Welcome to iGEM 2021
Environmental protection has always been one of the hot topics of the iGEM project, and the plastic topic is even more popular. A lot of work experience could be shared with the future iGEM team. The contribution work we have done this year mainly includes adding new data to existing parts BBa_K3039004, sharing the modeling method of the Michaelis-Menten equation, and lists of references and materials related to PETase and MHETase.
Add new characterization data to existing part (BBa_K3039004)
This year we developed an enhanced “plastic eating” biofilm system, including strengthen biofilm, PETase and MHETase. The basic part of MHETase expression is BBa_K3039004 which constructed by 2019 Exeter. It was said that the modified MHETase expression part was unable to express enough to characterize the enzyme. Thus, the characterization data of MHETase protein expression was added to this part registry page. We found that it needs longer time to express MHETase protein in E. Coli. Please click the part number BBa_K3039004 for more details.
Introduction and Experience about Michaelis-Menten equation
The classical Michaelis-Menten equation is a basic equation of enzyme kinetics. Analyzing the derivation of this equation yields the fact that its good performance of approximating real reaction processes is due to the Michaelis-Menten curve. Such a curve is derived from Quasi-Steady- State Assumption (QSSA), which has been proved always true and called Quasi-Steady-State Law introduced by Banghe Li et al . It was reported that a polynomial equation was found that gives a more accurate approximation of the reaction process in two aspects: during the quasi-steady-state of the reaction, Michaelis–Menten curve approximates the reaction well, while this equation gives a better approximation; near the end of the reaction, this equation approaches the end of the reaction with a tangent line the same to that of the reaction process trajectory simulated by mass action, while Michaelis-Menten curve does not. In addition, our equation differs from the Michaelis-Menten curve less than the order of 1/S3 as S approaches +∞, where S is the substrate concentration.
If the iGEM team in the future needs to use a more reasonable model to analyze the characteristics of the enzyme kinetic model, you can refer to it.
 Banghe Li et al. Quasi-steady state laws in enzyme kinetics, J. Phys. Chem. A 112(11) (2008) 2311-2321.
Lists of References and Materials Related to PETase and MHETase
List of previous iGEM projects using PETase and MHETase (with IvyMaker-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 ASTWS-China: https://2021.igem.org/Team:ASTWS-China
2021 IvyMaker-China: https://2021.igem.org/Team: IvyMaker-China
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 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 UMaryland: https://2018.igem.org/Team:UMaryland
2018 Yale: https://2018.igem.org/Team:Yale
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
List of reference papers about PET degradation (with IvyMaker-China)
Improving the degradation efficiency
Xu Han. et al. Structural insight into catalytic mechanism of PET hydrolase. Nat Commun. 2017;8(1):2106.
Yuan Ma. et al. Enhanced Poly(ethylene terephthalate) Hydrolase Activity by Protein Engineering. Engineering. 2018;888-893.
Harry P. et al. Characterization and engineering of a plastic-degrading aromatic polyesterase. PNAs. 2018; 115(19): E4350–E4357
V Tournier. et al. An engineered PET depolymerase to break down and recycle plastic bottles. Nature. 2020;580(7802):216-219.
Joo S. et al. Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nat Commun. 2018;9(1):382.
Harry P Austin. et al. Characterization and engineering of a plastic-degrading aromatic polyesterase. Proc Natl Acad Sci USA. 2018;115(19):E4350-E4357.
Taniguchi I. et al. Biodegradation of PET: current status and application aspects. ACS Catal. 2019;9:4089–4105.
Improving thermal stability of PETase
Ikuo Taniguchi. et al. Biodegradation of PET: Current Status and Application Aspects. ACS Catal. 2019;9:4089−4105.
Surface display of PETase
Smith MR, Khera E, Wen F. Engineering Novel and Improved Biocatalysts by Cell Surface Display. Ind Eng Chem Res. 2015 Apr 29;54(16):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. 2012 Aug;95(3):577-91.
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. 2020 Mar 20;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. 2018 Mar;102(6):2543-2561.
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. 2019 Jun;103(11):4253-4268.
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. 2021 Jan 13;11(3):1340-50.
Manual of different medias for live broadcasting (with IvyMaker-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:
How to broadcast using 小鹅通:
For more, could follow the wechat official account of 小鹅直播助手 （wechat account: gh_6b47eeda13f5)
List of laws and regulations about PET recycling (with IvyMaker-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)
n 《中华人民共和国环境保护法》(<Environmental Protection Law of The People's Republic of China>)
n 《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>)
n 《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>)
n 《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>)
n 《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>)
n 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):
n 《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.
n 《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.
n 《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.
n 《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:
n GB 8978 integrated wastewater discharge standard
n GB 12348 emission standard for environmental noise at boundary of industrial enterprises
n GB 14554 emission standard for odor pollutants
n GB 16297 integrated emission standard of air pollutants
n GB 18599 pollution control standard for general industrial waste storage and disposal sites
n GB 31572 emission standard of pollutants for synthetic resin industry