iGEM 2021 — Team HK_GTC

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# Team:HK GTC/Notebook

HK\_GTC 2021 Homepage

[# HK\_GTC](https://2021.igem.org/Team%3AHK_GTC)

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* [Home](https://2021.igem.org/Team%3AHK_GTC)
* Team
* Project
* [Parts](https://2021.igem.org/Team%3AHK_GTC/Parts)
* [Safety](https://2021.igem.org/Team%3AHK_GTC/Safety)
* Human Practice
* [Deep Learning](https://2021.igem.org/Team%3AHK_GTC/Deep_learning)

# Team

* [Team Members](https://2021.igem.org/Team%3AHK_GTC/Team)
* [Attributions](https://2021.igem.org/Team%3AHK_GTC/Attributions)

# Project

* [Description](https://2021.igem.org/Team%3AHK_GTC/Description)
* [Engineering Success](https://2021.igem.org/Team%3AHK_GTC/Engineering)
* [Proof of Concept](https://2021.igem.org/Team%3AHK_GTC/Proof_Of_Concept)
* [Notebook](https://2021.igem.org/Team%3AHK_GTC/Notebook)
* [Experiments and Results](https://2021.igem.org/Team%3AHK_GTC/Results)
* [Implementation](https://2021.igem.org/Team%3AHK_GTC/Implementation)
* [Contribution](https://2021.igem.org/Team%3AHK_GTC/Contribution)

# Human Practice

* [Integrated Human Practice](https://2021.igem.org/Team%3AHK_GTC/Human_Practices)
* [Collaborations](https://2021.igem.org/Team%3AHK_GTC/Collaborations)
* [Partnership](https://2021.igem.org/Team%3AHK_GTC/Partnership)
* [Education & Communication](https://2021.igem.org/Team%3AHK_GTC/Communication)

[expand\_less](#the-top)

# Notebook

# August 2020

* [06/08/2020](#1)
* [26/08/2020](#2)

# September 2020

* [09/09/2020](#3)

# October 2020

* [24/10/2020](#24/10/2020)

# November 2020

* [28/11/2020](#28/11/2020)

# December 2020

* [28/12/2020](#4)

# January 2021

* [21/01/2021](#5)
* [25/01/21](#6)
* [29/01/21](#7)

# February 2021

* [03/02/21](#8)
* [10/02/21](#9)

# March 2021

* [19/3/2021](#19/3/2021)

# April 2021

* [26/04/21](#10)
* [30/04/21](#11)

# May 2021

* [05/05/21](#12)
* [24/05/21, 28/05/21](#24/05/21, 28/05/21)
* [31/05/21](#13)

# June 2021

* [24/06/21](#14)

# July 2021

* [13/07/21](#15)
* [16/07/21](#16)
* [17/07/21](#17)
* [19/07/21](#18)
* [20/07/21](#19)
* [20/07/21](#20/07/21)
* [20/7/21 – 24/7/21](#20)
* [21/07/21](#21)
* [25/07/21](#25/07/21)
* [26/07/21](#22)
* [30/07/21](#23)

# August 2021

* [02/08/21](#24)
* [04/08/21](#25)
* [09/08/21](#26)
* [11/08/21](#27)
* [20/8/21](#28)

# September 2021

* [13/09/21 – 16/09/21](#29)
* [15/09/21](#30)
* [30/09/21](#31)

# October 2021

* [15/10/21](#32)
* [16/10/21](#16/10/21)

## August 2020

### 06/08/2020

Protein extraction of WT PETase and mutant PETase, S245I from *E. coli*

### 26/08/2020

Determination of protein concentration using Bradford assay

## September 2020

### 09/09/2020

SDS-PAGE for PETase-S245I and PETase(WT)

## October 2020

### 24/10/2020

Plastic photography in Lei Yu Mun

## November 2020

### 28/11/2020

Plastic photography in Cheung Chau

## December 2020

### 28/12/2020

**Bacterial transformation**
Transformation of MHETase, WT PETase-MHETase, and S245I PETase-MHETase sequence within PET21b vector into
*E. coli* C41(DE3) and TOP10 competent cells by heat shock of 42°C

## January 2021

### 21/01/2021

PET film digestion at 30°C for 96 hours

### 25/01/21

Digested PET film processing

### 29/01/21

Examination of digested PET films under SEM

## February 2021

### 03/02/21

HPLC of solution from digestion of PET film

### 10/02/21

Colony PCR
used MHETase primer
Colony PCR of constructs transformed into C41(DE3) competent cells

## March 2021

### 19/3/2021

Conducted survey pilot testing in G.T. College

## April 2021

### 26/04/21

Interview with Professor Jacky Ngo, CUHK

### 30/04/21

Interview with Professor T.F. Chan, CUHK

## May 2021

### 05/05/21

Protein extraction of WT PETase, S245I PETase (1) and (2), MHETase, WT PETase-MHETase and S245I PETase-MHETase

### 24/05/21, 28/05/21

Conducted physical full-scale survey and plastic tagging activity in G.T. College

### 31/05/21

Interview with Mr. Sidhant Gupta and Mr. Utkarsh Goel, Clearbot

## June 2021

### 24/06/21

SDS-PAGE of extracted protein constructs on 05/05/21

## July 2021

### 13/07/21

Bradford Assay of WT PETase, S245I PETase (1) and MHETase

### 16/07/21

PET film digestion at 30°C for 96 hours

### 17/07/21

Protein extraction of WT PETase, S245I PETase and WT PETase-MHETase chimera

### 19/07/21

SDS PAGE of extracted proteins from 17/07/21

### 20/07/21

Digested PET film processing
PET film digestion using enzyme cocktails on 16/07/21

### 20/07/21

Virtual meetup with Cornell Team

### 20/7/21 – 24/7/21

Large scale protein extraction (400mL bacterial culture)

### 21/07/21

HPLC of solutions after PET film digestion using enzyme cocktails

### 21/07/21

Physical meetup with HKUST team

### 21/07/21

Plastic photography in Lantau Island

### 25/07/21

Virtual meetup with Stony Brook team

### 26/07/21

SDS PAGE of extracted proteins from 21 – 24/7/21

### 26/07/21

Physical meetup with HKU team

### 26/07/21

PET film digestion

### 30/07/21

PET film processing

## August 2021

### 02/08/21

HPLC of solutions after PET film digestion using chimeric and single enzymes

### 04/08/21

Interview with Ms. Dana Winograd, Plastic Free Seas

### 09/08/21

Sharing session by Ms. Dana Winograd, Plastic Free Seas

### 11/08/21

iGEM Symposium 2021, HKUST

### 20/8/21

**SEM**
We examined the digested PET film using 16μL chimeric proteins under the SEM.

## September 2021

### 13/09/21 – 16/09/21

SDS-PAGE and Western blot

### 15/09/21

iGEM sharing session for G7 and G8 GT school students

### 30/09/21

Interview with Mr. Nigel Mattraver, New Life Plastics

## October 2021

### 15/10/21

Interview with Mr. Hamlet Yu, HKSTP, Mr. Sam Chow and Mr. Kelvin Pong from BioArchitect

### 16/10/21

Hong Kong High School iGEM Symposium 2021

# GT COLLEGE iGEM2021

![](https://static.igem.org/mediawiki/2021/7/78/T--HK_GTC--logo.png)

# Follow Us!

# Contact:

igemteam.gt@gmail.com

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

# Team:HK GTC/Engineering

HK\_GTC 2021 Homepage

[# HK\_GTC](https://2021.igem.org/Team%3AHK_GTC)

*arrow\_back*

* [Home](https://2021.igem.org/Team%3AHK_GTC)
* Team
* Project
* [Parts](https://2021.igem.org/Team%3AHK_GTC/Parts)
* [Safety](https://2021.igem.org/Team%3AHK_GTC/Safety)
* Human Practice
* [Deep Learning](https://2021.igem.org/Team%3AHK_GTC/Deep_learning)

# Team

* [Team Members](https://2021.igem.org/Team%3AHK_GTC/Team)
* [Attributions](https://2021.igem.org/Team%3AHK_GTC/Attributions)

# Project

* [Description](https://2021.igem.org/Team%3AHK_GTC/Description)
* [Engineering Success](https://2021.igem.org/Team%3AHK_GTC/Engineering)
* [Proof of Concept](https://2021.igem.org/Team%3AHK_GTC/Proof_Of_Concept)
* [Notebook](https://2021.igem.org/Team%3AHK_GTC/Notebook)
* [Experiments and Results](https://2021.igem.org/Team%3AHK_GTC/Results)
* [Implementation](https://2021.igem.org/Team%3AHK_GTC/Implementation)
* [Contribution](https://2021.igem.org/Team%3AHK_GTC/Contribution)

# Human Practice

* [Integrated Human Practice](https://2021.igem.org/Team%3AHK_GTC/Human_Practices)
* [Collaborations](https://2021.igem.org/Team%3AHK_GTC/Collaborations)
* [Partnership](https://2021.igem.org/Team%3AHK_GTC/Partnership)
* [Education & Communication](https://2021.igem.org/Team%3AHK_GTC/Communication)

[expand\_less](#the-top)

# Engineering Success

# Engineering Success

* [Summary](#1)
* [Engineering achievement](#2)
* [Research](#3)
* [Design and Build](#4)
* [Test, Learn and Improve](#5)
* [Future Prospect](#6)
* [References](#7)

## Summary

Chemical recycling of PET is considered to be one of the best ways to close the loop on the plastic
supply chain and contribute towards the concept of circular economy in plastics. The technology is
evolving rapidly in order to allow more efficient PET degradation into its constituting monomers,
terephthalic acid (TPA) and ethylene glycol (EG). In our project, we developed a dual enzyme system to
synergize existing PET depolymerization processes which use a single enzyme, PETase. We went through a
cycle of engineering (Research → Design → Build → Test → Learn → improve). In 2019, our team created two
successful PETase mutants, which were shown to exhibit higher enzymatic activity than that of WT PETase.
This year, we performed PET film digestion with WT PETase and PETase mutants, S245I PETase in order to
study its performance. Scanning Electron Microscope (SEM) images of digested PET films showed surface
erosion. However, High-Performance Liquid Chromatography (HPLC) profiles of products released from PET
film digestions revealed considerable amounts of intermediate product, mono(2-hydroxyethyl) terephthalic
acid (MHET). It suggested that PET cannot be completely depolymerized by WT PETase and its mutant alone.
Then we started the process of researching and brainstorming possible ways to achieve complete PET
degradation.

From literature reviews, we found that MHETase catalyzes hydrolysis of MHET into the monomers of PET, TPA
and EG. Thus, we hypothesized that the addition of a second enzyme, MHETase, in the PET depolymerization
system synergizes the degradation rate of PET into its constituting monomers. The concept of developing a
dual-enzyme system was supported by the existence of a natural PET degradation system in a bacterium,
*Ideonella sakaiensis*, which produces both PETase and MHETase to degrade and assimilate PET [1]. Also, a
recent finding of improvement in PET depolymerization with a dual-enzyme PETase/MHETase system using
chimeric proteins offered further insight for us to design a chimeric protein of WT PETase and MHETase
[2]. Then we design a chimeric construct WT PETase-MHETase cloned into the expression vector pET-21b,
which has a C-terminal His-Tag for protein purification. However, we found that the protein induction and
extraction protocol we used previously were not suitable for new constructs, and several attempts were
made to optimize the conditions. In the end, chimeric protein was successfully expressed and purified
despite the presence of multiple bands. The hydrolytic activity of purified chimeric protein was tested
by PET film digestion and analyzed by HPLC and SEM. Since the purified chimeric proteins used in PET film
digestion contained a mixture of other proteins, only trace amounts of PET degradation monomer, TPA, were
detected. The protein extraction protocol needed to be further optimized. To gain a more comprehensive
view on the action of two-enzyme systems, we also developed the system in the form of enzyme cocktail in
which purified proteins of WT PETase and MHETase were mixed in a single reaction. Thus, we designed the
MHETase construct, which was also cloned into expression vector pET-21b for protein purification. We
demonstrated that PET degradation achieved by enzyme cocktail of WT PETase and MHETase was impaired
compared with that achieved by single enzyme WT PETase. We speculated that the fusion of MHETase to
PETase impaired activity of PETase and subsequent PET depolymerization.

## Engineering achievement

* We engineered WT PETase, which was shown to exhibit hydrolytic activity in PET film digestion.
  However, PET depolymerization was incomplete as shown by HPLC analysis. (Research)
* We developed a dual-enzyme system for complete PET degradation by engineering a chimeric protein of
  WT PETase and MHETase and a single protein of MHETase. (Design and Build)
* We purified WT PETase-MHETase chimeric protein and a MHETase protein and studied their hydrolytic
  activity in PET film digestion. (Test, Learn and Improve)
* We characterized a New Part ([BBa\_K3787001](http://parts.igem.org/Part%3ABBa_K3787001))
* We aimed to improve an Existing Part ([BBa\_K2982000](http://parts.igem.org/Part%3ABBa_K2982000))

## Research

After the digestion of polyethylene terephthalate (PET) plastic for 96 hours at 30°C using WT PETase
designed two years ago, we analyzed the eluent with High-Performance Liquid Chromatography (HPLC) and
noticed that the monomer mono-(2-hydroxyethyl)terephthalic acid (MHET) was present in high
concentrations, suggesting that PET is not fully depolymerized. On the other hand, the bacteria *Ideonella
sakaiensis* not only secretes PETase, but also MHETase, which can digest MHET. Since MHETase can digest
MHET, one of the intermediate products of PET digestion into constituent monomers, we created a chimeric
protein, linking WT PETase and MHETase, with the aim to further synergize the PET depolymerization
process by completely depolymerizing PET.

## Design and Build

Our experiments focus on two ways of incorporating two enzymes into a single reaction: in the form of
protein chimeras and enzyme cocktails. To construct the chimeric proteins, PETase and MHETase are
covalently attached by a linker. To engineer the chimeric protein, we used the pET-21b plasmid as our
expression vector. Following the sequence from the 5’ end to the 3’ end, we have the T7 promoter, the
lactose operon (LacO), the ribosome binding site (RBS), the coding sequence of PETase, a 12-amino-acid
flexible serine glycine linker, the coding sequence of MHETase and a 6x His-tag before a stop codon
(Figure 1). The T7 promoter induces faster gene expression, which leads to a higher yield of our chimeric
protein. The LacO reduces basal expression of PETase in normal conditions and prevents leaky expression
as it is highly effective. Our PETase-MHETase chimeric protein would only be expressed when IPTG is added
to the culture medium, so it prevents the overexpression which may lead to death of bacterial cells in
uninduced conditions.

![](https://static.igem.org/mediawiki/2021/b/b0/T--HK_GTC--eng_fig1.png)

Figure 1. The basic backbone of a chimeric construct, PETase-MHETase.

![](https://static.igem.org/mediawiki/2021/1/1f/T--HK_GTC--eng_fig2.png)

Figure 2. Amino acid sequence of a linker connecting PETase and MHETase.
G: Glycine; S: Serine

In this project, we are using a 12 amino acid serine-glycine linker (Figure 2) to connect the C-terminus
of PETase and the N-terminus of MHETase.

### Why 12 amino acids?

Referring to Knott (2020), linkers with 8, 12, and 20 amino acid lengths were put in test to determine
which one exhibited the best performance. It was demonstrated that using a 12 amino-acid linker to link
MHETase with PETase will give the highest PET depolymerization activity [2]. In the study, the amino acid
sequence of the 12-amino-acid linker used was GGGSGGSGGGSG, and we use it as our linker sequence.

### Why flexible linker?

Linkers consisting of only serine and glycine residues are called flexible linkers. Flexible linkers are
usually applied when the joined domains require a certain degree of movement or interaction. They are
generally composed of small, non-polar (e.g. glycine) or polar (e.g. serine or thymine) amino acids. The
small size of these amino acids provides flexibility and allows for mobility of the connecting functional
domains. Besides, suitable space between two proteins will improve protein folding and thus activity [3].

### Why serine-glycine linker?

Serine and glycine are small amino acids. They provide good flexibility due to their small sizes, and
also help maintain stability of the linker structure in the aqueous solvent through formation of hydrogen
bonds with water. As serine is polar, its incorporation can maintain the stability of the linker in
aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces the
unfavourable interaction between the linker and the protein moieties.

## Test, Learn and Improve

### (A) Protein expression and purification of WT PETase-MHETase

Proteins of PETase-MHETase chimeras were expressed in *E. coli* C41(DE3). The chimeric protein was induced
by the addition of 0.5mM final concentration of IPTG in a 100mL LC medium shake at room temperature for
16 – 20h. This protein extraction protocol was optimized for WT PETase protein previously. However, this
protocol didn’t work for PETase-MHETase. Since these single proteins have smaller size (30kDa), we were
able to express and purify WT PETase and S245I PETase in the same round of protein induction and
extraction. We believe that the bigger size of WT PETase-MHETase might be one of the factors affecting
the expression (Figure 3).

![](https://static.igem.org/mediawiki/2021/0/04/T--HK_GTC--eng_fig3.png)

Figure 3. SDS PAGE of purified WT PETase, S245I PETase and WT PETase-MHETase proteins.

We learned that the *E. coli* strain, temperature, design of linker and IPTG concentration might affect the
expression of chimeric protein. Therefore, we tried to optimize the temperature of protein induction.
Since the induction temperature of protein expression at 12°C – 16°C can improve the folding and increase
the solubility of the chimeric protein, we lowered the temperature from room temperature to 16°C and
changed the culture volume from 100mL to 400mL, increased the time of shaking from 16 – 20h to 28 – 30h,
and used the same concentration of IPTG (0.5mM). Our chimeric protein is successfully expressed and
purified, giving a thick band in the area corresponding to their mass in the SDS-PAGE result (Figure 4).

![](https://static.igem.org/mediawiki/2021/c/c5/T--HK_GTC--eng_fig4.png)

Figure 4. SDS-PAGE of purified WT PETase, S245I PETase, MHETase and WT PETase-MHETase protein.
Arrow showing the correct size of WT PETase-MHETase (95kDa)

Despite the presence of multiple bands using the revised protocol, the results of SDS-PAGE demonstrated
that the PETase-MHETase chimeras are successfully expressed and purified. The protein induction and
extraction protocol will be further optimized in the future in order to obtain a single band of chimeric
proteins.

### (B) HPLC profile of the products released from the PET films

Products released from PET films after digestion with WT PETase-MHETase were detected and quantified by
HPLC.

#### (i) TPA Standard

Maximum intensity: 2600 cps at 4.64 min

![](https://static.igem.org/mediawiki/2021/e/ef/T--HK_GTC--eng_fig5.png)

Figure 5. HPLC profile of 0.397µM TPA standard

#### (ii) Products released from the PET film incubated with WT PETase-MHETase

Maximum intensity: 680 cps at 4.69 min

![](https://static.igem.org/mediawiki/2021/b/b4/T--HK_GTC--eng_fig6a.png)

Figure 6a. HPLC profile of products released from PET film digestion using 8µL PETase-MHETase

Maximum intensity: 1340 cps at 4.67 min

![](https://static.igem.org/mediawiki/2021/8/88/T--HK_GTC--eng_fig6.png)

Figure 6b. HPLC profile of products released from PET film digestion using 16µL PETase-MHETase

#### (iii) Products released from the PET film incubated with buffer only

Maximum intensity: 990 cps at 4.65 min

![](https://static.igem.org/mediawiki/2021/1/1d/T--HK_GTC--eng_fig7.png)

Figure 7. HPLC profile of products released from PET film digestion with buffer only

#### (iv) Quantification of TPA released from PET film digestion with WT PETase-MHETase

| Purified proteins | Calculated concentration (ng/mL) |
| --- | --- |
| 8μL PETase – MHETase | 0.0921 |
| 16μL PETase – MHETase | 0.0655 |
| Buffer only | 0.0135 |

Table 1. Calculated concentration of TPA released from PET film digestion with WT PETase-MHETase

HPLC profiles demonstrated that the detection peak representing TPA monomer has a retention time at 4.64 minutes (Figure 5). 8μL and 16μL of extracted chimera protein was used to digest PET film in order to investigate their PET depolymerization activity. HPLC data showing that trace amounts of TPA were detected in eluents of PET film digestion with PETase-MHETase, suggesting that the chimeras exhibit PET depolymerization activity (Table 1). However, the concentration of chimeric proteins added were different due to the presence of impurities as indicated by multiple bands observed in SDS-PAGE. Therefore, their degradation rate could not be compared.

### (C) SEM of digested PET film

#### (i) PET film incubated with PETase-MHETase chimera

![](https://static.igem.org/mediawiki/2021/e/e9/T--HK_GTC--eng_fig8.png)

Figure 8. PET film after incubation with 16μL WT PETase-MHETase chimera

#### (ii) PET film incubated with buffer only

![](https://static.igem.org/mediawiki/2021/3/3b/T--HK_GTC--eng_fig9.png)

Figure 9. PET film after incubation with 16μL buffer only

Consistent with the result from HPLC in which chimeric proteins exhibited PET depolymerization activity and released trace amounts of TPA, the pitting of PET film surface resulting from the digestion of PETase-MHETase was observed (Figure 8). No pitting of PET film surface was observed when PET film was incubated with buffer-only solution (Figure 9).

### (D) SDS-PAGE and western blot analysis

Since a few bands were obtained in our purified chimeric protein WT PETase-MHETase, SDS-PAGE and western blot were performed to confirm the identities of purified proteins. All the genes encoding WT PETase-MHETase were cloned into the expression vector pET-21b, which has a C-terminal His-Tag. Therefore, His-Tag antibody was used for the verification of expressed protein.

![](https://static.igem.org/mediawiki/2021/0/0c/T--HK_GTC--eng_fig10.png)

Figure 10. SDS-PAGE of purified proteins. Figure 11. Western blot of purified proteins.

Lane 4: PETase-MHETase (95kDa). Arrows show PETase-MHETase purified proteins.

Our SDS-PAGE results showed that the purified WT PETase-MHETase protein was expressed (Figure 10). Clear bands with correct sizes were observed in the western blot of all purified proteins (Figure 11).

These results demonstrated that our chimeric protein, PETase-MHETase, was successfully expressed and purified. It exhibited PET depolymerization activity as seen by the presence of TPA detected in HPLC analysis and the degraded PET film surfaces observed under SEM.

## Future Prospect

This engineering cycle has led us to the challenge to test different combination of amino acid length and sequence of protein linker in the chimeric protein in order to improve protein folding and thus its expression. Also, we will optimize our protocol, such as the imidazole concentration used in our wash and elution buffers. After obtaining our purified WT PETase-MHETase protein, we can develop a more efficient system for complete PET depolymerization.

## References

[1]: Yoshida, S., Hiraga, K. et al. (2016). A bacterium that degrades and assimilates poly(ethylene terephthalate). Science,351(6278), 1196-1199. doi:10.1126/science.aad6359
[2]: Knott B. C. (2020). Characterization and engineering of a two-enzyme system for plastics depolymerization, pnas.org. DOI:10.1073/pnas.2006753117
[3]: Chen, X., Zaro, J. L., & Shen, W. C. (2013). Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews, 65(10), 1357–1369. https://doi.org/10.1016/j.addr.2012.09.039

# GT COLLEGE iGEM2021

![](https://static.igem.org/mediawiki/2021/7/78/T--HK_GTC--logo.png)

# Follow Us!

# Contact:

igemteam.gt@gmail.com

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

# Team:HK GTC/Experiments

HK\_GTC 2021 Homepage

[# HK\_GTC](https://2021.igem.org/Team%3AHK_GTC)

*arrow\_back*

* [Home](https://2021.igem.org/Team%3AHK_GTC)
* Team
* Project
* [Parts](https://2021.igem.org/Team%3AHK_GTC/Parts)
* [Safety](https://2021.igem.org/Team%3AHK_GTC/Safety)
* Human Practice
* [Deep Learning](https://2021.igem.org/Team%3AHK_GTC/Deep_learning)

# Team

* [Team Members](https://2021.igem.org/Team%3AHK_GTC/Team)
* [Attributions](https://2021.igem.org/Team%3AHK_GTC/Attributions)

# Project

* [Description](https://2021.igem.org/Team%3AHK_GTC/Description)
* [Engineering Success](https://2021.igem.org/Team%3AHK_GTC/Engineering)
* [Proof of Concept](https://2021.igem.org/Team%3AHK_GTC/Proof_Of_Concept)
* [Notebook](https://2021.igem.org/Team%3AHK_GTC/Notebook)
* [Experiments and Results](https://2021.igem.org/Team%3AHK_GTC/Results)
* [Implementation](https://2021.igem.org/Team%3AHK_GTC/Implementation)
* [Contribution](https://2021.igem.org/Team%3AHK_GTC/Contribution)

# Human Practice

* [Integrated Human Practice](https://2021.igem.org/Team%3AHK_GTC/Human_Practices)
* [Collaborations](https://2021.igem.org/Team%3AHK_GTC/Collaborations)
* [Partnership](https://2021.igem.org/Team%3AHK_GTC/Partnership)
* [Education & Communication](https://2021.igem.org/Team%3AHK_GTC/Communication)

# 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:HK GTC/Results

HK\_GTC 2021 Homepage

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# Experiment & Results

# Research Team

* [1. Confirmation of PET hydrolytic activities of WT PETase and engineered mutant, S245I
  PETase studied in iGEM 2019](#1)
* [2. Confirmation of the presence of insert DNA in plasmid constructs](#2)
* [3. PET hydrolytic activities of PETase and MHETase cocktails](#3)
* [4. PET hydrolytic activities of PETase-MHETase chimeras](#4)
* [5. SDS-PAGE and Western blot analysis](#5)

## 1. Confirmation of PET hydrolytic activities of WT PETase and engineered mutant, S245I PETase studied in iGEM 2019

In 2019, our team created a successful PETase mutant, S245I, which exhibits higher enzymatic activity
than that of WT PETase using 4-nitrophenyl dodecanoate as a substrate. To explore further insights of the
performance of our engineered PETase mutant, this year, we perform PET film digestion to compare the PET
hydrolytic activities of both WT PETase and S245I PETase. After incubation of equal amount (9 μg) of
purified proteins with PET film at 30°C for 96h, we performed High Performance Liquid Chromatography
(HPLC) analysis of PET digestion eluents and Scanning Electron Microscope (SEM) of digested PET film.

### (A) SEM images of degraded PET films

![](https://static.igem.org/mediawiki/2021/e/e4/T--HK_GTC--1a.jpeg)

Figure 1a. PET film after incubation with WT PETase

![](https://static.igem.org/mediawiki/2021/a/af/T--HK_GTC--1b.jpeg)

Figure 1b. PET film after incubation with the PETase mutant, S245I

![](https://static.igem.org/mediawiki/2021/0/01/T--HK_GTC--1c.png)

Figure 1c. Buffer-only control of PET film

The pitting resulting from the digestion of PETase mutant, S245I, is much more significant than that from
the digestion of WT PETase. Consistent with enzyme assay results shown in 2019, S245I PETase exhibits a
higher depolymerization activity.

### (B) HPLC profile of the products released from the PET films

Products released from PET films after digestion with WT PETase and S245I PETase were detected and
quantified by HPLC.

![](https://static.igem.org/mediawiki/2021/d/dc/T--HK_GTC--2a.png)

Figure 2a. HPLC spectrum of TPA standard.

![](https://static.igem.org/mediawiki/2021/5/58/T--HK_GTC--2b.png)

Figure 2b. HPLC spectrum of MHET standard.

![](https://static.igem.org/mediawiki/2021/2/2e/T--HK_GTC--2c.png)

Figure 2c. HPLC spectrum of products released from the PET film digested with WT PETase.

![](https://static.igem.org/mediawiki/2021/6/60/T--HK_GTC--2d_new.png)

Figure 2d. HPLC spectrum of products released from the PET film digested with S245I PETase.

|  |  |  |  |
| --- | --- | --- | --- |
| Construct of PETase | Amount of protein added (μg) | MHET conc. (ng/mL) | TPA conc. (ng/mL) |
| S245I | 9 | 232 | 1510 |
| WT | 9 | 185 | 1390 |

Table 1. Quantification of products released from PET film digestions

HPLC profiles demonstrated that the detection peaks representing TPA monomer and MHET intermediate
product formed during PET film digestion have retention times at 4.64 minutes (Figure 2a) and 5.17
minutes (Figure 2b) respectively.

HPLC profiles of products released from PET film digestions using a single enzyme, WT PETase or S245I
PETase revealed incomplete PET hydrolysis as considerable amounts of intermediate product, MHET, which
showed a peak at 5.07 minutes were detected (Figure 2c and Figure 2d).
Thus, we hypothesized that the presence of a second enzyme, MHETase, which hydrolyzes MHET into TPA, in
the
PET depolymerization system synergizes the degradation rate of PET into its constituting monomers.
Therefore, to completely depolymerize PET, we aimed to develop dual-enzyme systems consisting of PETase
and
MHETase in this project.

## 2. Confirmation of the presence of insert DNA in plasmid constructs

A dual-enzyme system of PETase and MHETase is developed in the form of chimeras or enzyme cocktails.
For the chimeric constructs covalently linking PETase and MHETase, C-terminus of both WT PETase and
S245I PETase were linked to the N-terminus of MHETase using a 12 amino acid serine-glycine linker,
forming WT PETase-MHETase and S245I PETase-MHETase chimeric constructs. The chimeric constructs were
then cloned into the expression vector pET-21b digested with NdeI and XhoI. pET-21b has a C-terminal
His-Tag for subsequent protein purification. Besides, gene encoding MHETase from *Ideonella sakaiensis*
was also cloned into NdeI and XhoI digested pET-21b for subsequent protein purification and used in
enzyme cocktail experiments.

Colony PCR
We transformed the three recombinant plasmids, MHETase-pET-21b, WT PETase-MHETase-pET21b
and S245I PETase-MHETase-pET 21b in *E. coli* C41 (DE3) and TOP10 competent cells. We performed colony
PCR to confirm the presence of insert DNA in plasmids after transformation.

![](https://static.igem.org/mediawiki/2021/1/1b/T--HK_GTC--3.png)

Figure 3. Colony PCR screening of constructs transformed in C41(DE3) competent cells.

![](https://static.igem.org/mediawiki/2021/4/42/T--HK_GTC--9%283%29.png)

Figure 4. Colony PCR screening of constructs transformed in TOP10 competent cells.

Figure 3 and Figure 4 showed that PCR products with the correct size of 498 bp were amplified in all
constructs confirming the presence of MHETase, WT PETase-MHETase and S245I PETase-MHETase in the
expression vector pET-21b.

## 3. PET hydrolytic activities of PETase and MHETase cocktails

### (A) Protein expression and purification of WT PETase, S245I PETase and MHETase

Proteins of WT PETase, MHETase and S245I PETase were expressed in *E. coli* C41(DE3) and induced by the
addition of 0.5mM final concentration of IPTG in a 100mL LC medium shake at room temperature for 16 –
20h. Proteins were extracted and purified. The size of WT PETase and S245I PETase is 30kDa and that of
MHETase is 65kDa.

![](https://static.igem.org/mediawiki/2021/7/7e/T--HK_GTC--5.png)

Figure 5. SDS PAGE of purified MHETase, WT PETase and S245I PETase proteins.

### (B) Bradford protein assay

We performed Bradford protein assay to determine the concentration of our purified proteins.
The resulting concentrations of WT PETase, S245I PETase, and MHETase are at 1.35 mg/mL, 1.61 mg/mL,
and 0.77 mg/mL respectively.

![](https://static.igem.org/mediawiki/2021/6/6b/T--HK_GTC--6.png)

Figure 6. BSA standard curve. The line of best fit is drawn, with the R2 value being 0.994.

### (C) PET film digestion

To analyze the degradation rate of PET by enzyme cocktails, commercial PET film was used as the
substrate for enzyme assay. The PET film was prepared in a square form with a length of 6 mm. It was
then soaked in 300μL of pH 9.0 glycine-NaOH buffer with 4μg or 8μg of enzymes.

In the enzyme cocktails, 4μg WT PETase or S245I PETase were mixed with 4μg and 8 μg MHETase. A
solution with only 4μg WT PETase and a solution with only S245I PETase was used as a control to
compare the PET degradation rate of single-enzyme systems and dual-enzymes systems.

|  |  |  |  |
| --- | --- | --- | --- |
| Reaction | Concentration of WT PETase (μg) | Concentration of S245I PETase (μg) | Concentration of MHETase (μg) |
| 1 | 4 | 0 | 4 |
| 2 | 4 | 0 | 8 |
| 3 | 4 | 0 | 0 |
| 4 | 0 | 4 | 4 |
| 5 | 0 | 4 | 8 |
| 6 | 0 | 4 | 0 |
| 7 | 0 | 0 | 0 |

Table 2. Combinations of WT PETase, S245I PETase and MHETase in different enzyme cocktails.

The reaction mixture was incubated at 30°C for 96h. The reaction mixture was terminated by dilution of
the aqueous solution with 160mM phosphate buffer (pH 2.5) containing 10% (v/v) DMSO followed by
heating at 85°C for 15 min, after which the PET film was removed from the reaction mixture. Then the
samples were centrifuged at 13,200 rpm for 10 min. 20μL of the supernatant was analyzed by HPLC. The
film was washed with 1% SDS and 20% ethanol in distilled water.

### (D) HPLC of eluents of digested PET film with different enzyme cocktails

#### (i) TPA Standard

Maximum intensity: 36000cps at 4.62 min

![](https://static.igem.org/mediawiki/2021/2/2f/T--HK_GTC--7.png)

Figure 7. HPLC profile of 25μM TPA standard

#### (ii) Products released from the PET film incubated with WT PETase and MHETase cocktails

Maximum intensity: 1736.7 cps at 4.64 min

![](https://static.igem.org/mediawiki/2021/1/1b/T--HK_GTC--8a.png)

Figure 8a. HPLC profile of products released from PET film digestion using 4 μg WT PETase and 4
μg MHETase

Maximum intensity: 3986.7 cps at 4.62 min

![](https://static.igem.org/mediawiki/2021/a/ac/T--HK_GTC--8b.png)

Figure 8b. HPLC profile of products released from PET film digestion using 4 μg WT PETase and 8
μg MHETase

Maximum intensity: 16000 cps at 4.63 min

![](https://static.igem.org/mediawiki/2021/e/ec/T--HK_GTC--8c.png)

Figure 8c. HPLC profile of products released from PET film digestion using 4 μg WT PETase only

#### (iii) Products released from the PET film incubated with S245I PETase and MHETase cocktails

Maximum intensity: 7756.7 cps at 4.63 min

![](https://static.igem.org/mediawiki/2021/3/37/T--HK_GTC--9a.png)

Figure 9a. HPLC profile of products released from PET film digestion using 4 μg S245I PETase and
4 μg MHETase

Maximum intensity: 8166.7 cps at 4.62 min

![](https://static.igem.org/mediawiki/2021/9/93/T--HK_GTC--9b.png)

Figure 9b. HPLC profile of products released from PET film digestion using 4 μg S245I PETase and
8 μg MHETase

Maximum intensity: 4906.7 cps at 4.63 min

![](https://static.igem.org/mediawiki/2021/0/02/T--HK_GTC--9c.png)

Figure 9c. HPLC profile of products released from PET film digestion using 4 μg S245I PETase
only

#### (iv) Products released from the PET film incubated with buffer only

Maximum intensity: 2193.3 cps at 4.63 min

![](https://static.igem.org/mediawiki/2021/a/a6/T--HK_GTC--10.png)

Figure 10. HPLC profile of products released from PET film digestion without using enzyme
(negative control)

#### (v) Quantification of TPA released from PET film digestion with different enzyme cocktails

![](https://static.igem.org/mediawiki/2021/6/69/T--HK_GTC--11.jpeg)

Figure 11. A standard curve of TPA determined by HPLC.

![](https://static.igem.org/mediawiki/2021/d/d3/T--HK_GTC--fig12.jpg)

Figure 12. Concentration of TPA released from PET film digestion using different enzyme
cocktails.
S1: 4μg WT PETase and 4μg MHETase; S2: 4μg WT PETase and 8μg MHETase: S3: 4μg WT PETase only
S4: 4μg S245I PETase and 4μg MHETase;
 S5: 4μg S245I PETase and 8μg MHETase: S6: 4μg S245I
only
S7: buffer only

HPLC profiles demonstrated that the detection peak representing TPA monomer has a retention time at
4.62 minutes (Figure 7).

HPLC profiles of products released from PET film digestions using WT PETase and MHETase demonstrated
that a mixture of WT PETase and MHETase in a 1:2 ratio exhibited better depolymerization performance
than that in a 1:1 ratio as more monomers were released (Figure 8a and Figure 8b). However, the extent
of depolymerization achieved by WT PETase alone was the highest (Figure 8c) suggesting that the
addition of MHETase inhibited or slowed down the PET degradation. We cannot exclude the possibility
that the fusion of MHETase to WT PETase impaired activity of WT PETase and subsequent PET
depolymerization. Thus, the experimental result needs to be further investigated.

On the other hand, the enzyme cocktail with S245I PETase and MHETase synergizes PET degradation. A
mixture of S245I PETase and MHETase in a 1:2 ratio shows similar degradation activity with that in a
1:1 ratio but both exhibited better degradation activity than the mixture with S245I PETase alone as
more TPA were determined in the cocktails (Figure 9a – c).

A standard curve of TPA was obtained by HPLC analysis of 9 standard solutions (0.098μM, 0.195μM,
0.397μM, 0.78μM, 1.56μM, 3.125μM, 6.25μM, 12.5μM and 25μM) using serial dilution (Figure 11). PET film
digestion eluent has a background intensity of 2193 cps as determined by HPLC (Figure 10). TPA
concentration released from PET film digestion using different enzyme cocktails demonstrated that the
mixture with S245I PETase only released 2.02 ng/mL TPA, while the mixture with 1:1 and 1:2 ratio of
S245I PETase and MHETase released 3.49 ng/mL and 3.44 ng/mL (Figure 12). Comparing the extent of PET
degradation by S245I PETase alone, addition of MHETase synergizes depolymerization process by
increasing constituent monomer, TPA, up to 1.7 folds. The findings revealed that our engineered
mutant, S245I PETase, which outperforms WT PETase, further synergizes PET depolymerization in the
presence of MHETase.

### (E) SEM of digested PET film incubated with different enzyme cocktails

#### (i)PET film incubated with WT PETase and MHETase cocktails

![](https://static.igem.org/mediawiki/2021/a/ae/T--HK_GTC--13a.jpeg)

Figure 13a. PET film after incubation with 4μg WT PETase and 4μg MHETase

![](https://static.igem.org/mediawiki/2021/e/ee/T--HK_GTC--13b.png)

Figure 13b. PET film after incubation with 4μg WT PETase and 8μg MHETase

![](https://static.igem.org/mediawiki/2021/2/2b/T--HK_GTC--13c.jpeg)

Figure 13c. PET film after incubation with 4μg WT PETase only

#### (ii) PET film incubated with S245I PETase and MHETase cocktails

![](https://static.igem.org/mediawiki/2021/d/de/T--HK_GTC--14a.png)

Figure 14a. PET film after incubation with 4μg S245I PETase and 4μg MHETase

![](https://static.igem.org/mediawiki/2021/d/dc/T--HK_GTC--14b.png)

Figure 14b. PET film after incubation with 4μg S245I PETase and 8μg MHETase

![](https://static.igem.org/mediawiki/2021/f/fb/T--HK_GTC--14c.jpeg)

Figure 14c. PET film after incubation with 4μg S245I PETase only

#### (iii) PET film incubated with buffer only

![](https://static.igem.org/mediawiki/2021/6/6c/T--HK_GTC--15.png)

Figure 15. PET film after incubation with buffer only

Consistent with the result from HPLC, the pitting of PET film surface resulting from the digestion of
WT PETase only is much more significant than from the digestion of WT PETase and MHETase cocktails
(Figure 13a – c). Similarly, the pitting of PET film surface resulting from the digestion of 1:1 and
1:2 ratio of S245I PETase and MHETase is much more significant than from the digestion of S245I PETase
only (Figure 14a – c). Consistent with enzyme assay results shown in 2019, S245I PETase exhibits a
higher depolymerization activity. No pitting of PET film surface was observed in mixture with buffer
only (Figure 15). Our HPLC and SEM results demonstrated that cocktail mixtures of 1:1 or 1:2 ratio of
S245I PETase and MHETase synergize PET depolymerization process compared with S245I PETase alone.

## 4. PET hydrolytic activities of PETase-MHETase chimeras

### (A) Protein expression and purification of WT PETase-MHETase and S245I PETase-MHETase

Proteins of PETase-MHETase chimeras were expressed in *E. coli* C41(DE3). Using the same protein
extraction
method as that of PETase and MHETase as described in section 3(A) in which chimeric protein was
induced
by
the addition of 0.5mM final concentration of IPTG in a 100mL LC medium shake at room temperature for
16 –
20h, no WT PETase-MHETase chimeric protein was expressed. The size of WT PETase-MHETase is 95kDa.

![](https://static.igem.org/mediawiki/2021/5/59/T--HK_GTC--16.png)

Figure 16. SDS-PAGE of purified WT PETase, S245I PETase and WT PET-MHETase

To optimize the condition for protein expression, we changed the induction condition in which the
400mL
culture medium was induced with 0.5mM IPTG and shook at 16°C for 24 - 30h.

Protein extraction protocol was also revised. After induction, 400 mL bacterial culture was divided
into
two
200 mL solutions and harvested the cells by centrifugation at 5,000 rpm at 4°C for 15 minutes. The
cell
pellets were resuspended by adding 20 mL of lysis buffer. We further sonicated the suspension 5 times
for
15
cycles; each cycle consists of 10s with sonication followed by 10s without sonication. The sonication
power
is 6-8 W. After centrifuging at 13,000 rpm for 20 min at 4°C, 20mL supernatants were collected. 2 mL
Ni-NTA
resin was washed with lysis buffer for 5 times with short spins at 3,000 rpm and kept on ice. Then, 10
mL
of
the supernatant was mixed with 1 mL Ni-NTA resin and then shook on ice for 1 hour at 50 rpm. After
rinsing
the Nickel column 3 - 4 times with lysis buffer, the mixture was loaded to a column. The column was
then
washed 3 times with wash buffer and 3 times with 2 mL elution buffer. For SDS-PAGE, we mixed 15 µL of
purified proteins and 5 µL 4x loading dye and loaded into the wells. The size of PETase-MHETase
chimera
is
95kDa, with PETase and MHETase are 30kDa and 65 kDa respectively. If our proteins are successfully
expressed
and purified, they should give a thick band in the area corresponding to their mass in the SDS-PAGE.

![](https://static.igem.org/mediawiki/2021/5/51/T--HK_GTC--ex_2_2.png)

Figure 17. SDS-PAGE of purified WT PETase, S245I PETase, MHETase, WT PET-MHETase and
S245I PETase-MHETase. Arrows showing the correct size of new constructs, MHETase,
WT PETase-MHETase and S245I PETase-MHETase

The results of SDS-PAGE demonstrated the presence of purified WT PETase, S245I PETase, MHETase WT
PETase-MHETase, and S245I PETase-MHETase. It also showed that PETase-MHETase chimeras are successfully
expressed and purified despite the presence of multiple bands. The protein induction and extraction
protocol
will be optimized in the future to obtain a single band of chimeric proteins.

### (B) PET film digestion

To analyze the degradation activity of engineered chimeras, commercial PET film was used as the
substrate
for enzyme assay. The PET film was prepared in a square form with a length of 6 mm. It was then soaked
in
300μL of pH 9.0 glycine-NaOH buffer with 8µL and 16µL of enzymes, MHETase, WT PETase-MHETase and S245I
PETase-MHETase. A mixture with buffer only was used as a negative control. We incubated the mixtures
for
96h
at 30°C.

### (C) HPLC of eluents of digested PET film with different chimeras

We analyzed the eluent after the PET film digestion, and showed that the solution contained the
constituent
monomer of PET, TPA

#### (i) TPA Standard

Maximum intensity: 2600 cps at 4.64 min

![](https://static.igem.org/mediawiki/2021/9/9b/T--HK_GTC--18.png)

Figure 18. HPLC profile of 0.397µM TPA standard

#### (ii) Products released from the PET film incubated with MHETase only

Maximum intensity: 1050 cps at 4.71 min

![](https://static.igem.org/mediawiki/2021/3/37/T--HK_GTC--19a.png)

Figure 19a. HPLC profile of products released from PET film digestion using 8µL MHETase

Maximum intensity: 1050 cps at 4.66 min

![](https://static.igem.org/mediawiki/2021/9/96/T--HK_GTC--19b.png)

Figure 19b. HPLC profile of products released from PET film digestion using 16µL MHETase

#### (iii) Products released from the PET film incubated with S245I PETase-MHETase

Maximum intensity: 1160 cps at 4.66 min

![](https://static.igem.org/mediawiki/2021/b/bf/T--HK_GTC--20a.png)

Figure 20a. HPLC profile of products released from PET film digestion using 8µL S245I
PETase-MHETase

Maximum intensity: 1080 cps at 4.67 min

![](https://static.igem.org/mediawiki/2021/9/9f/T--HK_GTC--20b.png)

Figure 20b. HPLC profile of products released from PET film digestion using 16µL S245I
PETase-MHETase

#### (iv) Products released from the PET film incubated with WT PETase-MHETase

Maximum intensity: 1340 cps at 4.67 min

![](https://static.igem.org/mediawiki/2021/0/05/T--HK_GTC--21a.png)

Figure 21a. HPLC profile of products released from PET film digestion using 8µL WT PETase-MHETase

Maximum intensity: 990 cps at 4.65 min

![](https://static.igem.org/mediawiki/2021/0/0f/T--HK_GTC--21b.png)

Figure 21b. HPLC profile of products released from PET film digestion using 16µL WT PETase-MHETase

#### (v) Products released from the PET film incubated with buffer only

Maximum intensity: 680 cps at 4.69 min

![](https://static.igem.org/mediawiki/2021/a/aa/T--HK_GTC--22.png)

Figure 22. HPLC profile of products released from PET film digestion with buffer only

#### (vi) Quantification of TPA released from PET film digestion with different chimeras

![](https://static.igem.org/mediawiki/2021/6/6b/T--HK_GTC--Table.png)

Table 3. Calculated concentration of TPA released from PET film digestion with chimeras

Equal volume of extracted chimera proteins was used to digest PET film in order to investigate their
PET
depolymerization activity. HPLC data showing that trace amounts of TPA were detected in eluents of PET
film digestion with WT PETase-MHETase and S245I PETase-MHETase suggesting that chimeras exhibit PET
depolymerization activity (Table 3). However, their degradation rate could not be compared as the
concentration of chimeric proteins added were different due to the presence of impurities as indicated
by
multiple bands observed in SDS-PAGE.

#### (D) SEM of digested PET film with different chimeras

##### (i) PET film incubated with MHETase only

![](https://static.igem.org/mediawiki/2021/9/93/T--HK_GTC--23.png)

Figure 23. PET film after incubation with 16μL MHETase

##### (ii) PET film incubated with S245I PETase-MHETase chimera

![](https://static.igem.org/mediawiki/2021/f/f9/T--HK_GTC--24.png)

Figure 24. PET film after incubation with 16 μL S245I PETase-MHETase chimera

##### (iii) PET film incubated with WT PETase-MHETase chimera

![](https://static.igem.org/mediawiki/2021/7/7d/T--HK_GTC--25.png)

Figure 25. PET film after incubation with 16μL WT PETase-MHETase chimera

##### (iv) PET film incubated with buffer only

![](https://static.igem.org/mediawiki/2021/7/71/T--HK_GTC--26.jpeg)

Figure 26. PET film after incubation with 16μL buffer only

Consistent with the result from HPLC in which chimeric proteins exhibited PET depolymerization
activity
and released trace amount of TPA, the pitting of PET film surfaces resulting from the digestion of
S245I
PETase-MHETase and WT PETase-MHETase was observed (Figure 24 and Figure 25). No pitting of PET film
surface was observed when PET film was incubated with buffer only solution (Figure 26). However,
pitting
of PET film could also be observed in the PET film digested with MHETase only (Figure 23). This result
was not consistent with HPLC data which showed negligible amounts of TPA. The effect of digestion of
PET
film using MHETase will be further investigated.

## 5. SDS-PAGE and Western blot analysis

Since a few bands were obtained in purified chimeric proteins, WT PETase-MHETase and S245I
PETase-MHETase, SDS-PAGE and western blot were performed to confirm the identity of purified proteins.
The purified proteins, WT PETase, S245I PETase and MHETase were also included in this experiment. All
the
genes encoding PETase, MHETase and their chimeras were cloned into the expression vector pET-21b which
has a C-terminal His-Tag. Therefore, His-Tag antibody was used for the verification of expressed
proteins.

![](https://static.igem.org/mediawiki/2021/7/75/T--HK_GTC--27_28_new.png)

Lane 1: WT PETase (30kDa)
Lane 2: S245I PETase (30kDa)
Lane 3: MHETase (65kDa)
Lane 4: WT PETase – MHETase (95kDa)
Lane 5: S245I PETase – MHETase (95kDa)

Our SDS-PAGE results showed that all purified proteins were expressed (Fig. 27). Clear bands with
correct
sizes were observed in the western blot of all purified proteins (Figure 28). In addition to the band
of
96kDa, which matched the size of chimeric proteins, an extra band with the size matching to that of
MHETase was observed in the chimeric proteins. This suggests that some chimeric proteins were cleaved
during the process of protein purification. Since MHETase were linked to the C-terminal of WT PETase
and
S245I PETase in the WT PETase-MHETase and S245I PETase-MHETase chimeric constructs respectively,
MHETase
was attached to His-Tag which was located near the C-terminal of expression vector pET 21b.Thus
cleaved
MHETase was detected in both chimeric proteins using His-Tag antibody.

These results demonstrated that our chimeric proteins, WT PETase-MHETase and S245I PETase-MHETase were
successfully expressed and purified. They exhibited PET depolymerization activity as seen by trace
amounts of TPA detected in HPLC analysis and the degraded PET film surfaces observed under SEM.

To further characterize the chimeric proteins, protein induction and extraction protocols need to be
further optimized in order to obtain single purified proteins for subsequent PET digestion
experiments.
Besides, the length and amino acid sequence of protein linker also affect chimeric protein production.
An
effective protein linker provides suitable space between two proteins which will decrease their
intrusion, improve folding and ultimately improve protein production and activity. In our project, a
12
amino acid serine-glycine linker was used to covalently link C terminus of WT PETase or S245I PETase
and
N terminus of MHETase to form chimeric proteins. To improve production efficiency and reduce cleavage
between two proteins, linkers of different amino acid lengths and combination need to be tested in
order
to find out the proximity of the two enzymes which would provide the highest PET hydrolytic activity.

Taken together, our data suggest that single enzyme of PETase mutant, S245I exhibits a higher
depolymerization activity than that of WT PETase. The detection of considerable amounts of
intermediate
products, MHET after PET film digestion extend our idea to the development of dual enzyme system for
complete PET depolymerization. The PET degradation achieved by enzyme cocktails of WT PETase and
MHETase
was inhibited while that of S245I PETase and MHETase was synergized compared with that achieved by
single
enzyme, WT PETase or S245I PETase alone. Therefore, it clearly shows that our engineered mutant, S245I
PETase which outperforms WT PETase, further synergize PET depolymerization in the presence of MHETase.
Additionally, chimeric proteins of PETase and MHETase were successfully expressed and purified.
Preliminary studies suggest that they have PET depolymerization activity.

These findings suggest that enzymatic synergism in dual enzyme PET degrading system is of importance
to
industrial use. Improved hydrolysis rate compared to single enzyme reduce the use of enzymes and
hydrolysis time and thus the cost of PET depolymerization. More importantly, rapid and complete PET
degradation leads to ways of closing the circle from production to waste.

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