Poster: ASTWS-China

Team Achievement

This is the poster of Team ASTWS-China 2020. This year, our project is focusing on PET microplastics degradation, designing a genetically enhanced biofilms to promote PET microplastic degradation efficiency through the proximity effect of the enzyme and substrate. Here is the summary of our achievement in this iGEM season.

When Microplastics Meet Biofilms: Enhanced Degradation of Environmental Plastics by Biosorption

Presented by: Team ASTWS-China 2020

Team Roster:

PIs: Shan Dong, Ruyi Shi
Instructors: Zhou Tan, Yunfen Hua
Advisor: Xixi Song
Team members: Hengyi Zhang, Xinyi Chen, Junran Zhao, Yunqi Wang, Qianqian Wang, Yunfei Kan, Shufu Zha, Jinyan Wang, Ji Wu

Abstract

Microplastics from household waste are easily entered into our water system, which is one of the most difficult environmental pollutants to degrade. The newly developed biodegradation technology can effectively degrade highly polyester contaminants, such as PET plastics, which can be efficiently degraded using PETase. On the other hand, biosorption can effectively collect microplastic particles. Therefore, in this study, we hypothesize that genetically enhanced biofilms can promote PET microplastic degradation through the proximity effect of the enzyme and substrate, thus benefiting future environmental governance applications.

Introduction and Inspiration

Our world is overwhelmed in plastics pollutions. With the help of agricultural runoff, wastewater treatment plants, floods, river systems, etc., nano/microplastics are transported to freshwater systems or into the ocean and further diffused under the action of water currents, wind, and turbulence.

Concerns about environmental issues and news related to nano/ microplastic pollution inspired us to start this project. Our mission is to degrade nano/microplastics (PET) in the water body by genetically modified E.Coli biodegradation system that produces PETase and strengthens the biofilm system. This combined biodegradable system can not only degrade PET plastics but also attach degraded microplastics, so it will not flow away, thereby preventing it from further pollution.

Design

Our biofilm systems mainly include three parts:

1. Strengthening biofilm system (OmpR234 expression)
2. PETase expression system
3. Double fluorescence reporting systems: To ensure the proximity effect between PETase and the biofilm, the GFP-mCherry double fluorescence reporting system was introduced for the co-expression.

Results

This year, we successfully verified three systems, ie., four main parts, was worked as we expected. The main works are summarized as follows.

1. Expression of OmpR234 Increases Biofilm Formation (BBa_K3576000: strengthening biofilm system)

2. Enzyme activity test of PETase (BBa_K3576001: PETase Expression)

3. Co-expression Verification of double reporting system (BBa_K3576002 + BBa_K3576003)

4. Improvement degradation activity verification of co-expressed system (BBa_K3576002 + BBa_K3576003)

5. Quantitatively model predication for enhancement rate of biofilm on PETase degradation activity

6. Proof of degradation ability of our system to real PET plastic fragments

7. Possible improvement process consideration for microplastic treatment in the real sewage treatment plant

Separately Verification

Expression of OmpR234 Increases Biofilm Formation

Compared with the control groups, LB medium and E. Coli (BL21), the third group which overexpressed OmpR234 demonstrates a stronger red color, which indicated higher production of biofilm.

Enzyme Activity Test of PETase:

With the extension of reaction time, the OD405 value of p-NP gradually increased, which indicates that the degradation activity of the PETase.

Figure A Congo red assay stains biofilm after overexpressing of OmpR234 (BBa_K3576000); B OD405 of pNPB hydrolysis by overexpressed PETase (BBa_K3576001).

Co-expression Verification

Co-expression Verification of Double Fluorescence Reporting System

We transformed three sets of plasmids (OmpR-mCherry, eGFP-PETase, OmpR-mCherry + eGFP-PETase) into E. Coli and observed them with a fluorescence microscope. when the two expressed together, the microscopic image shows yellow, which also qualitatively proves the effective co-localization of PETase and enhanced biofilm.

Figure Fluorescence micrograph of engineered E. Coli transformated with (A) eGFP-PETase, (B) OmpR-mCherry, (C) and OmpR-mCherry + eGFP-PETase.

Degradation Activity Test of Co-expressed System

The p-NP assay was used to further test the effects of the strengthened biofilm on the degradation activity of PETase. It was demonstrated that, with the extension of time, the OD405 value of the co-expression system is higher than that of PETase alone. In other words, with the help of enhanced biofilm, the degradation activity of PETase could be improved.

Figure OD405 of pNPB hydrolysis by overexpressed PETase and PETase+OmpR

Model Prediction

To quantitatively predict the enhancement rate of biofilm on PETase degradation activity, a model was constructed based on the typical enzyme-catalysed reaction, which can be described by classic Michaelis-Menten equation. The enzyme activity of PETase was performed by p-NP assay which is a common way to quantify hydrolytic activity (pNPB as the substrate).

Solving of Km and Vmax

We plot 1/V0 and 1/S then preform linear fitting. The corresponding Michaelis-Menten equation was obtained in the end.

Prediction of Enhancing Rate

According to Michaelis-Menten equation of PETase and PETase enhanced by OmpR, the calculated enhancing rate is up to 62.2%. Encouragingly, from the HPLC results of PET degradation, we found the enhancing rate is about 66%, which is close to the prediction. The detailed results will be introduced in the Proof of Concept section.

Improvement of Michaelis-Menten Equation

We also developed an improved Michaelis Menten equation, which help describe the enzyme-catalysed reaction influenced by biofilm.

Human Practices

Public Survey

Aware of the seriousness of plastic pollution
Inadequate understanding of microplastic pollution
59.32% of people think that our idea is very promising.

IHP: Inspiration in Science communication activity

Field Trip

In depth background learning
Confirm the necessity of developing promising degradation treatment

Professional Interview

Preventive measures are as important as degradation techniques
More inspiration in implementation
Inspiration of strategies to improve PETase degradation efficiency

Proposed Implementation

We add three more steps and design the new treatment process based on our system.

1. A biocarrier is used for carrying our engineered bacteria. It may help remove the microparticles and biodegrade PET microplastics at same time.

2. A filter tank is also added after secondary sedimentary tank, which can help physically remove remaining microparticle from water by ultrafiltration membrane (Ultrafiltration system).

3. Activated sludge method is also planning to treat the microplastics (especially PET) from sludge (sludge treatment process) because most of the microplastics, about 80 ~ 99.9%, are retained in sludge disposal.

4. Meanwhile, biosafety is also being serious considered (Ultraviolet light disinfection process).

Example of commercial biocarriers is from Hangzhou Nihao Environmental Tech Co. Ltd.
Ultrafiltration system is from Dongying Waterwork Company (Dongying City, Shandong Province, China, Picture retrieved from https://www.meipian.cn/1kjcuyr9 at 2020/10/17)
The photo of ultraviolet light disinfection process is from Hangzhou city-west (Jiangcun) sewage treatment plant (West Lack District, Hangzhou, Zhejiang Province, China)

Proof of Concept

Our ASTWS sewage treatment system needs to be proved in three aspects:

1. Whether PETase can effectively degrade PET plastics；
2. Whether Biofilm can capture microplastics in water;
3. Whether the degradation efficiency of PETase and Biofilm system is higher than that of the single PETase.

For the second, we can see the biofilm (Congo red staining test) produced by the engineered bacteria was successfully attached to the surface of the plastic pieces (the inserted photo in Figure B).

For the first and third, the real PET plastic fragments degradation rates of PETase and PETase enhanced by biofilm was measured by HPLC. Figure A is the HPLC results of MHET (Mono-(2-hydroxyethyl) terephthalic acid), the PET degradation product, in the solution.

The relationship between peak area from HPLC results and MHET concentration was plotted to further analyze the degradation efficiency. The peak area increment of the co-expressed system is reached to 66.33% of the overexpressed PETase individually, which is further verified the effectiveness of our model.

Figure (A) HPLC results of real PET plastic fragment detection; (B) Standard curve of MHET standard (inserted is photo of biofilm (Congo red staining test) on the large plastic fragment); (C) HPLC results of MHET concentration in real sample test.

Future Work

Considering the thermal stability of PETase, the thermal stability improvement of PETase is on the way.
Purifying the protein to better demonstrate its degradability to ensure the biosafety and convenience to use in real world.
Building a PET degradation system with higher degradation efficiency and higher thermal stability.

Acknowledgement

Special thanks to our families and all other people involved in helping make us a successful iGEM team in this special year.

General Support:
Hangzhou Sipu Education and Technology, Co., Ltd.
Hangzhou Precision Medicine Research Center (Hprecision )

Expert Interview:
Dr. Ji Rong, Nanjing University
Dr. Lai Chunyu,The University of Queensland
Dr. Yuan Zhihua, Institute of Urban Environment, Chinese Academy of Sciences
Mr. Chen Xiaohong, Department of ecological environment of Zhejiang Province

WIKI Support:
Mr. Yunjie Zhang

Partnership:
TJUSLS_China 2020 iGEM team

Team:ASTWS-China/Poster