatalysts by Cell Surface Display.” Ind Eng Chem Res, volume 53, issue 16, 29 April 2015, pp. 4021-4032.
- Tanaka T, Yamada R, Ogino C, Kondo A. “Recent Developments in Yeast Cell Surface Display toward Extended Applications in Biotechnology.” Appl Microbiol Biotechnol, volume 75, issue 3, August 2012, pp. 577-591.
- V Tournier. et al. “An Engineered PET Depolymerase to Break Down and Recycle Plastic Bottles.” Nature, volume 580, issue 7802, 2020, pp.216- 219.
- Xu Han. et al. “Structural Insight into Catalytic Mechanism of PET Hydrolase.”Nature News, volume 8, issue 1, 2017, pp.2106.
- Yuan Ma. et al. “Enhanced Poly(ethylene terephthalate) Hydrolase Activity by Protein Engineering.” Engineering, 2018, pp. 888-893.
Contribution of list of laws and regulations about PET recycling （with ASTWS-China）
- 1. Whether the plastics sold on the market will be marked if using raw materials or recycled plastics, relevant regulations (non mandatory):
- 1.1 《废塑料回收与再生利用污染控制技术规范》 (Technical Specification for Pollution Control of Waste Plastics Recycling and Reuse) HJ / T 364-2007: recycled products shall be marked with recycling marks.
- 1.2 《塑料制品易回收易再生设计评价通则》(General Rules for Design Evaluation of Easily Recyclable and Recyclable Plastic Products) 2021: the "return" mark is officially launched to represent easily recyclable and recyclable plastic products.
- 1.3 《塑料、再生塑料》(Plastics and Recycled Plastics) GB / T 40006-2021 specifies the marking method for recycled plastic particles: for example, high density polyethylene (PE-HD) recycled plastics (REC), blue (B2), cylindrical (c), without filler, for extrusion of sheet (E), MFR 190 / 2.16 (d) is 0.38g/10min (010), density 950kg / m3 (95): name GB / T 40006.1-pe-hd (REC) - b2-c-1, E,D010-95PCR (post-consumer recycled) ISO 14021:2016
- 1.4 《RCS回收声明标准》（RCS Recovery Statement Standard): it is the recovery standard launched by the textile trading organization in 2013. This standard includes recycled materials with a content of at least 5% in all products processed, manufactured, packaged, labeled, sold and used.
- 1.5 (Global Recycling Standard (GRS)): an international, voluntary and comprehensive product standard that specifies third-party certification requirements for recycling content, production and marketing chain of custody, social and environmental practices and chemical restrictions. The proportion of recycled components in the product should be greater than 20%; If the product plans to hang GRS logo, the proportion of recycled components shall be greater than 50%.
- 2. What obstacles will the company encounter if it wants to establish treatment facilities in waste recycling stations or factories, and what factors will the company consider:
- 2.1 due diligence of Free to Operation (FTO): analysis and investigation on whether there is any risk of infringing the patent right of a third party when using, producing or selling products containing the Licensor's patent in the licensed area
- 2.2 environmental impact assessment (engineering analysis, environmental risk assessment, pollution control measures, economic profit and loss analysis, environmental management and testing), environmental protection facility design, completion environmental protection acceptance, pollutant discharge permit, cleaner production audit, safety supervision and fire control
- 2.3 Relevant laws and local policies and regulations to follow (examples)
- 《中华人民共和国环境保护法》(Environmental Protection Law of The People's Republic of China)
- 《中华人民共和国水污染防治法》( Law of The People's Republic of China on The Prevention and Control of Water Pollution)
- 《中华人民共和国大气污染防治法》(Law of The People's Republic of China on The Prevention and Control of Air Pollution)
- 《中华人民共和国噪声污染防治法》(Law of The People's Republic of China on The Prevention and Control of Noise Pollution)
- 《中华人民共和国固体污染环境防治法》(Law of The People's Republic of China on The Prevention and Control of Solid Pollution)
- 《中华人民共和国固体污染环境防治法》(Law of the People's Republic of China on the Promotion of Cleaner Production>)
- 2.4 Technical Specifications to follow (examples):
- 《废塑料回收与再生利用污染控制技术规范》(Technical Specification for Pollution Control of Waste Plastics Recycling and Reuse) HJ / T 364-2007: new plastic recycling projects shall not be built in urban residential areas, commercial areas and other environmentally sensitive areas. The site selection and subsequent construction of recycled plastic projects shall be with fences, and the plant area shall be divided according to functions, including management area, raw material area, product storage area and pollution control area. Measures such as wind, rain, seepage and fire prevention shall be taken.
- 《废塑料综合利用行业规范条件》(Industrial Standard Conditions for Comprehensive Utilization of Waste Plastics) and 《废塑料综合利用行业规范条件公告管理暂行办法》(Interim Measures for The Administration of Announcement of Industrial Standard Conditions for Comprehensive Utilization of Waste Plastics) (2015 No. 81): the comprehensive new water consumption of PET recycled bottle chip enterprises and waste plastic crushing, cleaning and sorting enterprises should be less than 1.5 tons per ton of waste plastics, and the comprehensive new water consumption of plastic recycled granulation enterprises should be less than 0.2 tons per ton of waste plastics.
- 《废塑料污染控制技术规范(征求意见稿) 》(Technical Specification for Pollution Control of Waste Plastics (Exposure Draft)) DB13 / T 5361-2021: production enterprises need to enter the industrial park for unified sewage treatment. Unprocessed pyrolysis oil and processed products that do not meet relevant product quality standards shall be managed as hazardous waste.
- 《废塑料再生利用技术规范》 (Technical Specification for Recycling of Waste Plastics) (GB / T 37821-2019) stipulates that recycled plastics must meet the following standards:
- GB 8978 integrated wastewater discharge standard
- GB 12348 emission standard for environmental noise at boundary of industrial enterprises
- GB 14554 emission standard for odor pollutants
- GB 16297 integrated emission standard of air pollutants
- GB 18599 pollution control standard for general industrial waste storage and disposal sites
- GB 31572 emission standard of pollutants for synthetic resin industry
