iGEM 2021 — Team IvyMaker-China

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# Team:IvyMaker-China/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:IvyMaker-China/Engineering

IvyMaker-China Model Page

[![](https://static.igem.org/mediawiki/2021/3/3c/T--IvyMaker-China--logo.jpeg)](https://2021.igem.org/Team%3AIvyMaker-China)

* Team
  + [Team](/Team%3AIvyMaker-China/Team)
  + [Attributions](/Team%3AIvyMaker-China/Attributions)
  + [Collaborations](/Team%3AIvyMaker-China/Collaborations)
  + [Partnership](/Team%3AIvyMaker-China/Partnership)
* Project
  + [Description](/Team%3AIvyMaker-China/Description)
  + [Implementation](/Team%3AIvyMaker-China/Implementation)
  + [Contribution](/Team%3AIvyMaker-China/Contribution)
* Wet Lab
  + [Results](/Team%3AIvyMaker-China/Results)
  + [Engineering Success](/Team%3AIvyMaker-China/Engineering)
  + [Proof of Concept](/Team%3AIvyMaker-China/Proof_Of_Concept)
  + [Parts](/Team%3AIvyMaker-China/Parts)
* [Model](/Team%3AIvyMaker-China/Model)
* Human Practice
  + [Overview](/Team%3AIvyMaker-China/Human_Practices_Overview)
  + [Human Practices](/Team%3AIvyMaker-China/Human_Practices)
* [Education and Communication](/Team%3AIvyMaker-China/Communication)
* [Excellence in Another Area](/Team%3AIvyMaker-China/Excellence_In_Another_Area)
* [![](https://static.igem.org/mediawiki/2021/9/97/T--IvyMaker-China--trophy.png)](/Team%3AIvyMaker-China/Awards)

[Overview](#overview)

[Part 1 Construction of Surface Display System](#1)

[1.1 Background](#1.1)
[1.2 Research](#1.2)
[1.3 Imagine](#1.3)
[1.4 Design](#1.4)
[1.5 Build](#1.5)
[1.6 Test](#1.6)
[1.7 Learn](#1.7)
[1.8 Improve](#1.8)

[Part 2 Pet Degradation](#2)

[2.1 Research](#2.1)
[2.2 Imagine](#2.2)
[2.3 Design](#2.3)
[2.4 Build](#2.4)
[2.5 Test](#2.5)
[2.6 Learn and Improve](#2.6)

[References](#3)

# ENGINEERING SUCCESS

### Overview

* The core of our project was to develop a whole-cell biocatalyst by displaying PETase and MHETase on the surface of yeast cells (*Candida tropicalis*). With careful consideration and effective improvement, we finally succeeded in achieving the purpose of our project step by step. We screened a promoter (BBa\_K3829001: P-GAPDH), a terminator (BBa\_K3829000: T-GAPDH), and a reporter (BBa\_K3829002: yeGFP), which could effectively screen anchor proteins.

### Part 1 Construction of surface display system

##### 1.1 Background

* Surface display technology can expose peptides or proteins on the cell surface, which is a powerful tool used in many fields such as medicine. It has been reported that the enzymes anchored on the cell surface have better activity and stability compared with free enzymes. Furthermore, the decrease of enzyme activity is not obvious after the completion of enzymatic recycling. The surface display system can also eliminate the step of enzyme purification, making industrial applications more feasible. Recently, some researchers have displayed PETase enzyme on the surface of *pichia pastoris* and its activity is 36 times as much as that of free enzyme, proving that PETase enzyme is suitable for functional display on yeast.
* At present, cell surface display technology mainly focuses on some model strains and some specific anchor proteins. To expand the application value of the technology, non-model strain display platforms and more efficient anchor proteins should be developed.

##### 1.2 Research

* Chassis microorganism
* *Candida tropicalis* is capable of absorbing alkanes and fatty acids as carbon sources and surviving in environments containing acids and phenolic substances. Compared with other surface display systems, the surface display technology on *Candida tropicalis* has not been fully developed. Therefore, it is worthy to establish the system on *Candida tropicalis*. In addition, *Candida tropicalis* is maturely used in the production of long-chain diacid, which lays a good foundation for its industrial applications.

* Anchor protein
* GPI-anchored proteins have three main characteristics: (1) N-terminal contains secretion signal peptide sequence (2) Mature protein has no transmembrane domain (3) C-terminal contains GPI signal peptide. Based on these characteristics, bioinformatics analysis was carried out to predict related anchoring proteins.

##### 1.3 Imagine

* We can build a surface display system for *Candida tropicalis*. BioBricks are promoters, reporters, and terminators. First, we need to ensure that reporter GFP can express fluorescence. Then GFP is fused with GPI-anchored proteins to reflect protein expression through fluorescence.

##### 1.4 Design

* According to the reported characteristics of GPI-anchored proteins, anchor protein candidates were screened from the *Candida tropicalis* genome using bioinformatics methods. Then, the yeast green fluorescent protein (yeGFP) was fused with the potential GPI-anchored protein for preliminary identification. Subsequently, the subcellular location was determined by immunofluorescence and Western bolt. Finally, GPI-anchored proteins that could be used to display proteins were screened and flow cytometry was used to evaluate the display efficiency of the screened GPI-anchored proteins.

* Screening of anchored proteins
* yeGFP (yeast enhanced green fluorescent protein) was detected to obtain the bacterial strain that displayed fluorescence on the outer side of the cell periplasm. Then immunofluorescence and Western Bolt were used to further screen the strains located in the cell wall.

##### 1.5 Build

* In order to determine that the protein can be successfully expressed, we have to select a promoter that can efficiently initiate transcription of downstream genes in *Candida tropicalis*. Therefore, so the promoter of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was chosen and the corresponding terminator was selected. To facilitate the display of protein expression, we chose GFP as the reporter. In addition, we have performed codon optimization (BBa\_K3829002) for the existing part-reporter GFP (BBa\_K3402050) to improve the expression efficiency in *Candida tropicalis*.
![](https://static.igem.org/mediawiki/2021/1/16/T--IvyMaker-China--Lab-03.jpg)* Fig.1 Composition of the basic Bio-bricks.

##### 1.6 Test

* The bacteria were collected, resuspend in buffer, and then observe with a confocal microscope. The results were shown in the figure 2. Compared with the negative control, that is, the wild strain (URA3-KO), the fluorescence of yeGFP (BBa\_K3829002) in the positive control can be expressed.
![](https://static.igem.org/mediawiki/2021/2/27/T--IvyMaker-China--Lab-19.png)* Fig.2 Representative images of yeGFP expression.

##### 1.7 Learn

* It has been proved that yeGFP (BBa\_K3829002) can be expressed intracellularly. On this basis, we fused a signal peptide (ss) to the N-terminus of yeGFP, so that downstream genes can be guided through the cell membrane to the periplasm.
![](https://static.igem.org/mediawiki/2021/d/df/T--IvyMaker-China--Lab-04.jpg)* Fig.3 Composition of the gene circuit after adding signal peptide (SS).

##### 1.8 Improve

* The ability of the new gene circuit to display the protein on the cell surface is also affected by different anchor proteins. Therefore, 129 anchor proteins were measured and merely 25 anchor proteins were found to work (Figure 4).
![](https://static.igem.org/mediawiki/2021/c/c0/T--IvyMaker-China--Lab-13.jpg)* Fig.4 Representative images of 25 screened anchor proteins.

* Then immunofluorescence was performed to verify that if the protein was successfully displayed outside the cell membrane. The results suggested that nine of the 25 anchor proteins worked well. The results of 4609 were shown in figure 5.
![](https://static.igem.org/mediawiki/2021/4/49/T--IvyMaker-China--Lab-14.jpg)* Fig.5 Representative images of 4609 (further screened anchor protein).

### Part 2 PET Degradation

##### 2.1 Research

* In 2016, Japanese scientist Yoshida discovered the bacterium *Ideonella sakaiensis* 201-F6 from the sludge of a bottle recycling plant in Osaka., was able to decompose PET plastic. The bacterium could completely degrade the low-crystallinity PET film in 6 weeks under the reaction conditions of 30℃, which was currently known to be the best bacteria for PET degradation. PETase and MHETase were found to be the key enzymes. PETase was able to degrade polyethylene terephthalate (PET) into bis-hydroxyethyl terephthalate (BHET), mono-(2-hydroxyethyl) terephthalic acid (MHET) and terephthalic acid (TPA). MHETase was able to further degrade MHET into TPA and ethylene glycol (EG).
![](https://static.igem.org/mediawiki/2021/e/ed/T--IvyMaker-China--Lab-18.png)* Fig.6 Enzymes and their products involved in PET degradation

##### 2.2 Imagine

* PETase and MHETase can be fused with GPI-anchored proteins with high surface display efficiency respectively. Afterwards, enzyme activity of the targeted proteins can be determined. Subsequently, the efficiency of the surface display system should be optimized, and the PET film can be used as the substrate to evaluate the ability of the dual-enzyme display system to degrade plastics in whole cells. Finally, *Candida tropicalis* should be further modified to convert the hydrolyzed substrates TPA and EG into high value-added products, such as protocatechuic acid (PCA) and glycolic acid (GLA) etc.

##### 2.3 Design

* We obtained the candidate anchor proteins through modeling analysis. The analysis steps were as follows. First, analyze the 10221 amino acid sequences of the genome (*Candida tropicalis* ATCC 20336). Then combined with the database, the sequences without secretion signal peptide and without GPI anchor signal were eliminated. Furthermore, proteins without transmembrane structure would be removed. Then, the sequences were further analyzed to obtain the targeted proteins. The anchor protein screening system was further designed to verify their functions. Finally, yeGFP would be replaced with PETase and MHETase.

##### 2.4 Build

* GPI-anchored proteins 5105 and 4609 were selected as the target proteins for surface display system. Then, the strains respectively displaying PETase and MHETase were constructed. The schematic diagram of gene expression boxes was shown in figure 7.
![](https://static.igem.org/mediawiki/2021/c/c3/T--IvyMaker-China--Lab-02.jpg)
![](https://static.igem.org/mediawiki/2021/a/a4/T--IvyMaker-China--Lab-01.jpg)* Fig.7 The composition of the genetic circuit after replacing yeGFP with PETase and MHETase

##### 2.5 Test

* After the targeted gene was successfully integrated into *Candida tropicalis* genome, we verified the gene function. Due to the advantages of the surface display system, we only need to add the same amount of cell suspension for measurement of enzyme activity instead of enzyme extraction. First, preliminary measurement of enzyme activity was conducted. The coefficient of determination was 0.9997, indicating that the standard curve was reliable and could be used to calculate the activity.
![](https://static.igem.org/mediawiki/2021/d/d2/T--IvyMaker-China--Lab-22.jpg)* Fig.8 standard curve of p-Nitrophenol.

* We determined the enzyme activity of the wild strain ATCC20336 (Negative control), which knocked out the URA3 gene, the strain expressing PETase intracellularly (cytPET), the strain displaying PETase 4609 (PET-4609), and the strain showing PETase 5105. (PET-5105). The catalytic activity of 4-Nitrophenyl acetate (CAS:830-03-5) at 30°C and 37°C was measured respectively. The results were shown in figure 9. Notes-The catalytic product was p-Nitrophenol, and the amount of p-Nitrophenol generated was calculated according to the standard curve.
![](https://static.igem.org/mediawiki/2021/e/ec/T--IvyMaker-China--Lab-23.jpg)* Fig.9 Determination of enzyme activity at different temperatures.

##### 2.6 Learn and Improve

* In the experiment, the PET film used was 0.175mm which was too thick. It took time finish the degradation cycle and the effect was not detectable. Therefore, we started to try PET powder or thinner plastic film (0.0025mm) in our experiment. Finally, it was found that PET powder had a better effect.

##### References

1. Tanasupawat, Somboon; Takehana, Toshihiko; Yoshida, Shosuke; Hiraga, Kazumi; Oda, Kohei. ”*Ideonella sakaiensis* sp. nov., isolated from a microbial consortium that degrades poly(ethylene terephthalate).” International Journal of Systematic and Evolutionary Microbiology, August 2016, volume 66, issue 8, pp. 2813-2818, ISSN 1466-5034. PMID 27045688. doi:10.1099/ijsem.0.001058.

![](https://static.igem.org/mediawiki/2021/3/39/T--IvyMaker-China--01_IK_logo.png)
![](https://static.igem.org/mediawiki/2021/3/3b/T--IvyMaker-China--02_%E9%85%B5%E6%AF%8D%E5%AE%9D%E5%AE%9Dlogo.png)
![](https://static.igem.org/mediawiki/2021/0/02/T--IvyMaker-China--03_%E7%94%9F%E5%B7%A5logo.jpg)

* **WeChat**: ivymakerigem
* **INS**: ivymakerchina
* **bilibili**: IvyMaker\_China
* **YouTube**: IvyMaker-China 2021

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# Team:IvyMaker-China/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:IvyMaker-China/Results

IvyMaker-China Results Page

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* Team
  + [Team](/Team%3AIvyMaker-China/Team)
  + [Attributions](/Team%3AIvyMaker-China/Attributions)
  + [Collaborations](/Team%3AIvyMaker-China/Collaborations)
  + [Partnership](/Team%3AIvyMaker-China/Partnership)
* Project
  + [Description](/Team%3AIvyMaker-China/Description)
  + [Implementation](/Team%3AIvyMaker-China/Implementation)
  + [Contribution](/Team%3AIvyMaker-China/Contribution)
* Wet Lab
  + [Results](/Team%3AIvyMaker-China/Results)
  + [Engineering Success](/Team%3AIvyMaker-China/Engineering)
  + [Proof of Concept](/Team%3AIvyMaker-China/Proof_Of_Concept)
  + [Parts](/Team%3AIvyMaker-China/Parts)
* [Model](/Team%3AIvyMaker-China/Model)
* Human Practice
  + [Overview](/Team%3AIvyMaker-China/Human_Practices_Overview)
  + [Human Practices](/Team%3AIvyMaker-China/Human_Practices)
* [Education and Communication](/Team%3AIvyMaker-China/Communication)
* [Excellence in Another Area](/Team%3AIvyMaker-China/Excellence_In_Another_Area)
* [![](https://static.igem.org/mediawiki/2021/9/97/T--IvyMaker-China--trophy.png)](/Team%3AIvyMaker-China/Awards)

[Overview](#1)

[Part 1 Bioinformatics prediction and analysis of anchor proteins](#1)

[1.1 Selection of a robust host](#1.1)

[1.2 Prediction of GPI-anchored proteins](#1.2)

[Part 2 Identification of biological functions](#2)

[2.1 GFP was used to screen suitable anchor proteins](#2.1)

[2.2 Construction of plasmids](#2.2)

[Part 3 Surface display of PETase and MHETase](#3)

[Part 4 Evaluation of degradation effect](#4)

[4.1 Determination of enzyme activity](#4.1)

[4.2 Determination of degradation products with HPLC](#4.2)

[4.3 Surface display of MHETase to improve the degradation effect](#4.3)

[4.4 Application Scenarios](#4.4)

[References](#5)

![](https://static.igem.org/mediawiki/2021/2/2c/T--IvyMaker-China--wetlab1.jpg)

### Overview

![](https://static.igem.org/mediawiki/2021/6/6e/T--IvyMaker-China--Lab-34.png)

### Part 1 Bioinformatics prediction and analysis of anchor proteins

##### 1.1 Selection of a robust host

* Previous researches have compared the surface display system of different hosts as shown in Table 1. *Candida Tropicalis* was chosen as the host strain with strong viability, good safety and high industrial value.
![](https://static.igem.org/mediawiki/2021/4/48/T--IvyMaker-China--Lab-45.jpg)* Table 1 Comparison of surface display enzyme hosts
* [1] D. Gercke, C. Furtmann, I. E. P. Tozakidis, J. Jose, Highly Crystalline Post-Consumer PET Waste Hydrolysis by Surface Displayed PETase Using a Bacterial Whole-Cell Biocatalyst[J].ChemCatChem 2021, 13, 3479
* [2] Chen Z, Wang Y, Cheng Y, et al. Efficient biodegradation of highly crystallized polyethylene terephthalate through cell surface display of bacterial PETase [J]. Science of The Total Environment, 2019, 709:136138.

* **Strong viability**
* *Candida Tropicalis* is capable of absorbing alkanes and fatty acids as carbon sources and surviving in environments containing acids and phenolic substances.

* **Good safety**
* *Candida Tropicalis*is a level one biosafety strain. (https://www.atcc.org/products/20336)
  We used CRISPR-CAS9 technology to knock out the key genes of uracil synthesis in wild strains, which turned them into uracil nutrition-deficient strains to prevent leakage to the environment. Here are strains on the plate with different concentrations of uracil. It was found that the uracil nutrition-deficient strain constructed could not grow in solid medium without uracil, and the colony number increased with the increase of uracil concentration.
![](https://static.igem.org/mediawiki/2021/6/69/T--IvyMaker-China--Lab-30.jpg)* Fig.1 Growth of uracil nutritionally deficient strain in solid medium with different concentrations of uracil. (a) Growth medium without uracil. (Coating) (b) Growth medium with 0.006 g/L uracil. (Coating) (c) Growth medium supplement with 0.06 g/L uracil. (Coating) (d) Growth medium without uracil. (Plate streaking). (e) Growth medium with 0.006 g/L uracil. (Plate streaking). (f) Growth medium supplement with 0.06 g/L uracil. (Plate streaking)
* **High industrial value**
* Compared with other surface display systems, the surface display technology on *Candida Tropicalis* has not been fully developed. Therefore, it is worthy to establish the system on *Candida Tropicalis*. In addition, *Candida Tropicalis* is maturely used in the production of long-chain diacid, which lays a good foundation for its industrial applications.

##### 1.2 Prediction of GPI-anchored proteins

* Our experiment focused on selecting the best potential anchor protein for PETase surface display system. To achieve this goal, an anchor protein screening system was used to predict feasible candidates for our surface display system. The screening system included different online protein databases. For surface display systems, each polypeptide follows the same structure with a signal peptide on the N terminus to lead the protein out of the cell, a serine/threonine to support the structure, and a GPI-anchored protein on the C terminus to bind to the inner cell membrane. Figure 1 illustrated the workflow of screening process.
![](https://static.igem.org/mediawiki/2021/5/5e/T--IvyMaker-China--Lab-11-new.png)* Fig.2 Prediction model of GPI-anchored Protein.

* **The link of resources for GPI-anchored protein prediction were listed below.**
* Secreted signal peptide prediction database:
* http://www.cbs.dtu.dk/services/SignalP/
* GPI modification site prediction in fungi:
* http://mendel.imp.ac.at/gpi/fungi\_server.html
* Prediction of transmembrane helices in proteins:
* http://www.cbs.dtu.dk/services/TMHMM/
* Basic protein properties prediction database:
* https://web.expasy.org/protparam/
* Protein subcellular localization prediction tool:
* https://www.genscript.com/psort.html
* Website for conservative domain prediction:
* https://www.ncbi.nlm.nih.gov/Structure/wrpsb.cgi
* Predictions of mucin type GalNAc O-glycosylation sites in mammalian proteins:
* http://www.cbs.dtu.dk/services/NetOGlyc/

* **Results**
* 129 GPI-anchored proteins (alleles included) were obtained, including *ALS, Zan, HYR, HYR3, PHR, PHR2 and RHD3* gene families. The main information of several potential GPI-anchored proteins is presented in Table 2

* Table 2 Putative GPI-anchored proteins in *C. tropicalis*
![](https://static.igem.org/mediawiki/2021/5/59/T--IvyMaker-China--Lab-35.png)* Take 4609 as an example to explain the workflow.
* First, the big-PI Fungal Predictor was used to predict GPI modification sites and it was found that there are potential C-terminal GPI-Modification Sites at the position of amino acid residue 438. Then use the website SignalP 5.0 to predict the protein sequence numbered 4609, and the results show that Cleavage site between pos. 18 and 19: VSA-SY. Probability: 0.8063. Finally, the transmembrane domain prediction was performed by TMHMM. Results showed that there was no transmembrane domain and other related websites were used to predict its basic properties at the same time. In summary, the 4609 protein was preliminarily inferred to be a GPI-anchored protein.
![](https://static.igem.org/mediawiki/2021/4/40/T--IvyMaker-China--Lab-38.png)* Fig.3 Prediction of GPI modification sites

![](https://static.igem.org/mediawiki/2021/6/65/T--IvyMaker-China--Lab-36.png)* Fig.4 Signal peptide prediction for 4609

![](https://static.igem.org/mediawiki/2021/c/c8/T--IvyMaker-China--Lab-37.png)* Fig.5 Prediction of transmembrane domain

### Part 2 Identification of biological functions

##### 2.1 GFP was used to screen suitable anchor proteins

![](https://static.igem.org/mediawiki/2021/1/16/T--IvyMaker-China--Lab-03.jpg)
![](https://static.igem.org/mediawiki/2021/d/df/T--IvyMaker-China--Lab-04.jpg)* Fig.6 Structure of composite parts.
  Promoter--GAPDH gene promoter
  Terminator--GAPDH gene Terminator
  yeGFP--Enhanced Green Fluorescent Protein
  V5-tag: V5 tag is a short peptide tag for detection and purification of proteins. The V5 tag can be fused/cloned to a recombinant protein and detected in ELISA, flow cytometry, immunoprecipitation, immunofluorescence, and Western blotting with antibodies and Nanobodies.
* The structures of control and test groups were shown in Figure 3. Both groups involve GFP and V5-tag. GFP was used to indicate protein expression and V5-tag was used for protein purification. In the test group, a signal peptide was added upstream of the GFP which will allow the polypeptide to be exposed to the outer cellular space.

##### 2.2 Construction of plasmids

* Plasmid construction was completed in *E. coli*, and then plasmid was extracted and verified by enzyme digestion and further confirmed by sequencing. Subsequently the purified fragments were then transformed into *Candida tropicalis* following a previously protocol. Finally, the transformants were selected and further seeded in the liquid medium. After 24 to 36 hours, the genome was extracted as a template to verify the purpose fragments.
![](https://static.igem.org/mediawiki/2021/8/81/T--IvyMaker-China--Lab-08.jpg)
![](https://static.igem.org/mediawiki/2021/e/e4/T--IvyMaker-China--Lab-32.jpg)* Fig.7 Construction of the strain 4609. Plasmid map of Ts-CAT2-gda324-URA3-P-SS-yeGFP3-V5-4609-T (Left). Verification of recombinant plasmids with restriction enzyme digestion.
  M:DL 15000 DNA Maker(left); 1：Plasmid Ts-CAT2-gda324-URA3-P-SS-yeGFP3-V5-4609-T double enzyme digestion (EcoR Ⅰ &Xba Ⅰ) (Right).
![](https://static.igem.org/mediawiki/2021/e/ed/T--IvyMaker-China--Lab-09-new.jpg)
![](https://static.igem.org/mediawiki/2021/f/f5/T--IvyMaker-China--lab31.jpg)* Fig.8 Construction of the strain 5105. Plasmid map of Ts-CAT2-gda324-URA3-P-SS-yeGFP3-V5-5105-T (Left). Verification of recombinant plasmids with restriction enzyme digestion. M: DL 15000 DNA Maker; 1: Plasmid Ts-CAT2-gda324-URA3-P-SS-yeGFP3-V5-5105-T digested by EcoRⅠ& XbaⅠ(Right).

* **Results**
* Green fluorescence was detected in 9 of the anchored proteins with confocal laser microscopy, indicating that they could function normally.
* ![](https://static.igem.org/mediawiki/2021/c/c5/T--IvyMaker-China--Lab-12.jpg)
* ![](https://static.igem.org/mediawiki/2021/c/c0/T--IvyMaker-China--Lab-13.jpg)
* Fig.9 Representative images of yeGFP.
* Fig.10 Representative images of 25 screened anchor proteins.

![](https://static.igem.org/mediawiki/2021/6/6b/T--IvyMaker-China--Lab-07.jpg)
![](https://static.igem.org/mediawiki/2021/4/49/T--IvyMaker-China--Lab-14.jpg)* Fig.11 Representative images of 4609 (further screened anchor protein).

### Part 3 Surface display of PETase and MHETase

![](https://static.igem.org/mediawiki/2021/c/c3/T--IvyMaker-China--Lab-02.jpg)* Fig.12 Structures of the gene circuits.

![](https://static.igem.org/mediawiki/2021/b/bb/T--IvyMaker-China--Lab-10.jpg)
![](https://static.igem.org/mediawiki/2021/8/89/T--IvyMaker-China--Lab-42.png)* Fig.13 Construction of the surface display system of PETase. Plasmid map of Ts-CAT2-gda324-URA3-P-PETase-V5-4609-T (Left). Verification of recombinant plasmids with restriction enzyme digestion. M:DL 15000 DNA Maker; 1：Plasmid Ts-CAT2-gda324-URA3-P-SS-yeGFP3-ALS3-T; 2：Plasmid Ts-CAT2-gda324-URA3-P-SS-yeGFP3-ALS3-T digested by EcoRⅠ& ApaⅠ(Right).

### Part 4 Evaluation of degradation effect

##### 4.1 Determination of enzyme activity

![](https://static.igem.org/mediawiki/2021/d/d2/T--IvyMaker-China--Lab-22.jpg)
![](https://static.igem.org/mediawiki/2021/e/ec/T--IvyMaker-China--Lab-23.jpg)* Fig.14 Determination of enzyme activity of PETase.
* The overall enzyme activity of PETase was measured. Firstly, a crude test was carried out to show the enzyme activity at different temperatures. Then, p-nitrophenol absorption was measured because PETase could also catalyze substrates into p-nitrophenol. The results showed that PET-4609 and 5105 performed better than the wild type ATCC20336 and cytPET (Figure 14).

##### 4.2 Determination of degradation products with HPLC

* To characterize the enzyme degradation efficiency more accurately, HPLC method was adopted. Purified TPA, MHET, and BHET standards were run as control. For the treated group, we incubated yeast with PET powder and tested for its degradation. The data showed that PETase was able to degrade PET into TPA and MHET and at the same time (Figure 15). According to the HPLC detection results, the product content of the degraded PET powder of each strain were plotted (Figure 16). It was demonstrated that target products were not detected in the group of ATCC 20336. Compared with the control strain, degradation products were obviously presented in the strain PET-4609 and PET-5105, indicating PETase is displayed on the surface of *Candida tropicalis* with high enzyme activity. However, several products were detected in the strain cytPET which should not be detected theoretically. It was speculated that a part of PETase is released due to the lysis of cells.
* In conclusion, these results demonstrated that the surface display system was indeed able to degrade PET, which was consistent with the previous results.
![](https://static.igem.org/mediawiki/2021/d/de/T--IvyMaker-China--Lab-20.jpg)
![](https://static.igem.org/mediawiki/2021/a/a9/T--IvyMaker-China--Lab-21.jpg)* Fig.15 Determination of degradation products with HPLC

![](https://static.igem.org/mediawiki/2021/2/20/T--IvyMaker-China--Lab-39-new.png)* Fig.16 Hydrolysate content of PET powder. Content of PET powder: 10 mg, thallus: OD=5, reaction system: 1 mL, reaction time: 18 h.

##### 4.3 Surface display of MHETase to improve the degradation effect

* In order to further improve the degradation effect of PET powder, *Candida tropicalis* was used to display MHETase. First of all, we measured the activity of MHETase.
* Methods
* Conditions: 200 mg/L MHET as the substrate,1 mL reaction system, bacterial mass OD=1, buffer: 50 mM glycine-NaOH (pH 9.0), 30 °C, 900 rpm.
  Then centrifuge at 15000 g for 1 min and the supernatant was taken. The product was detected by HPLC.
* We set different reaction times, namely 10, 20, 30, 60, and 90 min. The HPLC results showed that the substrate decreased sharply within 60 min, and the degradation was almost complete at 60 min. Besides, MHET could not be detected at 90 minutes, indicating that the display of MHETase was successful.
![](https://static.igem.org/mediawiki/2021/b/bd/T--IvyMaker-China--Lab-43.png)* Fig.17 Determination of enzyme activity of MHETase.
* After confirming that *Candida tropicalis* showed a good degradation effect of MHETase, we used different proportions of cells (expressing PETase and MHETase, respectively) to degrade PET powder, and then respectively measured and calculated the content of different degradation products.
  Reaction conditions for degrading PET powder:
* 10 mg PET powder-the substrate
  the reaction system-1 mL
  bacteria displaying PETase OD=5
  bacteria displaying MHETase-different proportions
  the buffer：50 mM glycine-NaOH (pH 9.0)
  30 °C，for 18 h, 900 rpm.
* The reaction was terminated by heating at 100°C for 10 min. The supernatant was centrifuged at 15000 g for 1 min, and the product was detected by HPLC.
* The products of PETase degradation of PET are MHET, BHET and TPA. And MHETase can degrade MHET (one of the products of PETase degradation) into TPA and EG. Therefore, the content of MHET can reflect the activity of PETase, and the content of TPA reflects the result of co-catalysis.
* The results showed that when the displaying MHETase cells were added, MHET could be completely degraded after 18 hours. The results showed that the amount of MHETase added was not proportional to the degradation effect. When the ratio of PETase and MHETase cells was 2:1, the degradation effect was the best.
![](https://static.igem.org/mediawiki/2021/9/91/T--IvyMaker-China--Lab-26.jpg)
![](https://static.igem.org/mediawiki/2021/8/8b/T--IvyMaker-China--Lab-25.jpg)* Fig.18 HPLC determination of the degradation effect of different proportions of PETase and MHETase. Determination of MHET (Left). Determination of TPA (Right). P represents the cell displaying PETase. M represents the cell displaying MHETase. P+M represents the mixing of the two kinds of cells, and the OD600 of P is controlled at 5.

##### 4.4 Application scenarios

* To make our research closer to reality, we made a PET film with low crystallinity, which was used to quickly detect the degradation effect of PET enzyme.
* Methods:
* Cut the Coca-Cola bottle into PET flakes (⌀6 mm, 10 mg) and then dissolve them in 0.5 mL 1,1,1,3,3,3-hexafluoro-2-propanol solution. Amorphous PET can be generated by the volatilization of the solution.
* Results:
![](https://static.igem.org/mediawiki/2021/b/b7/T--IvyMaker-China--Lab-24.png)* Fig.19 The process of making PET films. (a). 10 mg PET plastic bottle with high crystallinity. (b). PET treated with hexafluoro-isopropyl alcohol. (c). PET film with low crystallinity

##### References

1. Sagong, Hye-Young, et al. "Decomposition Of The PET Film By MHETase Using Exo-PETase Function." ACS Catalysis, volume 10, issue 8, 2020, pp.4805-4812.
2. Knott, Brandon C., et al. "Characterization And Engineering Of A Two-enzyme System For Plastics Depolymerization." Proceedings of the National Academy of Sciences, volume 117, issue 41, 2020, pp.25476-25485.
3. Kim, Dong Hyun, et al. "One-Pot Chemo-bioprocess Of PET Depolymerization And Recycling Enabled By A Biocompatible Catalyst, Betaine." ACS Catalysis, volume 11, issue 7, 2021, pp.3996-4008.
4. Meng, Xiangxi, et al. "Protein Engineering Of Stable IsPETase For PET Plastic Degradation By Premuse." International Journal of Biological Macromolecules, volume 180, 2021, pp. 667-676.

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