Solving a problem generally requires three steps: discover the problem, analyze the problem, and deal with
the problem in an appropriate way. In each step, problems arising from the connection between theory and
reality will be encountered. The road of scientific research is not smooth, but we can never forget our
source and the value of final application promotion in this journey. We answered the core questions of the
team project, including environmental friendliness and responsibility, social technological innovation,
social tolerance, and scientific feasibility. We must ensure that our projects can provide certain value to
the world and society and can be used for reference in human life and even the future.
How can we gather a holistic perspective about the public responses and holding the social
investigation?
Since we want to deal with the microplastics in the environment, the most accumulated microplastics are still
in the downstream sewage treatment and other links. We contacted the Chunliuhe Sewage Treatment Plant for
interviews and investigations. As a more professional sewage monitoring and treatment institutions. In the
view of the treatment characteristics of sewage treatment plants, we mainly investigate the microplastic
composition of water waste in sewage treatment plants. In order to obtain more professional knowledge
guidance, we consulted with teachers whose research direction is similar to that of plastic degradation.
However, it was important to realize that there was a target question we need to solve, namely the
application of our innovation in the real world.
What kind of factors should be considered in the practice of project application?
We have to consider the effects of a number of factors in the process of environment application. Because our
study subjects are not single in the environment but co-existing with toxic substances, we must consider the
possible effects of the presence of other substances on enzyme activity and effects. The nature of the
degradation products and the interaction with the unknown environment are in urgent need of research and
in-depth understanding. We also need to consider the cost in order to apply the extensiveness in the
practical application. Material recovery and disposal should also be taken into account and we must ensure
that it can be recovered timely so that it doesn't impose a burden on the environment in case of the event
of unforeseen circumstances.
What other ethical considerations should we take into account in our project?
Having the strict technical requirements and standards in the production process, the products used in the
land will not produce microplastics are significantly important, which effectively avoiding affecting soil
natural-bacterial consortium populations. Different production companies will choose to add different raw
materials according to the cost, such as plasticizers. We wonder whether the company considers the
degradation of plastics when processing, and whether they have taken measures to minimize the environmental
burden that it may bring. The Basel Convention adopted not long ago made us wonder whether the relevant
staff consciously started to improve the regulations after discovering problems. We set out to interview the
Golden Land Company to understand the impact of plastics on planting soil and crop growth. Our team took a
stakeholder-focused approach for our project at every stage. To gain a better sense of the overall space and
get advice from industry professionals, we talked to several individuals at some of the famous plastic
processing technology companies in China such as JIN TU DI Group and Chunliu River Sewage Treatment Ltd. We
also talked to individuals in academia to understand some of the crucial variables that we had to consider
when designing and optimizing our genetic circuits.
From talking to these stakeholders, we came to realize that there was a broader picture here, namely the
deployment of our innovation in the real world. In designing our degradable microplastics system, we were
able to meet the requirements of both companies and sewage treatment plant, including environmental
friendliness, high efficiency and thoroughly degradation. The one factor that we were still unable to meet
was specific delivery method as the vector we used is Escherichia coli. One of our goals going forward will
be to further promote our technology to more vectors and meet this requirement.
How can we become better scientists? How to popularize and promote synthetic biology through education?
We need to communicate more with other colleagues and learn from each other to make progress. We formed New
Pollutants Alliance with Ocean University of China, Shanghai Jiaotong University, Nanjing Foreign Languages
School, and Zhejiang University. We organized an online workshop with the theme of New Pollutants together.
We invited teachers in the field of synthetic biology and ecotoxicology, enterprises in the field of rapid
inspection and students with relevant legal backgrounds to share and discuss online solutions to decrease
pollutants. We also co-planned with OUC-IGEM to write popular science articles on "new pollutants", focusing
on the hazards of antibiotics and microplastics to today's environment. In addition, we also exchanged and
promoted projects with students from 18 schools including Tongji University, Beijing Institute of Technology
and Tianjin University who participated in the iGEM competition.
Our team knew that brilliant idea couldn't come up alone: it is only through communications and discussions
from co-teams that we are able to be inspired and move forward. As a result, we completed our core
design-Hydrophobin from the sharing meeting with China Pharmaceutical University in May. That's also one of
the main spirits of iGEM.
What are the central values of our project to society and life?
There are currently 4.9 billion tons of plastic waste. Only 9% of the world's plastics are recycled, and the
rest of the garbage ends up in landfills or scattered in our land and oceans. If we continue or the current
plastic consumption continues to grow, by 2050, there will be 12 billion tons of plastic waste. The shocking
thing is that 91% of all plastics are disposable. Since plastics became common use about 60 years ago, the
speed of mass production has resulted in 8.3 billion metric tons of plastic pollution. Although people are
becoming more aware of its adverse effects on the environment, it will still double in the next 20 years.
Putting all these facts together, it is clear that we are in the gloomy depths of the global plastic crisis.
This problem is so serious that the United Nations has identified single-use plastics as one of the biggest
environmental challenges in the modern world.
Vladimir Moshkalo, the head of the United Nations Environment Programme (UNEP) in the Russian Federation,
said:"We must fundamentally rethink the way we produce and consume. If these trends continue, by 2050,
there
will be more plastic in the ocean than fish. A sustainable future requires cooperation from all levels of
society", he said.
Based on what we investigated, we designed our program to solve this urgent and serious problem in the
world.
More important information is provided in the Integrated Human Practice part.
Integrated Human Practices
1 Background
Synthetic polymers pervade all aspects of modern life, due to their low cost, high durability, and
impressive range of tunability. Originally developed to avoid the use of animal-based products, plastics
have now become so widespread that their leakage into the biosphere and accumulation in landfills is
creating a global-scale environmental crisis. Currently, large amounts of plastic debris are entering
landfills or are left as waste accumulated in natural world, and it's a public emergency to prevent its
release and accumulation in the environment via appropriate biological, chemical, and physical
treatments. Among them, wide spreading and utilization of plastic polyethylene terephthalate (PET) in
the world has caused a large quantity of environmental challenges and gained much attention. PET is one
of the major plastics, in addition to polyethylene, polypropylene, polystyrene, polyvinyl chloride, and
polyurethane, and its worldwide production amounted to more than 106 million tons in 2020. Synthetic
plastic polymers Polyethylene terephthalate (PET) is one of the most widely used forms of synthetic
plastics in several industrial fields, which has been reported spreading as particles less than 5 mm in
environments and being captured from environments with low efficiencies. Furthermore, in addition to the
microplastics found in the environment, PET is frequently used in household products, which is severely
accumulated in the landfill and even worse, natural environments. Thus, plastic waste may have a
negative effect on human health and reveal a serious threatening to environments.
In August 2021, the United Nations Biodiversity Organization released the document "The global plastic
crisis is imminent". More than 180 countries and regions passed the plastic waste amendment of the Basel
Convention, calling on human beings to minimize the generation of plastic waste and carry out
environmentally sound management.
In China, as one of the world's top 10 producers and consumers of plastic products, Chinese natural
environment and city are faced with severe plastic pollution and high recycling pressure. In 2020, China
produced 70.302 million tons of plastics and imported nearly 450 thousand tons. The total consumption of
plastics in China in 2019 was about 90.877 million tons, of which millions of tons were used for
packaging. Most of the plastic used for packaging is discarded in the environment in the form of waste
film, plastic bags and cutlery. In terms of consumption level, the consumption of bottle-grade PET in
China continues to grow from 6.33 million tons in 2016 to 9.49 million tons in 2020. In 2020, the total
consumption of PET bottles in China reached 9.49 million tons. Although the export volume decreased to
2.34 million tons due to the epidemic in 2020, accounting for 24.7% of the total consumption, the
domestic demand continued to expand, reaching 7.15 million tons. All these demands and consumptions have
caused severe challenges in environment, especially for over accumulation of frequently used plastic
form, PET, in biosphere.
2 What can we do?
Plastics pollution represents a global environmental crisis. In response, microbe Ideonella
sakaiensis
was evolved corresponding mechanism that is capable of secreting two efficient enzymes to deconstruct
PET polymers in mild temperature. However, this two-enzyme system degradation capacity is highly limited
by inhibition effects, diffusion of intermediates and PET surface physicochemical properties. According
to previously reported experimental results, the direct linking combination of these two different
enzymes effectively improved the reaction rate. However, this new strategy is still limited by enzymatic
activity loss caused by direct linking method and low expression ratio. Simultaneously, the hydrophobin
protein is discovered to be capable of attaching to plastic surface via its hydrophobic part and expose
its hydrophilic part to aqueous environment. This unique feature may effectively make enzymes more
accessible to hydrophobic surface of PET. Therefore, inspired by nature, we designed a delicate
multicomplex enzyme system, in which short peptide tags (Redesigned RIAD and RIDD derived from natural
proteins) are applied to create scaffold-free modular enzymes assemblies. Here, in order to effectively
degrade microplastic PET particles, we construct enzymes of IsPETase and MHETase and protein
hydrophobin
together in our complex system via scaffold modular part, which may reveal higher catalytic efficiency
in mild temperature and avoid high possible loss of enzymes activity. Therefore, we created an
innovative strategy to improve PET degradation at mild temperature via biosynthetic factories and
artificially designed proteins system that do not exist in the nature.
3 Overview
While completing our project design and experimental parts, our ultimate goal is to put scientific
research results into real practice. We've been thinking, what is the significance of our project? What
problems can be solved? What kind of value can be created? Are there any risks? As a team, we always
believe that it is vital to have a lasting impact on others and society through our project. This is why
we try our best to understand the needs of stakeholders in the process of promoting the project, and we
will consult the implementation of bio-based enzymes degradation feedback from experts, doctors,
manufacturers and the public to promote our project, while ensuring that our project is biosafe,
responsible, feasible and sustainable. In the following content, you will learn about our progress,
thinking process and how we integrate expert opinions.
4 Expert interviews
The first stage
At this stage, we interviewed many relevant stakeholders regarding the plastic including PET pollution in
China, the problems and challenges faced at current stage. To better understand the situation of PET
accumulation with the corresponding impact on the economy, society and environment. In this way, we can
understand our problems from multiple angles and think about how our projects can better affect
society.
Name: JIN TU DI Group
JIN TU DI Group is a famous company that dedicated to the products of agricultural film, plastic
products, plastic plates, tubes and waste plastic recycling processing. It is the largest agricultural
plastic industry company in the northwestern part of China. It is fortunate for us to communicate with
this company's chief manager Mrs. Sun and her student Qi Liu about the current industry situation
related to plastic and corresponding national manage laws. Company officials told us that the type of
plastic is mainly PE and PET material, in which the current PE material plastic degradation method
invented by JIN TU DI Group has become matured. However, PET recovery rate is only around 10%, and PET
common recycling technology involves high temperature and high pressure, strong acid and alkali and
other issues, is likely to cause further pollution. According to JIN TU DI Group market investigation,
PET degradation at mild temperature especially in agricultural land is still a great challenge. Plastics
caused by agricultural plastics cannot be effectively recycled and be buried in the agricultural land,
resulting severe soil fertility damage. Soil contamination makes it impossible to grow the ideal crops
and the cost is huge. Although government has made corresponding laws to limit plastic using and
spreading in relevant fields, the effects are still limited. The PET degradation at mild temperature
still needs to be improved.
Take away: Making sure that PET degradation at mild temperature is a truly emergency and
meaningful
direction and our project focus point should be dedicated to solve this tough problem among other
plastic pollutions problems. The key points for solving current challenges in plastic pollution mainly
lies in mild temperature degradation, effective degradation rate and good performances in its endurances
for long period using and degradation, which would be our project designing core. Although China has
issued relevant strict policies on controlling plastic pollution, the current law on managing PET and
other material is still at early stage and not consummated. Thus, new method for accelerating PET
plastic at mild condition is needed and more comprehensive and mature Legislative Proposals are
necessary.
Name: Kaiserslautern University 2019 iGEM team
We communicate with iGEM alumni from Kaiserslautern 2019, Germany, who is also dedicated to focus on the
PET pollution threatening. Through this online communication, we obtain an overview situation of plastic
degradation and current limitations, which significantly function in our final project formation. Their
work mainly related to revolutionizing plastic degradation by introducing Chlamydomonas
reinhardtii as a
eukaryotic secretion platform, which effectively secret two degradation enzymes and adhere to
microplastic surface so that PET can be continuously exposed to enzymes degradation. Our online meeting
fully discussed all previous efforts in this field and possible future strategies for further
accelerated PET decomposing in environment. Through this discussion, we have also discovered the
limitation of current PET degradation: 1. High temperature that may not be applicable to current mild
enzymes like IsPETase. 2. Newly discovered enzymes with prefect performance at high temperature
may not
be able to convert accumulated MHET into final monomers TPA that possess economical values.
3.Hydrophobic surface of PET makes it hard for enzymes effectively adhere to it. Therefore, after this
communication, we recognized that one of the core challenges in this field would be to degrade
accumulated PET at mild temperature with higher rate. Most PET accumulated in environment can only be
degraded at room temperature and involvement of industrial method at high temperature may cause the
further pollution to natural environment. Although at this stage, our team was experiencing the
brainstorm section and the new possible ideal was still being formed.
Take away: Recognizing one of the core challenges is degrade PET effectively at mild temperature
because
most plastic accumulated in environment can only be degraded at room temperature. With their shearing of
PET-related fields information, we further understand the current stage of PET degradation key
limitations and decide to apply IsPETase discovered in 2016 rather than LCC enzymes reported in
2020 to
our project because IsPETase is potentially applicable to degrade PET at mild temperature and
combined
with MHETase to accelerate reaction rate.
Name: Chunliu River Sewage Treatment Ltd
Dalian Chunliu River Sewage Treatment Ltd was built in 1982 and put into use in 1986. It is the earliest
urban secondary sewage treatment plant built in the northeastern part of China. In 2017, the second
phase of the Dalian Chunliuhe River Sewage Treatment Ltd was upgraded with advanced sewage treatment
technology. The plant mainly uses corresponding physical, chemical and biological methods for sewage
treatment and purification. Our communications focus on sewage treatment processes and industry
standards related to wastewater waste, and in particular, we are more concerned about the industry
standards for plastics in wastewater treatment plants, but staff revealed that they have never known any
knowledge related to microplastic particles, and after our introduction, they said that existing
processes cannot remove plastics debris from water because relevant equipment was not initially
considered to remove accumulated plastic particles in water. Out of our expectations, even professional
workers didn't pay much attention to plastic processing in waste water. This means that plastics in
sewage are often overlooked, and whether they are effective in removing plastics from sewage is often
overlooked. Since many research teams around the world have detected the existence of plastics in many
types of water including the waste water processing system. However, the possible existence of plastic
in Chunliu River Sewage Treatment Ltd has been ignored, which may cause threatening to humankind health
and environment. Current technology on degrading plastics including PET material is still limited and
further processing at high temperature may not be applicable due to the further pollution and
inconvenience. Thus, the effective detection and degradation of accumulated PET at room temperature is
urgent.
Take away: Conversations with sewage treatment plants have exposed the problem that terminal
treatment
plants do not pay much attention to PET accumulated in water. Through this conversation and follow-up
data verification, we knew that there is no uniform standard for PET processing in water purification,
in which the uniform Legislative Proposals are needed. Therefore, this report directly encouraged us to
propose corresponding Legislative Proposals calling for paying more attention to eco-friendly remove PET
and other plastics accumulated in environment and our community. Simultaneously, this communication with
relevant industry directly strengthened and supported that our project focused problem is a true
emergency, which further conformed with our previous investigations, and our approach is really
important and needed for further solving PET plastic pollution challenge.
The second stage
In order to solve the problem of effectively degradation of accumulated PET at mild temperature, we hope
to find a safe and efficient approach. At first, we summarized and compared the certain methods reported
by previous literature. Among them, we regard artificially designed scaffolds combined with PET
degradation enzymes as the most potential method due to its neglected disturbances to enzymes structure
stability and possible improved efficiency. We believe it is of great practical significance to develop
this new ideal for PET degradation. For further understanding of its characteristics, advantages,
disadvantages and other related questions, we visited experts and researchers at this stage for
designing our project.
Name: Wei Kang
Occupation: Associate professor, School of Bioengineering, Dalian University of Technology
Field: Protein chemistry, synthetic biology, chemical biology
Associate professor, Mr. Kang expressed great interest in our project, which may be combined with his new
discovery of RIAD-RIDD dimer peptides interaction. These two types peptides possess strong binding
affinity to each other. Under his discovery inspiration, we proposed that IsPETase and MHETase
may can
be fused to RIDD and RIAD respectively so that the physical closer contact with these two enzymes active
site may significantly accelerate PET degradation via intermediate delivery. One previous research has
proved that the combination of IsPETase and MHETase has significantly accelerated PET
degradation.
Therefore, our new inspired designing may further accelerate enzymes degradation rate without disturbing
enzymes active site structure or enzymatic activity. Associate professor, Mr. Kang encourage us to make
use of his newly discovered system.
Take away: Through this key communication, we obtained inspiration of making use of RIDD-RIAD
system to
construct our artificial PET degradation enzyme that may significantly contribute to higher PET
degradation via intermediate delivery and, simultaneously, we also invited the Assistant Prof. Wei Kang
as the team advisor to guide our project. Deciding to apply this intricate RIDD-RIAD system into our
project.
Name: CHINA PHARMACEUTICAL UNIVERSITY
We communicate with the 2021 iGEM team from CHINA PHARMACEUTICAL UNIVERSITY on plastic degradation. Their
team is dedicated to degrade PE materials, which is also one of the frequently used forms of plastics.
During the communication, their team has mentioned the hydrophobic surface of plastic including PET and
PE is one of the significant factors that limit the degradation rate. Is there exist possible methods to
retard this limitation? Inspired by their team, we get accessed to a new functional protein called
hydrophobin, whose structure is highly flexible and able to adhere to hydrophobic plastic surface with
its hydrophobic surface part with maintaining the hydrophilic surface exposed to water phase. With the
involvement of this new functional protein, PET surface may become accessible to our enzymes.
Take away: Hydrophobin was once used for improving PET degradation rate, however, its efficiency
was
limited at that time due to the lack of highly efficient enzymes at mild temperature and therefore was
ignored by scientists for a long time. Through the discussion with the 2021 iGEM team from CHINA
PHARMACEUTICAL UNIVERSITY, we obtained the knowledge of hydrophobin protein and were inspired to
redesigned our project and apply this new protein via fusing it to RIAD and constructed via our
artificially designed scaffold system, in which this new involved protein may significantly improve the
designing our project. We also decided to further cooperate with the iGEM team from CHINA PHARMACEUTICAL
UNIVERSITY.
Name: TIANJIN UNIVERSITY 2021 iGEM team
Browsing through the previous iGEM wiki page, we noticed the 2020 iGEM team from TIANJIN UNIVERSITY was
dedicated to improve IsPETase enzymatic activity via protein evolution method and this team
continuously
participate in 2021 iGEM competition. We actively connect this team's student leader and student
responsible for dry experiment. From this connection, we obtained the information that IsPETase
possess
prefect ability at mild temperature, however, with limited thermal stability. Their work is to dedicated
to improve its thermal stability and enzymatic activity. They are willing to share us with their new
mutated protein sequence that possess more excellent features. We also simultaneously offered our
constructed engineering bacteria that express MHETase protein to their team. Their team is responsible
for providing the IsPETase sequence with good performances, and we are responsible for
constructing
improved IsPETase with MHETase via artificially designed scaffolds. Furthermore, our two team dry
experiment members communicate with each other since we both choose to apply molecular dynamics
simulation to explore and identify structure information that support project designation. We share the
valuable information about the correct force field choosing for different explicit simulation system and
the parameters setting. From our communication related to dry experiment, we both recognized that the C
termini of IsPETase is too near to enzymatic active center and the fusion of our artificially
designed
scaffolds to this location should be avoided. Lastly, their team invite us to visit their laboratory and
shared with information on human practice, in which we shed with them the information we obtained from
stakeholders. We decided to cooperate with TIANJIN UNIVERSITY 2021 iGEM team in different fields
including wet experiment, dry experiment, human practice and etc.
Take away: We decided to further cooperate with 2021 iGEM team from TIANJIN UNIVERSITY, by which
we
successfully co-finished the business plan and legislative proposals in the next several months. We also
shared the dry experiment parameters verification of molecular dynamics simulations with their team.
Sharing with information about PET stakeholders and relevant industry. Offered us necessary protocols
for establishing PET degradation detection method. We obtained the improved IsPETase sequence and
more
valuable structure information that significantly contribute to our artificially designed scaffolds
fusion orders decision of our project.
Name: Ziying Zhang
Occupation: Chief manager of Guangzhou Yinfo Information Technology Co., Ltd.
Field: Expert in macromolecular simulation and calculation
Fortunately, our team got a chance to discuss with Mr. Zhang for better using of molecular dynamics
simulation that support our project designing. He and his company are dedicated to make computational
biology more accessible to biological researches. All the necessary measurement methods for evaluating
the overall stability of our assembly system in the aqueous environment and Ca2+ binding site
maintenance were suggested by Mr. Zhang. He also offered us professional suggestions of parameters
setting and forcefield choosing in protein simulation. The obstacles of unreasonable energy minimization
caused by un-rational initial structure were overcome with the aid of Mr. Zhang. More detailed
information is offered in the model part. Furthermore, he and his company selflessly provide free high
speed and accurate super computational calculate resources to run our molecular dynamics simulations
since he thought our project may greatly contribute to solving accumulated PET pollution challenges.
Thanks for his selflessly support!
Take away: We obtained professional guidance and aid in molecular dynamic simulations and
available free
super computational calculate resources to run our molecular dynamics simulations. Meanwhile, we further
understood the significant roles of each domain in enzymes applied in our project and how do they
contribute to enzymatic activity function. We also decide to apply flexible GS linker because it
effectively minimizes the surface area of solvation and thus make each protein physically close to each
other with synergistic relationship, which has been demonstrated in our dry experiment part. More
importantly, we obtained a deep insight of how to make use of modern computational technology to design,
explain and improve our project.
Name: Zhiwei Zhu
Occupation: Professor, School of Bioengineering, Dalian University of Technology
Field: Protein chemistry, synthetic biology, genetic engineering
For avoiding the mismatch of three scaffolds components and maintain their expression level in general
same level, we discussed with Pro. Zhu. He strongly recommended us to use the same cis acting element
for both the 3 genes translate 3 different functional components. T7 promoter, RBS, lac operator, T7
terminator and His tag of pET28a (+) were available for each of the 3 target genes. Furthermore, Pro.
Zhu suggested to secret our enzymes into extracellular spaces so that un-penetrate PET material could be
effectively degraded by our designed enzymes. He said this suggestion may also avoid the mis-matching of
enzymes components because the peptides translated from mRNA will not be further folded before secreting
into the extracellular spaces because of the transporting proteins locking effects. After secretion, the
peptides will spontaneously fold into functional state.
Take away: We re-designed our plasmids for ensuring the rationality of enzymes components correct
assembly ratio and avoiding mismatching expression level of 3 unique components. Deciding to add signal
peptide in our system to secreting our newly designed proteins according to Prof. Zhu's suggestions, in
which the secreted proteins with large quantities of disulfide bonds will be much easier to be
successfully folded in the oxidizated extracellular environment and assembled in the extracellular
spaces. Meanwhile, we obtained a deep insight of how unmatured peptides is secreted into extracellular
spaces and folded to become matured functional proteins.
The third stage
Experts in the field of synthetic biology and protein engineering research have a deep understanding on
making use of genetic elements via synthetic biology methodology. So, we hope that our project will
become feasible and reasonable with their guidance. Simultaneously, their professional advices also
significantly support our project and experiment developed smoothly. At this stage, we initiate our
experiment sectors and many of our imaginations were becoming reality with the guidance of different
experts.
Name: Ziying Zhang
Occupation: Chief manager of Guangzhou Yinfo Information Technology Co., Ltd.
Field: Expert in macromolecular simulation and calculation
Our simulated proteins system was collapsed in explicit simulation due to unknown reasons. Therefore, we
discussed with Mr. Zhang for further help. After repeated checking, Mr. Zhang finally find out the crux.
The topology in our simulated document is not included disulfide bonds, which ultimately lead to the
collapse of our final simulation structure. Simultaneously, he also pointed out that the TIT3P may not
be accurate to simulate our system because it is a type of early 3 points water model that is too old
and imprecise to simulate our structure.
Take away: Reconstruct our structure topology that is applicable to be simulated via molecular
dynamics
simulations. Substitute original TIT3P water model with more advanced water model.
Name: Yanbin Feng
Occupation: Associate professor, School of Bioengineering, Dalian University of Technology
Field: Biocatalyst and biosynthesis guided by protein evolution
Our meeting with assistant professor Yanbin Feng was focused on the signaling peptide that has been
applied in our research. After one month period of experiments, our experiments result has revealed that
the pelB signaling peptide applied possess insufficient ability to secret PETase, MHETase and
Hydrophobin effectively. Therefore, we were looking for a new alternative solution to produce PETase,
MHETase, Hydrophobin as a complex and then we sought help from assistant professor Yanbin Feng, who
dedicated in protein designing. He suggested us that we should wipe off signaling peptide and synthesize
modular enzymes system in cytoplasm. The maintaining of signal peptide would prevent the further folding
of each enzyme according to signaling transportation mechanisms reported in previously published
article. Therefore, with signaling peptide in our sequence of enzymes, most enzymes would not
successfully fold and lagged in cytoplasm. Only few of these enzymes successfully secreted and then
folded. According to our experiment results, it may be inevitable to assembly these enzymes in the
cytoplasm of the cell and then broken the cell to obtain our target enzymes. Furthermore, the
feasibility of the signaling peptide applied in our research was only demonstrated in the secreting of
free PETase without RIDD and GSlinker that involved in our research. It may not be applicable to be
further used in the system of secreting MHETase and Hydrophobin.
Take away: The feasibility of maintaining pelB signaling peptide in our research need to be
reconsidered
according to previously published articles and our experiment results. The assembly complex released via
ultrasonic wave would be more applicable and effective to obtain our target enzymes complex.
Name: Chengba Zhu
Occupation: postdoc at RWTH Aachen University
Field: Microalgae engineering and plastic degradation
We were fortunate to discuss with Dr. Zhu about the enzymatic activity detection problem. In our
experiment, we found both the reference group and induced protein expression revealed possible enzymatic
activity, which made us feel confused. Dr. Zhu is also dedicated to research related to IsPETase
Therefore, we seek Dr. Zhu for further help and solutions. Finally, after listening our detailed
experiment procedures, he pointed out that our model substrate 4-(p-nitrophenyl) butyric ester may be
easily degraded to be final product when exposing to light and Tris-HCl buffer, which may account for
our abnormal experimental results. He suggested that we should held our experiment in dark place. We
finally verified that the exposure to light is the main cause of our experiment failure. With his help,
we finished our corresponding experiment.
Take away: We recognized the reason of our abnormal experiment results and finally finished our
experiment with the aid of Dr. Zhu. He pointed out that our model substrate 4-(p-nitrophenyl) butyric
ester may be easily degraded to be final product when exposing to light and Tris-HCl buffer, which may
account for our abnormal experimental results.
The fourth stage
We are currently trying to make use of artificially designed scaffold fused to degradation enzymes to
accelerate PET decomposing rate at mild temperature. We expect to apply synthetic biology to solve the
challenges faced in the application of its industry, including protein expression, collection and using.
From lab to industry, what are the challenges for future application and how can we maximize our
influence on society?
Project Sustainability
Name: Yu Fan
Occupation: Professor, School of Foreign Languages, Dalian University of Technology
Field: Language and diplomacy, international policies research
Pro. Fan from the School of Foreign Languages of Dalian University of Technology has nearly ten years of
experience in leading the Model United Nations and has a deep understanding of various United Nations
documents and backgrounds.
We had a conversation with Prof. Fan. We elaborated on the purpose and reality of the project. Prof. Fan
thought that our project was totally in line with the basic core of SDGs: SUSTAINABLE CITIES AND
COMMUNITIES and LIFE ON LAND. Our project is based on environmental care, solving human problems with
engineering thinking and methods, providing a convenient way for possible PET degradation, which is of
positive significance and value for preventing the accumulated PET materials in environment. Our project
is contributing to SDG goals in terms of SUSTAINABLE CITIES AND COMMUNITIES and LIFE ON LAND. But the
teacher also mentioned that the biosafety issues involved cannot be ignored, especially the potential of
engineering bacteria to spread resistance genes, pollute the environment and harm the human body. The
biosafety protection of SDG and the relevant documents of the United Nations on biosafety have paid
attention to related issues. We should have a deeper understanding of the connection between SDG and the
iGEM community and the connection between human beings as a species, and clarify the responsibility and
feelings of the concept of sustainable development.
Take away: Sustainable, biosafety, responsible
Name: JIN TU DI Group
At this stage, we re-interviewed with the member of researchers from JIN TU DI Group, who is professional
in current Chinese degradable plastic market and field. Their suggestion clearly mentioned the
significance of biosafety and real practical using efficiency. Any product needs to be ensured that they
possess corresponding function without safe risks. Many experiment products do not well serve for
society needs because of lacking of these necessary principles. We also visited their laboratory and
their producing line, which enhanced our understanding of real-sustainable production mode.
Simultaneously, they remind us of researching all the relevant laws requested by government to form our
possible products into a safe and responsible norm. Relevant laws and clauses for industrial production
for plastic degradation and recycling are offered by their company manager and well introduced by
professional engineers.
Take away: Through this visit, we obtained a new perspective as engineers and stakeholders for
sustainable production mode. Basic clauses and laws related to our project and relevant fields were well
introduced to us and we knew more about current industrial production technology and cooperation between
different departments. On these bases, we re-understood how to propagandize one new proposed product to
market, via which we draw up our business plan.
Public Level
Public Acceptance
We hope that our project can promote the research of PET degradation, and act as an alternative to
traditional methods coupled with high pollution. The public took a conservative and hesitant attitude
towards the emergence of new things. In order to further understand the public's acceptance to our
proposed methods and knowledge to current PET pollution condition, how difficult it is for our
engineered parts to be applied and promoted industrially, and what factors affect the public's
acceptance of new things, we conducted a detailed questionnaire survey. Detailed questionnaire results
and analysis can be found below.
We hope that our project will improve the urgent problem of plastic pollution and provide a more
effective, applicable and green biodegradation method. In the process of talking with enterprises and
the community, we realized that one of the challenges of our project is to make people really pay
attention to and understand the close connection between plastic pollution and their own lives,
especially in the process of talking with plastic companies, we found that the current plastic
components are mainly PE, PET, etc., but the degradation and recycling methods of PE are more mature,
but PET is not optimistic. And the current degradation method of PET plastic is highly polluting and
energy consuming, which is not the ideal degradation method. It is with this inspiration that our
project was born. A detailed questionnaire survey was conducted to further understand the public's
knowledge of plastic pollution and plastic composition, and to promote our project to the public. The
detailed questionnaire results and analysis are as follows.
Question 1: What is your job?
According to the results of the questionnaire, the occupational distribution of the respondents was
roughly balanced. The number of students was 219, the number of researchers was 167, and the number of
the general public was 182, which is roughly balanced and in line with our expectations. The
respondents' occupations are widely distributed, and the results can basically reflect the opinions of
people from various occupations in society.
Question 2: What do you usually use to carry your shopping items?
From the overall data, nearly one-third of people will use plastic bags provided by supermarkets in their
daily lives, plastic bags are widely distributed in people's daily use, and only one in ten people will
reuse plastic bags, which means that if there is no effective way to degrade plastic, plastic will
continue to accumulate in daily life.
Question 3: Would you mind if the garbage bag is degradable?
More than half of the respondents do not care whether the plastic is degradable or not. Although
degradable plastic has been popularized to a certain extent, people's awareness of using degradable
plastic in their life is not strong, which means most of the plastic used is still hard to degrade
plastic products. This gives us some inspirations: firstly, we can carry out publicity and
popularization of degradable plastics; secondly, we should find more widely applicable degradation
solutions for the existing plastic accumulation.
Question 4: Do you think PET plastic is easy to degrade?
After a brief science on PET plastic, more than half of the respondents think that PET plastic is easy to
degrade, which is very different from the current situation we are facing, and there is no efficient and
mild effective way to degrade PET plastic, which brings us the inspiration that people's inadequate
knowledge of plastic material misleads their attitude and way to use plastic products.
Question 5: What are the benefits of using biological methods to degrade PET plastic?
In terms of the benefits of biodegradation of PET, most people believe its fast degradation rate,
environmental benefits and low costs. This shows the public's recognition of biotechnology.
In contrast, "will not cause secondary pollution" has been suspected by some people. Therefore, we need
stronger evidence about the final product verification and safety to ensure that our product is accepted
and used.
Question 6: Do you think plastic degradation is very difficult or easy in nature?
What shocked us is that nearly half of the people think plastic degradation is very simple. The daily use
of plastics may make everyone take it for granted that it is easy to degrade, which ultimately leads to
the widespread phenomenon of plastic waste and serious pollution. While the project is committed to
degrading plastics, this data makes us also aware of the necessity of universal education. The increase
in public awareness will become an effective means of improving the environment.
Question 7: What is your acceptance of the method of linking multiple functional proteins
together, that
is, the
method of degrading plastics by multiple backbone proteins?
From the graph, testers do not have a high degree of recognition of the multi-frame protein degradation
method. The number of approved people is only 13.93%, and most people are not clear about the method or
have doubts. It can be seen that our project has a certain degree of innovation and difficulty. Although
relevant studies have proved that multi-skeleton proteins can better improve the efficiency of the
experiment, it is still necessary to fully demonstrate the feasibility of the experiment in the early
stage and in the process of the project to ensure that the experiment can be successfully completed and
recognized by the public.
Question 8: Where do you think plastic can be found?
From the overall data, the respondents have a relatively average understanding of the question plastic
distribution set answers, it is evident that the respondents still have a certain basis of knowledge of
the widespread distribution of plastic such as mountains, oceans medium and the location of the human
body, and recognize the urgency of the spread of plastic pollution and harm solution.
Question 9: If there is a mild and efficient plastic degradation method, but the cost is high,
would you
use it?
According to the questionnaire results, more than 80% of people would not use efficient but expensive
plastic degradation methods, indicating that people still give priority to economic issues such as
practicality and cost of degradation solutions. The distribution of answers to this question is
generally in line with our expectation, indicating that we still ultimately pursue the economic
feasibility of optimized methods.
Question 10: What do you think is the urgency of research in plastic degradation?
According to the results of the questionnaire, the perception of urgency of research on plastics is at a
slight extreme, with less than 30% of respondents not feeling a sense of urgency and more than 30%
believing that the current status of plastic degradation is already very urgent. In this regard, we
believe that we need to promote the knowledge of plastic degradation to the public, and at the same
time, we can actively explore and mobilize people with the same ambition to take action with us.
Conclusion:
The respondents were mainly practitioners and students in the biological industry, accounting for more
than 90%. The questionnaire mainly investigated the relative professional people's understanding of
plastic pollution and their views on the difficulty and advantages of biodegradation of plastic. The
respondents generally believe that the research on plastic degradation is very urgent, and the use of
biological degradation of plastic is beneficial to the environment and will not cause secondary
pollution, but they have a wait-and-see attitude towards the cost and ease of operation.
Word frequency statistics
Word
Frequency
Plastics
57 times
Degradation/degradable
15 times
Respondents
7 times
PET
7 times
Public
6 times
Pollution
6 times
Urgency
4 times
Effective
4 times
5 Computer algorithm applied in our work
The following is the modeling and analysis of the data. My idea is to build a decision tree or
neural network model, using specific data in the questionnaire as a dependent variable and other
data as an independent variable to establish an analysis and prediction model. For example, use
other factors on the questionnaire to analyze "do you care if garbage bags are degradable?" Or
"do you think PET plastic degrades easily?"
The feasibility of this is that due to the large scale of the data, there is some relevance among the
options in the problem.
We tried to draw a column chart for each column and observe their quantity distribution.
After carefully considering the data, we decided to establish a decision tree model based on
comprehensive learning (a machine learning method).
# Set independent and dependent variables
X = df.loc[:,df.columns != 'Think whether PET plastic is easy to degrade']
Y = df.loc[:,df.columns == 'Think whether PET plastic is easy to degrade']
Take "care about when the garbage bag is degradable" as an example, first divide the data set, then
automatically divide the training set and the test set by the train_test_split function
in sklearn.model_selection package, and finally determine 449 rows of data as
the training set and 150 rows of data as the test set.
The decision tree model was established by the DecisionTreeClassifier function
in the package sklearn.tree, and the decision tree model was automatically
fitted by the fit function. The
optimal parameters were determined by searching the optimal decision tree in the sklearn.model_selection
package.
The optimal hyperparameters were determined by optimizing the decision tree through grid
search.
Finally, cross-validation was used to calculate the accuracy of the optimization model, the accuracy
of the model prediction is 81.81092436974791%.
This is the visual structure of the decision tree model drawn by us with software Graphviz.
Similarly, "care about whether the garbage bag is degradable" was used as the dependent
variable. The method is similar, and the accuracy of model prediction is 75.8053221285155%.
