iGEM 2023 — Team njtech-china-a

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{% block title %}Notebook{% endblock %}
{% block lead %}Explain your model's assumptions, data, parameters, and results in a way that anyone could understand.{% endblock %}
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![](https://static.igem.wiki/teams/4724/wiki/csj.gif)

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## Laboratory notebook

## 2023/05/15-2023/05/20

## Soluble expression:

Construct plasmids with affinity tags:

Design primers for fusion tags (2 types) and signal peptides (2 types) on SnapGene, send them to a company for synthesis, determine the molecular partners to be used, and purchase the corresponding plasmids.

## 2023/05/21-2023/06/21

## Soluble expression:

Amplify fragments from the vector using PCR, verify and purify the DNA fragments through agarose gel electrophoresis and gel recovery, and store them in a -20°C freezer for subsequent use.

## 2023/06/21-2023/07/05

## Soluble expression:

Use Gibson assembly (one-step cloning) to connect the vector with fusion tags and signal peptide fragments.

Add the volumes of the corresponding fragments and vector, add ddH2O to make a 5 μL system, and add 5 μL of 2xClone Mix enzyme.

Heat shock transformation: Introduce the recombinant plasmids into competent cells *E. coli* BL21 (DE3) following specific methods and steps.

After heat shock transformation, perform preliminary screening on LB plates containing the corresponding plasmid resistance. Invert the plates and incubate at 37°C for 12 hours for colony picking and verification.

Mix the plasmids of the molecular partners with an equal amount of the original plasmid containing only LSPET.

Heat shock transformation: Introduce the plasmids into competent cells *E. coli* BL21 (DE3) following specific methods and steps.

After heat shock transformation, perform preliminary screening on LB plates containing the corresponding plasmid resistance. Invert the plates and incubate at 37°C for 12 hours for colony picking and verification.

## 2023/07/11-2023/07/21

## Domain:

Construct plasmids with mutants:

Sequencing results: Only six mutant plasmids were successfully constructed in the first attempt, so we re-constructed the remaining four mutants and finally successfully constructed ten mutant plasmids. (Refer to [experiment] for specific steps of construction)

## Enzyme activity assay:

Induce the production of the target protein, perform a series of operations such as protein extraction, disruption, and purification to obtain the enzyme solution, and react the purified enzyme with PET substrate. Liquid phase analysis showed that W159H had the best effect. In addition, *Is*PETaseS93\_I94insE, *Is*PETaseT116R, and *Is*PETaseQ119F had higher product concentrations than the wild-type *Is*PETase.

## Soluble expression:

Preliminary identification of transformants by colony PCR:

Pick a single colony, streak it on the corresponding resistance plate, and then place it in a prepared PCR system.

Use PCR to amplify the target fragment, verify the PCR product through DNA gel electrophoresis, select colonies with correct band sizes for sequencing verification, and save the correct strains.

## 2023/07/23-2023/07/30

## Domain:

Overlaying of mutant sites:

Using *Is*PETaseW159H as a template,overlay the other three mutation sites that enhance enzyme activity onto *Is*PETaseW159H.Successfully constructed *Is*PETaseS93\_I94insE/W159H,*Is*PETaseT116R/W159H,and *Is*PETaseQ119F/W159H.

## Enzyme activity assay:

Induce the production of the target protein, perform a series of protein extraction and purification operations to obtain the enzyme solution, and react to the enzyme with PET substrate. Liquid phase analysis showed that *Is*PETaseQ119F/W159H had the highest enzyme activity.

## Soluble expression:

## Protein induction:

Repeatedly culture and induce the bacteria with two fusion tags TrxA and NusA, two signal peptides DsbA and OmpA, and three molecular partner plasmids pTF16, pKJE7, and pGro7.

Culture → Transfer bacteria: Transfer the appropriate volume of bacterial liquid to LB medium → Induce bacteria: When the OD value is approximately 0.6-0.8, add IPTG and induce at 20°C for 19 hours.

![](https://static.igem.wiki/teams/4724/wiki/not1.png)

## Mutation:

Constructing plasmids with structural domains: (Refer to [experiment] for specific steps of the construction method)

Clone the fragments of the five structural domains from the plasmids stored in the laboratory using Polymerase Chain Reaction (PCR). Clone is from the synthesized pET22b-*Is*PETase plasmid. Perform double enzyme digestion on the pET-22b vector to linearize it.

Verify and recover the DNA nucleotides through agarose gel electrophoresis, including the five structural domain fragments, the linearized pET-22b vector, and *Is*PETase.

Fuse the five structural domains with *Is*PETase. Calculate the concentration of the fusion system based on the recovered gel results.

Use Gibson Assembly (one-step cloning) to connect the vector and the fused fragments. Transform the constructed five plasmids into five tubes of competent *E. coli* BL21 (DE3) cells, and spread the bacterial solution onto LB (AMP) solid medium for overnight incubation at 37°C.

Use T7 and reverse primers specific to the structural domains as the forward and reverse primers for colony PCR. Verify the PCR products through agarose gel electrophoresis. Mark the correct bands on the agar plate and pick single colonies from the corresponding lines to inoculate the LB liquid medium.

Send the bacterial cultures for sequencing to a company.

Using the same method as above, connect the *Is*PETaseQ119F/W159H mutant with the five structural domains to construct five recombinant plasmids.

Enzyme activity validation:

However, we found that after attaching the structural domains, most of the target proteins exist in a precipitated form. Therefore, we decided to introduce molecular chaperones to solve the problem of protein insolubility. Adding molecular chaperones resolved the issue of protein insolubility. The concentrations and reaction systems of the recorded enzyme solutions are shown in the figure.

![](https://static.igem.wiki/teams/4724/wiki/not2.png)

Multiple rounds of protein induction were performed to produce the target protein. After a series of protein extraction and purification steps, enzyme solutions were obtained. These enzyme solutions were then reacted with different substrates. We repeated the cultivation of multiple sets of bacteria, induced multiple groups of proteins, and performed multiple rounds of reactions.

![](https://static.igem.wiki/teams/4724/wiki/not3.png)

## Soluble expression:

Protein extraction:

Proteins were extracted from induced bacteria expressing 2 fusion tags TrxA and NusA, 2 signal peptides DsbA and OmpA, as well as 3 chaperone plasmids pTF16, pKJE7, and pGro7.

Bacterial collection → Protein extraction: After sonication and centrifugation, the supernatant containing the cell lysate was collected, and the pellet was resuspended in urea for denaturation.
We conducted multiple experiments and obtained the target proteins expressed by bacteria containing fusion tags TrxA, NusA, signal peptides DsbA, OmpA, and chaperone plasmids pTF16, pKJE7, pGro7.

## 2023/08/29-2023/09/02

## Mutation:

Testing enzyme degradation effect:

The reaction mixture was subjected to High Performance Liquid Chromatography (HPLC) analysis to obtain the peak area of the products, analyzing the effect of different enzymes on PET degradation.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Explain your model's assumptions, data, parameters, and results in a way that anyone could understand.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4724/wiki/csj.gif)

---

Engineer

* [Overview](#EN-Overview)
* [Research process for soluble expression](#eng1)
* [Signal peptides](#Signal-peptides)
* [molecular chaperone](#molecular-chaperone)
* [Research processes of mutation](#Research-processes-of-mutation)
* [Research processes of hydrophobic structural domains](#eng2)
* [Outlook](#EN-Outlook)

## Overview

Welcome to the Engineering page, where we present the progress and results of our project based on the concepts of synthetic biology. Here, we focus on the conceptual development and engineering cycle of the entire project and present our progress in the order of "Brief Introduction", "Design", "Build", "Test ", and "Learn" to present the progress of our project.

## Research process for soluble expression

## I. Fusion labelling

## Brief Introduction

Certain fusion tags are highly soluble and can facilitate soluble expression of proteins by binding and isolating aggregation-prone folding intermediates of their target proteins[1].

We chose the following two fusion tags to incorporate into the original target protein to address the inclusion body issue.

Thioredoxin A (TrxA): thermally stable, highly soluble and with solid folding properties.

N-utilization substance A (NusA): a highly soluble protein that binds and segregates the aggregation-prone folding intermediates of its target proteins, preventing them from self-association and aggregation.

We insert both the target protein and the DNA of the fusion tag into the plasmid, which is then transcribed and translated to produce the target protein attached to the fusion tag.

## Design

We synthesized the pET-22b(+)-LSPETase plasmid from the company and will construct the recombinant plasmid by attaching each of the above two fusion tags in front of the LSPETase.

Plasmid construct design: (Gibson assembly)

TrxA and NusA gene sequences were inserted into the vector pET-22b(+)-LSPETase, and the constructed recombinant plasmids were named pET-22b-TrxA-LSPETase; pET-22b-NusA-LSPETase.

Pairs of strains modified with were named TrxA-LSPETase; NusA-LSPETase.

The vector pET-22b(+)-LSPETase was first linearised by inverse PCR, and the fusion-tagged fragment was amplified by post-PCR, and the plasmid was mapped as follows:

![](https://static.igem.wiki/teams/4724/wiki/pet-22b-trxa-lspetase.png)

Fig. 1 Plasmid mapping of pET-22b-TrxA-LSPETase

![](https://static.igem.wiki/teams/4724/wiki/pet-22b-nusa-lspetase.png)

Fig. 2 Plasmid mapping of pET-22b-NusA-LSPETase

## Build

Section 1: The DNA fragment of the fusion tag is amplified by PCR and the vector is linearized by reverse PCR.

Section 2: After homologous recombination of the linearised vector and amplified fragments, heat-stimulated transformation is performed.

Section 3: Single colonies of bacteria grown on the transformed plates were picked and cultured, then colony PCR was performed to verify that the single colonies grown contained the recombinant plasmid.

Section 4: Bacteria with successful colony PCR validation were sent for testing, and bacteria with correct sequencing were cultured for induced expression. After induction of expression, the supernatant and precipitate after cell crushing were subjected to SDS-polyacrylamide gel electrophoresis, respectively.

## Test

![](https://static.igem.wiki/teams/4724/wiki/engineer3.jpg)

Fig. 3 SDS-PAGE of recombined LSPETase with fusion tag attached
(M: Marker; Lane 1: Supernatant of NusA-LSPETase slurry; Lane 2: Precipitate of NusA-LSPETase slurry; Lane 3: Supernatant of TrxA-LSPETase slurry; Lane 4: Precipitate of TrxA-LSPETase slurry; lane 5: Supernatant of original LSPETase slurry; lane 6: Precipitate of original LSPETase slurry.)

The molecular weight of LSPETase is 30.2 kDa, while NusA-LSPETase and TrxA-LSPETase are 87.0 kDa and 43.8 kDa, respectively.

As depicted in Fig. 3, original LSPETase (lanes 5 and 6) had less supernatant and more precipitation in the cell breakage solution, indicating that the soluble expression of the target protein was restricted. After the addition of fusion tag NusA (lanes 1 and 2), the concentration of the 87.0 kDa protein band (lane 1) in the supernatant of cell breakage solution was obviously improved, and the precipitated protein band (lane 2) was obviously lightened. And after the addition of fusion tag TrxA (lanes 3 and 4), the concentration of the 43.8 kDa protein band (lane 3) in the supernatant of cell breakage solution was obviously improved, and the precipitated protein band was obviously lightened.

Further optical density analysis was performed on protein gels at induction conditions of 20°C for 19 h to quantify the increase in protein expression, and the results were analysed as follows:

![](https://static.igem.wiki/teams/4724/wiki/engineer4.png)

Fig. 4 Histogram of the optical density analysis data of supernatant and precipitated protein bands from the SDS-PAGE gel of the bacterial cell breakage solution before and after the attachment of the fusion tag

![](https://static.igem.wiki/teams/4724/wiki/engineer5.png)

Fig. 5 Percentage comparison of the optical density analysis data of the supernatant and precipitated protein bands of the bacterial cell breakage solution before and after the attachment of the fusion tag

An examination of Figure 4 and Figure 5 revealed that, under the specified induction conditions, the protein solubility of TrxA-LSPETase and NusA-LSPETase, exhibited a remarkable enhancement, with a respective increase of 3.1-fold and 1.7-fold compared to LSPETase. Moreover, the density values of the protein bands in the precipitate exhibited a reduction. This provides additional validation for the inference that the incorporation of fusion tags effectively facilitates the solubility of the target protein.

## Learn

The utilization of a fusion tag positioned at the N-terminus of LSPETase aimed to enhance the solubility of the protein within *E. coli*. However, we seek to explore alternative approaches that may yield greater effectiveness. Through an extensive review of relevant literature, we have discovered that the intracellular reducing environment hampers the formation of protein disulfide bonds, resulting in the aggregation and formation of inclusion bodies in proteins expressed at high levels. Conversely, the oxidative environment present in the periplasmic space of *E. coli* promotes the formation and stable existence of disulfide bonds. Therefore, directing the LSPETase enzyme to the periplasmic space emerges as a promising strategy to achieve soluble expression.

Particular signal peptides possess the ability to convey synthesized proteins to the periplasmic compartment. The sequestration of proteins expressed in the periplasmic space offers advantages such as protein purification and prevention of interactions with cytoplasmic constituents.

Consequently, by employing a rational design approach and incorporating signal peptide sequences, our objective is to establish a system for co-expressing signal peptides and LSPETase enzymes, enabling the targeted transport of recombinant proteins to the periplasmic domain for secretory expression. This approach holds the potential to address the challenge of inclusion body formation during the expression of exogenous proteins.

## II. Signal peptides

## Brief Introduction

Disulfide Bond Formation Protein A (DsbA) and Outer Membrane Protein A (OmpA) represent two N-terminal signal peptides of approximately 20-30 amino acids. Both DsbA and OmpA possess the capability to facilitate the transfer of synthesized proteins to the periplasmic intermediates. Notably, DsbA exhibits an additional role in catalyzing the formation of disulfide bonds within newly synthesized proteins[2], thereby promoting their soluble expression. Hence, DsbA serves as a catalyst for the formation of disulfide bonds, enabling the soluble expression of nascent proteins.

## Design

In the initial design, since the signal peptide is a small molecule protein of 20-30 amino acids, we aimed to design primers to add the coding sequence of the signal peptide to the front of the coding sequence of the LSPETase enzyme by PCR. To achieve this, we employed a reverse PCR primer, positioned between the RBS and LSPETase enzyme sequences to append the signal peptide sequence to the primer.

Subsequently, as we constructed the plasmid through homologous recombination, we linearized the plasmid through reverse PCR and added signal peptide sequences during this process, ensuring that the two ends of the linearized plasmid fragment had approximately 20 bp homologous sequence regions, enabling it to achieve single fragment homologous recombination.

We synthesised the pET-22b(+)-LSPETase plasmid and constructed the recombinant plasmid by attaching each of the above two signal peptides in front of LSPETase.

Plasmid construct design: (Gibson assembly)

DsbA and OmpA gene sequences were inserted into the vector pET-22b(+)-LSPETase, and the constructed recombinant plasmids were named pET-22b-DsbA-LSPETase; pET-22b-OmpA-LSPETase.

Pairs of strains modified with were named DsbA-LSPETase; OmpA-LSPETase.

By using reverse PCR to linearize the vector pET-22b (+) - LSPETase and attach signal peptide fragments, a single fragment homologous recombination plasmid was constructed. The designed plasmid map is as follows:

![](https://static.igem.wiki/teams/4724/wiki/pet-22b-dsba-lspetase.png)

Fig. 6 Plasmid mapping of pET-22b-DsbA-LSPETase

![](https://static.igem.wiki/teams/4724/wiki/pet-22b-ompa-lspetase.png)

Fig. 7 Plasmid mapping of pET-22b- OmpA -LSPETase

## Build

The steps are similar to adding a fusion label at the N-terminal of LSPETase: the vector is linearized through reverse PCR, but at the same time, the linearized plasmid must be accompanied by a signal peptide sequence. The linearized plasmid with the signal peptide must undergo homologous recombination of a single fragment and undergo heat shock transformation. Cultivate the transformed bacteria to induce expression. The expressed protein was subjected to SDS-polyacrylamide gel electrophoresis to analyze whether the bands were correct and verify the effectiveness of the experiment.

## Test

![](https://static.igem.wiki/teams/4724/wiki/engineer8.jpg)

Fig. 8 SDS-PAGE of recombinant protein expression products in the supernatant of fermentation broth under induction conditions of 20°C for 19h
(M: Marker; lane 1: Supernatant of LSPETase slurry; lane 2: Precipitate of LSPETase slurry; lane 3: Supernatant of DsbA-LSPETase fermentation broth; lane 4: Supernatant of OmpA-LSPETase fermentation broth)

As depicted in Figure 8, it was observed that upon induction of expression, the LSPETase enzyme, when equipped with the N-terminal signal peptide, did not exhibit localization within the fermentation broth. Upon revisiting the relevant literature, it was hypothesized that the recombinant target protein was likely directed to the periplasmic compartment of *E. coli*.

Consequently, to further investigate this phenomenon, the fermentation broth was subjected to centrifugation, followed by fragmentation, and subsequent analysis using SDS-PAGE.

![](https://static.igem.wiki/teams/4724/wiki/engineer9.jpg)

Fig. 9 SDS-PAGE of the LSPETase and LSPETase fused with the signal peptide after induction at 20 ℃ for 19h
(M: Marker; lane 1: Supernatant of LSPETase slurry; lane 2: Precipitate of LSPETase slurry; lane 3: Supernatant of OmpA-LSPETase slurry; lane 4: Precipitate of OmpA-LSPETase slurry; lane 5: Supernatant of DsbA-LSPETase slurry; lane 6: Precipitate of DsbA-LSPETase slurry.)

The molecular weight of LSPETase is 30.2 kDa, while DsbA-LSPETase and OmpA-LSPETase are 32.3 kDa and 32.2 kDa, respectively.

As shown in Fig. 9, the soluble expression of LSPETase enzyme protein after the addition of the signal peptide was greatly enhanced. The comparison of the two signal peptides revealed that DsbA significantly reduced the insoluble expression of LSPETase enzyme in the precipitate, and the effect of OmpA, although it also reduced the insoluble expression of LSPETase enzyme in the precipitate, was not as obvious. The protein expression of OmpA-LSPETase in the supernatant was significantly higher than that of LSPETase and DsbA-LSPETase. It can be concluded that OmpA signal peptide can increase the soluble expression of LSPETase enzyme by increasing the soluble expression of LSPETase enzyme, but the effect is weaker than that of DsbA signal peptide in reducing the insoluble expression of LSPETase enzyme. Both signal peptides were effective in soluble expression of LSPETase.

Further optical density analysis was performed on protein gels at induction conditions of 20°C for 19 h to quantify the increase in protein expression, and the results were analyzed as follows:

![](https://static.igem.wiki/teams/4724/wiki/engineer10.png)

Fig. 10 Histogram of the optical density analysis data of supernatant and precipitated protein bands from the SDS-PAGE gel of the bacterial cell breakage solution before and after the attachment of the signal peptides.

![](https://static.igem.wiki/teams/4724/wiki/engineer11.png)

Fig. 11 Percentage comparison of optical density analysis data of the supernatant and precipitated protein bands of the bacterial cell breakage solution before and after the attachment of the signal peptides

An analysis of Figure 10 and Figure 11 showed that under the specified induction conditions, the protein solubility of DsbA-LSPETase and OmpA-LSPETase was increased by 1.7-fold and 1.4-fold respectively compared to the original LSPETase. The density values of the protein bands in the precipitate also decreased. This confirms the conclusion that the addition of signal peptides effectively facilitates the solubility of the target protein.

## Learn

In the field of synthetic biology, the selection of an optimal system requires careful consideration of multiple factors. To overcome the limitations associated with the current approach, our research team has developed a system for enhancing the soluble expression of the LSPETase enzyme in *E. coli* by incorporating two N-terminal signal peptides, namely DsbA and OmpA. Despite these efforts, the obtained results were not sufficiently significant and the operational process remained relatively cumbersome. Therefore, our objective is to identify a simplified approach that can substantially improve the expression level of the LSPETase enzyme.

In light of this, we conducted a thorough investigation of the existing literature and discovered the concept of molecular chaperones. By harnessing chaperone proteomes in individual plasmids, these chaperones collaborate to facilitate protein folding, thereby increasing the recovery rate of soluble proteins. This promising method offers the potential to enhance the expression of the LSPETase enzyme while ensuring ease of use.

## III. Molecular chaperone

## Brief Introduction

In our experiments we need a protein that can assist in the correct folding of proteins. Certain protein molecules in the cell recognise polypeptides being synthesised or partially folded polypeptides and bind to certain parts of the polypeptide, thus helping in the translocation, folding or assembly of these polypeptides; this class of molecules is not involved in the formation of the more final product itself, and is therefore called molecular chaperones[3].

The Chaperone Plasmid Set, a chaperone proteome, was selected to construct a new plasmid by accessing the target proteins, and the chaperone proteomes act synergistically and participate in protein folding together, which can increase the recovery rate of soluble proteins.

## Design

We chose the Chaperone Plasmid Set for the chaperone proteome, and selected different chaperone plasmids, pTF16, pKJE7, and pGro7. We introduced the dual plasmid, pET-22b-LSPETase plasmid and molecular chaperone-expressed plasmid, into *E. coli*, and the recovery of soluble proteins can be increased through the synergistic effect of each chaperone proteome and the joint participation in protein folding.

Pairs of strains modified with were named pTF16/LSPETase, pKJE7/LSPETase, and pGro7/LSPETase.

## Build

The procedure is the same as the above two experimental protocols: linearisation of the vector and amplification of the fusion-tagged DNA fragments by reverse PCR. After homologous recombination of the linearised vector and the amplified fragments, heat-stimulated transformation was performed. The transformed bacteria were cultured for induced expression. The expressed proteins were subjected to SDS-polyacrylamide gel electrophoresis to analyse whether the bands were correct or not, and to verify the validity of the experiment.

## Test

![](https://static.igem.wiki/teams/4724/wiki/engineer12.jpg)

Fig. 12 SDS-PAGE of LSPETase, pTF16/LSPETase, pKJE7/LSPETase, and pGro7/LSPETase
(M: maker; lane 1: Supernatant of LSPETase slurry; lane 2: Precipitate of LSPETase slurry; lane 3: Supernatant of pTF16/LSPETase slurry; lane 4: Precipitate of pTF16/LSPETase slurry; lane 5: Supernatant of pKJE7/LSPETase slurry; lane 6: Precipitate of pKJE7/LSPETase slurry; lane 7: Supernatant of pGro7/LSPETase slurry; lane 8: Precipitate of pGro7/LSPETase slurry.)

As depicted in Figure 12, the soluble expression of LSPETase enzyme after the addition of all three molecular chaperones was significantly enhanced, and the comparison of the three molecular chaperones revealed that pGro7/LSPETase was the most effective among them.

Further analysis of the protein gel was conducted to quantify the increase in protein expression using optical density measurements. The results were shown in Figure 13 and 14. Compared with LSPETase, the soluble protein expression levels of pTF16/LSPETase, pKJE7/LSPETase, and pGro7/LSPETase were increased by 2.3-fold, 1.8-fold, and 2.2-fold, respectively. The conclusion demonstrates that the addition of molecular chaperones significantly enhances the solubility of the target protein.

![](https://static.igem.wiki/teams/4724/wiki/engineer13.png)

Fig. 13 Histogram of the optical density analysis data of supernatant and precipitated protein bands from the SDS-PAGE gel of LSPETase, pTF16/LSPETase, pKJE7/LSPETase, and pGro7/LSPETase

![](https://static.igem.wiki/teams/4724/wiki/engineer14.png)

Fig. 14 Percentage comparison of optical density analysis data of supernatant and precipitated protein bands of cell breakage solution of LSPETase, pTF16/LSPETase, pKJE7/LSPETase, and pGro7/LSPETase

In conclusion, the above three different strategies for enhancing soluble expression laid the foundation for the subsequent efficient soluble expression of PETase enzymes.

## Research processes of mutation

## Brief Introduction

*Ideonella sakaiensis*[4] is a novel bacterium that can use polyethylene terephthalate (PET) as the main energy and carbon source, and it produces the enzyme *Is*PETase with PET hydrolysis activity. After a lot of literature research[5], we realized that mutation is a common method to increase the activity of *Is*PETase. Therefore, we decided to adopt the mutation strategy to improve the activity of *Is*PETase. By analyzing the structure and mechanism of *Is*PETase protein (Fig. 15), we selected 10 mutation sites,namely *Is*PETaseS93\_I94insE,*Is*PETaseT116R,*Is*PETaseQ119F,*Is*PETaseS121E,*Is*PETaseQ126L,*Is*PETaseM157A,*Is*PETaseM157S,*Is*PETaseW159H,*Is*PETaseW185F and *Is*PETaseA240\_S242del.

![](https://static.igem.wiki/teams/4724/wiki/engineer15.png)

Fig. 15 Tertiary structure of *Is*PETase

Table.1 Reasons for selection of mutation sites

![](https://static.igem.wiki/teams/4724/wiki/engineer16.png)

## Design 1

We designed primers for point mutation and amplified the pET22b-*Is*PETase plasmid as a template to obtain the single-point mutants (Fig. 16).

![](https://static.igem.wiki/teams/4724/wiki/engineer17.png)

Fig. 16 Mapping of point mutant plasmid

## Build 1

The PCR system for the mutation was given in [Experiment], and 5 µL was taken for nucleic acid gel electrophoresis verification after PCR was completed. The rest of the system was demethylated with DpnI and then transformed into *E. coli* BL21 (DE3) sensory state. Single colonies were picked from the colonies grown after transformation and colony PCR was performed. Fig.17 shows the colony PCR bands verified by nucleic acid gel electrophoresis. After nucleic acid electrophoresis verification, the corresponding single colonies with correct bands were transferred to an LB (AMP) liquid medium, and sequencing verified that all 10 mutant plasmids had been successfully constructed.

![](https://static.igem.wiki/teams/4724/wiki/engineer18.png)

Fig.17 Nucleic acid gel electrophoresis for PCR of colonies

## Test 1

Ten mutant strains were subjected to activation, expanded culture, and a series of protein purification operations to extract the target proteins using the method in [Experiment]. The volume of purified enzyme solution required for the 500 µL reaction system was determined based on protein concentration as in [Experiment]. The reaction with PET powder was carried out at 30℃, 37℃, and 45°C for 48 h, at the end of which the products were analyzed using High Performance Liquid Chromatography (HPLC).

![](https://static.igem.wiki/teams/4724/wiki/engineer19.png)
![](https://static.igem.wiki/teams/4724/wiki/engineer20.png)\
![](https://static.igem.wiki/teams/4724/wiki/engineer21.png)

Fig. 18 Concentrations of the products TPA, MHET of 500 nM WT, S93\_I94insE, T116R, Q119F, S121E, Q126L, M157A, M157S, W159H, W185F, and A240\_S242del reacted with PET powder at different temperatures for 48h. (A) and (B) are the product concentrations obtained at a reaction temperature of 30℃; (C) and (D) are the product concentrations obtained at a reaction temperature of 37℃; (E) and (F) are the product concentrations obtained at a reaction temperature of 45℃.

As shown in Fig.18, *Is*PETaseS93\_I94insE,*Is*PETaseT116R,*Is*PETaseQ119F and *Is*PETaseW159H product concentrations were higher than that of WT under the same conditions, indicating that the four mutants were more effective in degrading PET powders compared with WT.

At 30°C, the product concentrations of *Is*PETaseS93\_I94insE and *Is*PETaseQ119F products were about 2 times that of WT, and *Is*PETaseW159H was about 2.5 times that of WT. Thus at 30°C, *Is*PETaseW159H had the most significant increase in enzyme activity and the best degradation of PET powder.

At 37°C, *Is*PETaseS93\_I94insE,*Is*PETaseT116R, and *Is*PETaseQ119F were all elevated, but the effect was not significant. The concentration of *Is*PETaseW159H product was about 2.5 times that of WT. Therefore at 37°C, it was also *Is*PETaseW159H enzyme activity that was most significantly elevated.

At 45°C, the *Is*PETaseS93\_I94insE product concentration was about 9 times that of WT, while *Is*PETaseW159H was about 5 times that of WT, which indicated that *Is*PETaseS93\_I94insE is more adapted to higher temperatures than *Is*PETaseW159H, and this mutation site likely improves the enzyme's heat resistance.

## Learn 1

Because *Is*PETaseW159H has the most obvious enhancement effect relative to WT, we decided to use *Is*PETaseW159H as a template in the later experiments, on which we superimposed S93\_I94insE, T116R, and Q119F, respectively. we expected to obtain mutants with higher activity through superimposed mutations.

## Design 2

Similar to [Design 1], primers designed for previous single point mutations can be used directly due to the large spacing between the mutation sites of S93\_I94insE, T116R, Q119F W159H, and W159H.

## Build 2

The same method as [Build 1] was successfully constructed for *Is*PETaseS93\_I94insE/W159H, *Is*PETaseQ119F/ W159H. Multiple attempts to construct *Is*PETaseT116R/ W159H plasmid were unsuccessful, we will optimize the construction method later and continue the experiment.

## Test 2

Same method as [Test 1].

![](https://static.igem.wiki/teams/4724/wiki/engineer41.png)
![](https://static.igem.wiki/teams/4724/wiki/engineer42.png)
![](https://static.igem.wiki/teams/4724/wiki/engineer43.png)

Fig. 19 Concentrations of the products TPA, MHET of 500 nM WT, S93\_I94insE/W159H, and Q119F/W159H reacted with PET powder at different temperatures for 48h. (A) is the concentration of the products obtained at a reaction temperature of 30℃; (B) is the concentration of the products obtained at a reaction temperature of 37℃; (C) is the concentration of the products obtained at a reaction temperature of 45℃.

As depicted in Fig.19, the product concentration of *Is*PETaseQ119F/W159H was much higher than that of WT under the same conditions, whether at 30℃, 37℃ or 45℃, while the effect of *Is*PETaseS93\_I94insE/W159H was much lower than that of WT, suggesting that simultaneous mutation of both the S93\_I94insE and W159H sites reduces the enzyme activity.

At 30°C, the concentration of *Is*PETaseQ119F/W159H product was about 4 times that of WT; at 37°C, the concentration of *Is*PETaseQ119F/W159H product was about 5 times that of WT; at 45°C, the concentration of *Is*PETaseQ119F/W159H product was about 23 times that of WT. These indicate that the stacking mutant *Is*PETaseQ119F/W159H has much improved high temperature resistance compared with WT, but the activity of this mutant is higher at 37°C compared with 45°C.
Learn2

## Learn2

We found that the enzyme activity of the *Is*PETaseQ119F/ W159H stacking mutant was significantly higher than that of WT, in agreement with the results of our model analysis. The strategy of using mutation to increase the activity of *Is*PETase essentially changes the structure of the protein, in addition to this, we also consider changing the extrinsic properties to increase the activity of the enzyme.

## Research processes of hydrophobic structural domains

## Brief Introduction

The hydrophobicity of the PET surface prevents *Is*PETase from binding to PET, which prevents *Is*PETase from functioning well. Therefore, we would like to add a hydrophobic domain, CBM, to *Is*PETase, so that under the force of water molecules, *Is*PETase associated with the hydrophobic domain will be easier to bind to the PET surface.Through literature research[6], we selected the hydrophobic carbohydrate domain CBM3, as well as a series of carbohydrate domains CBM4, CBM11, LSChi4CBM and LSChi5CBM found in our laboratory preservation cultures.

![](https://static.igem.wiki/teams/4724/wiki/engineer23.png)

Fig. 20 Schematic diagram of the role of hydrophobic structural domains

## Design 1

Five recombinant plasmids were constructed by attaching the five structural domains mentioned above after *Is*PETase. However the direct fusion of *Is*PETase and CBM may lead to protein expression problems, so we added a linker when designing the primers, and the linker flexibly combines *Is*PETase and CBM when constructing the plasmids, as shown in Fig.21.

![](https://static.igem.wiki/teams/4724/wiki/engineer24.jpg)

Fig. 21 Schematic diagram of the reorganization element

## Build 1

We respectively constructed recombinant plasmids with the structural domains of CBM3, CBM4, CBM11, LSChi4CBM and LSChi5CBM. *Is*PETase was first cloned from the pET22b-*Is*PETase plasmid. CBM3, CBM4, CBM11, LSChi4CBM, and LSChi5CBM were cloned from the plasmids kept in the laboratory of the research group, and then *Is*PETase was respectively fused with the five structural domains mentioned above. The pET-22b (+) plasmid was used as a template, and the linear vector was obtained by double digestion with NdeI and XhoI. Five recombinant plasmids were obtained by ligating each of the above five fusion proteins to the linear vector using the Gibson assembly method. The plasmids were transformed into *E. coli* BL21 (DE3) sensory state and single colonies were picked out from the colonies grown after transformation. The T7 promoter and the post-primer of the amplified structural domain were used for colony PCR, as Fig.22.

![](https://static.igem.wiki/teams/4724/wiki/engineer25.png)

Fig. 22 Nucleic acid gel electrophoresis for colony PCR.(M: DL2000 Plus DNA Marker,1-5: pET22b-*Is*PETase-linker-CBM3,6-9: pET22b-*Is*PETase-linker-CBM4,10-14: pET22b-*Is*PETase-linker-CBM11,15-19: pET22b-*Is*PETase-linker-LSChi4CBM,20-22: pET22b-*Is*PETase-linker-LSChi5CBM.)

After nucleic acid gel electrophoresis verification, the corresponding single colonies with the correct bands were transferred to an LB(AMP) liquid medium and then sequenced to verify that the five recombinant plasmids were successfully constructed. The five recombinant plasmids were successfully constructed.

## Test 1

The above five recombinant engineering strains were subjected to activation, expanded culture, and a series of protein purification operations to obtain the target proteins using the methods in [Experiment]. The supernatant after cell lysis, the precipitate after cell lysis, and the purified
protein was kept for SDS-PAGE analysis (Fig. 23).

![](https://static.igem.wiki/teams/4724/wiki/engineer26.png)

Fig. 23 SDS-PAGE plots of supernatant, precipitated, and purified proteins of WT, *Is*PETase-CBM3, *Is*PETase-CBM4, *Is*PETase-CBM11, *Is*PETase-LSChi4CBM, and *Is*PETase-LSChi5CBM.(M: 180 kDa Prestained Protein Marker,1-3: Supernatant, precipitated, and purified enzyme solution of WT (protein size ~29kDa),4-6: Supernatant, precipitated, and purified enzyme solution of *Is*PETase-CBM3 (protein size ~46 kDa),7-9: Supernatant, precipitated, and purified enzyme solution of *Is*PETase-CBM4 (protein size ~39 kDa),10-12: Supernatant, precipitated, and purified enzyme solution of *Is*PETase-CBM11 (protein size ~39 kDa),13-15: Supernatant, precipitated, and purified enzyme solution of *Is*PETase-LSChi4CBM (protein size ~39kDa),16-18: Supernatant, precipitated, and purified enzyme solution of *Is*PETase-LSChi5CBM (protein size ~39kDa))

## Learn 1

SDS-PAGE analysis (Fig. 23) showed that most of the target proteins were precipitated after ligating *Is*PETase to the structural domains. To solve the problem of protein insolubility, we decided to introduce the five constructed recombinant plasmids and the most effective molecular chaperone identified in [soluble expression] section into *E. coli* BL21(DE3). The supernatant of cell lyses, precipitates from cell lyses, and purified proteins were analyzed by SDS-PAGE.

## Build 2

The five recombinant plasmids that were successfully constructed in [Construct 1] and recombinant *Is*PETases were respectively introduced into *E. coli* BL21 (DE3) with molecular chaperone pGro7.

![](https://static.igem.wiki/teams/4724/wiki/engineer29.png)

Fig. 24 SDS-PAGE plots of supernatant, precipitated, and purified proteins of WT, *Is*PETase-CBM3, *Is*PETase-CBM4, *Is*PETase-CBM11, *Is*PETase-LSChi4CBM, and *Is*PETase-LSChi5CBM after the introduction of the molecular chaperone pGro7.(M: 180 kDa Prestained Protein Marker,1-3: Supernatant, precipitated, and purified enzyme solution of WT (protein size ~29kDa),4-6: Supernatant, precipitated, and purified enzyme solution of *Is*PETase-CBM4 (protein size ~39 kDa),7-9: Supernatant, precipitated, and purified enzyme solution of *Is*PETase-CBM3 (protein size ~46 kDa),10-12: Supernatant, precipitated, and purified enzyme solution of *Is*PETase-CBM11 (protein size ~39 kDa),13-15: Supernatant, precipitated, and purified enzyme solution of *Is*PETase-LSChi4CBM (protein size ~39kDa),16-18: Supernatant, precipitated, and purified enzyme solution of *Is*PETase-LSChi5CBM (protein size ~39kDa).)

As shown in Fig.24, although there were bands in the precipitation, both the supernatant and the purified enzyme solution showed bands in the correct position. It indicates that our recombinant proteins were successfully expressed and purified.

Test 2

The purified protein concentration was recorded in [notebook], and the amount of purified enzyme solution required for the 500 μL reaction system was determined based on the protein concentration as described in [Experiment]. The reaction was carried out with PET powder at 30°C, 37°C, and 45°C for 48h, after which the products were analyzed by HPLC.

![](https://static.igem.wiki/teams/4724/wiki/engineer30.png)
![](https://static.igem.wiki/teams/4724/wiki/engineer31.png)
![](https://static.igem.wiki/teams/4724/wiki/engineer32.png)

Fig. 25 500nM of WT, *Is*PETase-linker-CBM4, *Is*PETase-linker-CBM3, *Is*PETase-linker-CBM11, *Is*PETase-linker-LSChi4CBM, *Is*PETase-linker-LSChi5CBM at different temperatures. The products TPA and MHET concentrations reacted with PET powder for 48h. (A) is the concentration of the products obtained at a reaction temperature of 30°C; (B) is the concentration of the products obtained at a reaction temperature of 37°C; (C) is the concentration of the products obtained at a reaction temperature of 45°C.

As depicted in Fig.25, only the product concentration of *Is*PETase-CBM11 was higher than that of WT at 30°C and 37°C; the product concentration of both *Is*PETase-CBM4 and *Is*PETase-CBM11 was higher than that of WT at 45°C and *Is*PETase-CBM4 was about 4 times that of WT. It suggests that CBM4 enhances the hydrophobicity of the enzyme and may also enhance the high-temperature resistance of the enzyme. The results also showed that CBM11 enhanced the hydrophobicity of the enzyme at all three temperatures.

## Learn 2

CBM4 was less active than WT at both 30 and 37°C, but CBM4 was more active than WT and CBM11 at 45°C. This indicates that CBM11 has higher enzyme activity than WT at both low and higher temperatures, and CBM4 has higher enzyme activity than WT at higher temperatures. Among the superimposed mutations, our results showed that *Is*PETaseQ119F/ W159H had the highest activity at 37°C; CBM11 hydrophobicity was best at 37°C after experimental screening at different temperatures. We expect to obtain a more active enzyme by ligating *Is*PETaseQ119F/ W159H to CBM11.

## Design 3

Connect *Is*PETaseQ119F/ W159H to CBM11 according to [Design 1].

## Build 3

The pET22b-*Is*PETaseQ119F/ W159H - CBM11 recombinant plasmid was successfully constructed. The method is the same with [Build 1].

## Test 3

The volume of purified enzyme liquid required for the 500 µL reaction system was determined based on protein concentration as in [Experimental]. After reacting with PET powder for 48 h at 37°C, we also performed the enzymatic reaction using PET film and industrial filter cloth as substrates. Since PET film and industrial filter cloth are more difficult to degrade due to their higher crystallinity than PET powder, we expected to use the most effective enzyme to attempt degradation. The products were analyzed using HPLC at the end of the process.

![](https://static.igem.wiki/teams/4724/wiki/engineer33.png)

Fig. 26 Concentrations of the products TPA, MHET of the reaction of 500nM *Is*PETaseQ119F/ W159H - CBM11 with PET powder, PET film, and filter cloth at 37°C for 48h.

As shown in Fig.26, *Is*PETaseQ119F/ W159H - CBM11 degraded PET powder at about 6 times the product concentration of WT, degraded PET film at about 5 times the product concentration of WT, and degraded filter cloth at about 6 times the product concentration of WT. However, it is clear that *Is*PETaseQ119F/ W159H - CBM11 is much more capable of degrading PET powder than PET film and filter cloth.

After 48h degradation, we observed the changes on the surface of the PET film under an electron microscope(Fig. 27).

![](https://static.igem.wiki/teams/4724/wiki/engineer34.png)

Fig. 27 (A) SEM images of PET films degraded by the enzyme-free system for 48h; (B) SEM images of PET films degraded by *Is*PETase for 48h; (C) SEM images of PET films degraded by *Is*PETaseQ119F/ W159H - CBM11 for 48h.

As depicted in Fig.27 and 28, the degree of degradation of the PET material can be reflected by the degree of surface depression in the SEM image. Since (A) was only immersed in buffer solution for 48h without degradation by enzyme addition solution, it presents a smooth surface in the SEM image. (B) is the effect after 48h of WT degradation, a slight depression can be seen on the surface, but the degree of depression is not obvious. (C) is the effect of *Is*PETaseQ119F/ W159H - CBM11 after 48h of degradation, it can be seen that some areas of depression are more obvious than the effect of WT.

![](https://static.igem.wiki/teams/4724/wiki/engineer36.png)

Fig. 28 (A) SEM images of the degradation of industrial filter cloth by the enzyme-free system for 48 h; (B) SEM images of the degradation of industrial filter cloth by *Is*PETase for 48 h; (C) SEM images of the degradation of industrial filter cloth by *Is*PETaseQ119F/ W159H - CBM11 for 48 h. The degradation degree of the PET material can be reflected by the roughness of the surface in the SEM images.

## Learn 3

Due to the high crystallinity of PET films and industrial filter cloths, *Is*PETaseQ119F/ W159H - CBM11 degrades PET films and industrial filter cloths much less effectively than it degrades PET powders. Therefore, it is necessary to pre-treat PET film and industrial filter cloth to reduce their crystallinity. We will optimize the experimental protocol and find a suitable pre-treatment method in the following experiments.

## Outlook

We will continue this experiment to screen more structural domains and stack more effective mutation sites to obtain more active enzymes. We hope our research can contribute to PET degradation and will continue to work on this in the future.

## References

1. Malik, A. Protein fusion tags for efficient expression and purification of recombinant proteins in the periplasmic space of E. coli. 3 Biotech 6, 44 (2016).

2. Debarbieux, L. & Beckwith, J. The reductive enzyme thioredoxin 1 acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc. Natl. Acad. Sci. U.S.A. 95, 10751-10756 (1998).

3. Farajnia, S. Effect of Molecular Chaperone on the Soluble Expression of Recombinant Fab Fragment in E. coli. International Journal of Peptide Research and Therapeutics (2020).

4. Yoshida, S. et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196-1199 (2016).

5. Cui, Y. et al. Computational Redesign of a PETase for Plastic Biodegradation under Ambient Condition by the GRAPE Strategy. ACS Catal. 11, 1340-1350 (2021).

6. Zhang, Y. et al. Interactions between type A carbohydrate binding modules and cellulose studied with a quartz crystal microbalance with dissipation monitoring. Cellulose 27, 3661-3675 (2020).

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