iGEM 2024 — Team uestc-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

Notebook

* [April](#April)
* [May](#May)
* [June](#June)
* [July](#July)
* [August](#August)
* [September](#September)

# **April**

## **Week 1**

After the school competition selections, students from different faculties joined the UESTC-China team, and everyone is full of energy and enthusiasm! So far, we've successfully recruited members responsible for the wet lab section, but we're still looking for more students to handle mathematical modeling, human practices, and design. We held our first team meeting, got to know each other, and elected our student leader and finance manager. In addition, we had an initial discussion with our PIs about the project's direction. Everyone spent quite some time reading papers to dive deeper into the research on PET degradation and detection. Meanwhile, our WIKI team is gearing up to learn web development basics, familiarizing themselves with frameworks to prepare for the website building phase later on.

## **Week 2**

This week was mainly focused on summarizing and discussing a massive amount of literature we've gathered. Reading papers and brainstorming can be exhausting, but thanks to our PIs' guidance, we quickly formulated an initial project idea. Now, we’re thinking about the specific technical roadmap. Our WIKI team continues to dive into web-related knowledge. It’s been a busy week, with everyone working tirelessly towards the same goal, sometimes barely getting any rest.

## **Week 3**

We successfully recruited new members for mathematical modeling, human practices, and design, making our team officially complete! After our routine Wednesday meeting, we took a group photo in front of the main building on campus. This week, our project design finally started taking shape. The team working on degradation is planning to solve the issue of plastic macromolecule breakdown using a surface display system. Meanwhile, the members responsible for the upcycling section have begun constructing a multi-enzyme cascade conversion system inside cells. The detection team has decided to pursue a colorimetric approach, and we've also discussed the feasibility of various techniques like gene editing. Our WIKI members are now gathering materials, and our HP team member has presented her overall plan for team activities—big things are definitely coming!

![notebook-fg1.png](https://static.igem.wiki/teams/5487/notebook-fg1.png)

Figure 1:iGEM-China team members in April

## **Week 4**

This week, the identification team confirmed the sequences of most expression vectors and conducted a review, steadily advancing.

The fastest-progressing degradation group continues to work on the construction of the surface display system.

The colleague in charge of upgrading and rebuilding the plate has designed a multi-enzyme cascade system and completed the construction of a three-plasmid system.

The colleagues in the gene editing module identified the target genes for knockout - aldosterone reductase and ethanol dehydrogenase

The graphic designer's colleagues have started to design team uniforms for the team.

![notebook-fg2.png](https://static.igem.wiki/teams/5487/notebook-fg2.png)

Figure 2:iGEM-China team uniform design drawing

---

# **May**

## **Week 1**

The start of this month was incredibly busy! We’re deep in vector construction, which means every day is basically a PCR marathon.

The degradation group successfully verified the construction of the surface display system, and the PET degradation experiments are steadily progressing.
Meanwhile, the upcycling team completed the first plasmid transformation and confirmed it using ampicillin resistance and gene sequencing. After successfully preserving the bacterial strains, they began working on the conversion of terephthalic acid into vanillin. The gene-editing team also designed a gene knockout strategy based on the λ-RED system.

The WIKI group’s progress remains steady, as they continue to learn the framework and gather materials.

## **Week 2**

This week, the detection team expressed four types of anchoring peptides fused with sfGFP and conducted affinity experiments with plastics. We also analyzed the preliminary results of fluorescence experiments. The degradation experiments are starting to take shape.
The detection techniques are being optimized step by step, with everyone testing various conditions—things are getting more intense in the lab!

The upcycling team began validating the expression of target proteins in E. coli transformed with the first plasmid. Using SDS-PAGE, they explored enzyme protein expression and, after finding poor results, decided to switch bacterial strains to improve protein yield.
The gene-editing team attempted three rounds of experiments based on the designed λ-RED knockout route, but all attempts unfortunately failed.

After multiple discussions during our team meetings, we finally nailed down the direction for our dry lab work. The dry lab team started looking for models suited to directed enzyme evolution.

## **Week 3**

The hectic pace continues, but this week was relatively quiet.

The detection team has been exploring the cleavage conditions for TEV protease, which will take more time.

Both the degradation and gene knockout teams are still combing through literature and adjusting experimental conditions.

The upcycling team continued refining experimental methods and, after switching strains, observed good protein expression. They then harvested the cells and began the upcycling process for terephthalic acid. However, liquid chromatography analysis didn’t detect the expected product, protocatechuic acid, prompting the team to further improve the degradation system.

The modeling group started testing models for protein evolution, while the HP team had a discussion with Professor Quan to finalize the framework structure. Meanwhile, the WIKI group made a surprising leap forward this week, officially starting the framework construction for the website.

## **Week 4**

The lab teams continued to adjust the conditions for the degradation experiments. Repeating experiments requires patience, but it feels like everyone is gaining more confidence with each attempt.

The detection team has completed the initial exploration of the TEV protease cleavage system, while the modeling team began testing molecular docking of the mutants.

The upcycling team is still working on refining the transformation system conditions. This week, they made significant adjustments to the degradation system components, but high-performance liquid chromatography still didn’t detect protocatechuic acid, so they’re continuing to tweak the experimental conditions.

The gene-editing team, after consulting protocols from Nature and various forums, tried changing electroporation conditions, cell recovery times, and medium components, but success remains elusive.

The HP and WIKI groups had a relatively smooth week. In particular, the WIKI team has started drafting some basic documentation, marking steady progress.

---

# **June**

## **Week 1**

As June begins, our experimental progress has really picked up speed. However, the detection team faced some setbacks this week—modifying the gold nanoparticles didn’t go as planned, and we found that the concentration of the anchoring peptide was still too low. To make things more challenging, we ran out of bacterial culture, so we need to re-express the target protein.

On a more positive note, the degradation team made some key adjustments and finally obtained critical data, which has everyone really excited!

The upcycling team has not stopped tweaking the transformation system. Unlike last time, this week they used ultrasound to lyse the cell suspension and successfully detected the target product. This narrowed the problem down to the transmembrane transport of terephthalic acid.

The upcycling team has not stopped tweaking the transformation system. Unlike last time, this week they used ultrasound to lyse the cell suspension and successfully detected the target product. This narrowed the problem down to the transmembrane transport of terephthalic acid.

The WIKI group is progressing steadily with development.

## **Week 2**

The lab was buzzing with activity again this week. The detection team successfully re-expressed and purified their proteins.

The degradation team continued adjusting experimental conditions, testing more optimization schemes.

The upcycling team, after numerous trials, finally nailed down the optimal transformation system. By adding 1% n-butanol, they successfully enabled the transmembrane transport of terephthalic acid and converted it into protocatechuic acid. This marked a major milestone in the success of the first step of the multi-enzyme cascade system.

Meanwhile, the gene-editing team started learning the principles and structure of the pEcCas gene knockout system.

The modeling team continued their experiments on protein mutations.

The WIKI team made solid progress by refining the structure of the website, and it looks like it’s going to be quite impressive when we showcase it later.

On the HP side, plans for upcoming outreach activities are already in the works.

## **Week 3**

This week, the degradation team’s experimental results gave us renewed hope! The data looks incredibly promising, and the focus will now shift to analyzing the results in depth.

The upcycling team successfully transformed the remaining two plasmids separately and used SDS-PAGE to analyze enzyme protein expression. The results showed that both plasmids could express independently.

The detection team also made progress, exploring new TEV protease cleavage conditions, resulting in a significant increase in concentration.

The gene-editing team started designing a technical route for the pEcCas gene knockout system, working on plasmid construction and other tools.

The modeling team continued testing various mutants—it’s a complex process, but the progress is steady.

The HP team began drafting promotional materials, while the WIKI team continued enhancing the website’s functionality.

## **Week 4**

This week was full of energy!

The degradation team’s preliminary data is in, and now they’re diving into a deeper analysis of the results.

The upcycling team completed the co-transformation of three plasmids, and after screening with antibiotics, they’re ready for the next phase of work.

The detection team finished preparing all the anchoring peptides. The modeling team is still focused on studying mutants, and although the process is tedious, the results are looking promising.

The gene-editing team conducted three experimental attempts based on the pEcCas gene knockout system’s technical route, but all resulted in false positives.

The HP team has finalized their promotional plan and is ready to put it into action.

![notebook-fg3.png](https://static.igem.wiki/teams/5487/notebook-fg3.png)

Figure 3:The design of human practice

---

# **July**

## **Week 1**

As July begins, our experiments are progressing exceptionally well.

The degradation team completed the final round of PET degradation experiments this week. Everyone is busy collecting and analyzing data, and the atmosphere in the lab is both tense and fulfilling.

The upcycling team moved on to verifying the second key pathway. Under new conditions, they successfully detected the final product, vanillin, confirming the feasibility of the second step in the multi-enzyme cascade pathway.

The detection team is also in full swing with their colorimetric experiments, continuously exploring optimal conditions for nanoscale modifications.

Unfortunately, the gene-editing team has faced a series of setbacks. They’re going back to square one, diving into literature and forums to find a workable gene-editing system.

The modeling team is applying mutant data to their models, preparing to provide more theoretical support for the experiments.

On the HP front, the team participated in the first Chengdu Metropolitan Ecological and Environmental Volunteer Service Competition and won the first prize in the Creative Group. This recognition of our earlier plans has been a great encouragement, and we’re hopeful for continued progress in our HP work moving forward!

![notebook-fg4.png](https://static.igem.wiki/teams/5487/notebook-fg4.png)

Figure 4:Won the first prize in Human Practice

## **Week 2**

The lab remains as busy as ever, with both the degradation and detection experiments reaching critical stages.

The upcycling team successfully induced expression using the final constructed strain. Under the optimized conditions, they once again detected vanillin as the final product, confirming the viability of the entire pathway.

This marks a major success for the upcycling project!

The detection team encountered some difficulties with their colorimetric experiments, but we’re confident that teamwork and collaboration will help overcome these challenges.

The gene-editing team made a breakthrough by discovering the PHCY-25A/PHCY-26D dual plasmid gene-editing system, allowing them to design a new technical route for targeted gene knockout.

The modeling team continued refining their models, and everyone is excited to see how well the experiments and theoretical models can come together.

Meanwhile, the HP team had a busy week as well. They organized a summer community event titled "Green Future: Reducing Plastic, Cutting Pollution" at Xinglin Community in Yongning Street. The activities included educational talks, DIY bottle-recycling crafts, talent shows, eco-protection poetry collages, and even an environmental song performance. The kids were highly enthusiastic, and everyone walked away with a sense of accomplishment!

![notebook-fg5.png](https://static.igem.wiki/teams/5487/notebook-fg5.png)

Figure 5:Group photo at a community event

## **Week 3**

The progress of our experiments is becoming smoother by the day! The degradation team is nearing the final stages of data collection, while the upcycling group has completed their experimental validation phase. We’re gaining more confidence in the stability of our experimental data.

The detection team finally achieved a breakthrough in their colorimetric experiments this week, and the next step will be to replicate the results for consistency.

The gene-editing team decided to abandon the ethanol dehydrogenase gene knockout and instead completed the design of sgRNAs for five aldehyde-ketone reductase genes, successfully introducing the vectors.

The modeling team has reached a point where their model can accurately predict the performance of various mutants, which is highly satisfying for everyone.

Despite the hectic pace in the lab, the WIKI preparations are progressing in parallel.

On the HP front, our team recently engaged with students from Malaysia to explore new solutions to microplastic pollution, enriching the research and learning experience while brainstorming innovative approaches to tackle this global issue.

![notebook-fg6.png](https://static.igem.wiki/teams/5487/notebook-fg6.png)

Figure 6:Human Practice members visit Malaysia

## Week 4

The experimental tasks for July are finally complete! The degradation and upcycling teams have successfully gathered all the necessary data and are now shifting their focus entirely to data analysis. The detection team wrapped up their experiments beautifully, with the colorimetric process now fully optimized and ready.

However, the gene-editing module hit a snag in the design of sgRNA plasmids, which will require some problem-solving moving forward.

The modeling team is in the final stages of fine-tuning their mutant models, and they’ll soon begin cross-verifying them with experimental data. The conclusion of this experimental phase brought a sense of relief to everyone, but we all know the next steps will be just as challenging.

On the WIKI and HP fronts, the teams are collaborating to discuss the design of our IP character and refine the storyline. More communication with the experimental groups will be needed to ensure that the designs align closely with the lab’s work.

---

# **August**

## **Week 1**

As we enter August, the experiments are entering their final stages. The degradation, upcycling, and detection teams have all shifted their focus to deep analysis of the experimental data. The teams discussed how the data performed under different conditions and are working to find the optimal solutions.

The gene-editing team made significant progress this week—after adjusting the recovery time, they successfully constructed the sgRNA plasmid based on the YahK gene.

The modeling team combined the experimental data with their mutant models, successfully validating the accuracy of their predictions.

At the start of August, the HP team held a collaborative exchange session with students from the University of Science and Technology of China (USTC). We learned about their innovative design ideas and strategies, leading to an exciting exchange of thoughts and inspiration that left both teams with valuable insights.

![notebook-fg7.png](https://static.igem.wiki/teams/5487/notebook-fg7.png)

Figure 7:Communicate with USTC

## **Week 2**

The analysis of experimental data continued this week. Members from the degradation, upcycling, and detection teams began summarizing the key findings of their experiments and started drafting technical documentation. The gene-editing team made attempts to construct sgRNA plasmids for four other aldehyde-ketone reductase genes but encountered repeated failures in obtaining the sgRNA plasmid for the YahK gene.

Meanwhile, the modeling team further refined their model to ensure consistency with experimental results.

We held a few internal meetings to discuss the best ways to present our experimental data, with everyone eagerly contributing ideas.

The HP and WIKI teams collaborated on designing the storyline and finalizing the project name, narrowing down their focus after multiple rounds of discussion and revisions.

## **Week 3**

This week’s main focus was organizing the experimental results and drafting the experimental reports. The degradation, upcycling, and detection teams completed their data analysis and plots, successfully addressing some of the critical issues encountered during the experiments. The gene-editing team successfully extracted the sgRNA plasmid for the YahK gene from the strain and began testing it in gene knockout experiments.

They also continued working on the sgRNA plasmids for the other aldehyde-ketone reductase genes.

The modeling team finished optimizing their model, and the next step will be to integrate these results into our project presentation. We aim to complete all reports and related materials by mid-September.

The HP team has been busy organizing a social media campaign to connect with universities, planning to complete their outreach posts by the end of August. They are also designing an online environmental and innovation competition, with plans for an offline event in September, which involved active discussions with the school.

## **Week 4**

This week, the degradation, upcycling, and detection teams meticulously organized all their experimental data.

The gene-editing team, after multiple setbacks, decided to focus on the YahK gene knockout. The experimental team collaborated closely with the HP and design teams to discuss the presentation of the project, creating detailed flowcharts and project designs. The modeling team incorporated their finalized data into the reports, ensuring the results were clear and convincing.

---

# September

## **Week 1**

The focus for September is the writing and organization of the WIKI documentation. The degradation, upcycling, and detection teams have fully organized their experimental results, and everyone is now focused on systematically integrating these findings into the WIKI.

The modeling team has also started drafting documentation for their mutant model, ensuring that both the experimental and theoretical components are well-represented in the WIKI.

The WIKI team has been instrumental in assisting with the document structure, ensuring that our team’s achievements are presented clearly and accurately.

Meanwhile, the gene-editing team successfully knocked out the YahK gene in E. coli BL21(DE3) and confirmed the knockout using nucleic acid electrophoresis.

## **Week 2**

This week, the WIKI writing process entered a critical phase. The experimental content from the degradation, upcycling, gene-editing, and detection teams has mostly been incorporated into the WIKI, while the modeling team’s documentation is beginning to take shape. The team is now focused on refining the language and structure to ensure that the content is not only scientifically rigorous but also easy to understand.

The WIKI team has been working hard to integrate the various modules, and overall, the progress is smooth.

On the HP front, the team hosted an online environmental design and knowledge competition. We’ve prepared a variety of exciting prizes for the participants, and I must say, I’ve taken a real liking to that adorable pillow!

![notebook-fg8.png](https://static.igem.wiki/teams/5487/notebook-fg8.png)

Figure 8:Poster of the Environmental Knowledge and Innovation Competition

## **Week 3**

The WIKI documentation is nearing completion. All experimental data, model results, and relevant discussions have been fully integrated into the document. We had several rounds of discussions with our PIs to ensure that the content is both comprehensive and scientifically accurate. The degradation, upcycling, gene-editing, and detection teams have carefully reviewed their sections multiple times to ensure nothing was missed or incorrectly presented. The modeling team also finalized their mutant model documentation, successfully linking the theoretical and experimental aspects of the project. The team has now moved on to the final stages of formatting and layout adjustments.

## **Week 4**

The final version of the WIKI documentation is complete!

All the content from the various experimental modules has been fully integrated, with the modeling team’s theoretical contributions seamlessly included.

The WIKI team has done an excellent job in formatting and proofreading the document, and the overall presentation of the project is highly professional and polished.

Thanks to everyone's hard work, the final document showcases our team’s efforts in a way that is both clear and impressive, highlighting the full scope of our accomplishments.

---

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
{% block page\_content %}

Engineering

* [Identification](#Identification)
  + [Cycle 1](#4Cycle1)
  + [Cycle 2](#4Cycle2)
* [PET Biodegradation](#PETBiodegradation)
  + [Cycle 1](#1Cycle1)
  + [Cycle 2](#1Cycle2)
  + [Cycle 3](#1Cycle3)
  + [Cycle 4](#1Cycle4)
* [Upcycling](#Upcycling)
  + [Cycle 1](#2Cycle1)
  + [Cycle 2](#2Cycle2)
  + [Cycle 3](#2Cycle3)
  + [Cycle 4](#2Cycle4)

# **Identification**

## **Cycle 1: Construction of Anchoring Peptide Expression Vector**

#### Design

The anchoring peptide gene was inserted into the plasmid pET-30a to create a fusion protein expression vector **(Fig 1)**.

#### Build

The linearized vector and the inserted fragment were cloned and transformed into DH5α via heat shock. Positive clones were screened by PCR and Sanger sequencing (Shanghai Sangon Inc.). The plasmid extracted from DH5α was then transformed into BL21(DE3). After induction, the anchoring peptide-GFP fusion protein was purified using a nickel column. The anchoring peptide-GFP fusion protein was cleaved with TEV protease (at 30°C for 4 hours), yielding the His-tagged anchoring peptide and GFP without the His-tag. The cleavage mixture was then passed through a His gravity column (Takara, Beijing, China) to collect the anchoring peptide.

#### Test

Using the BCA assay and SDS-PAGE analysis, we found that the concentration of the anchoring peptide was low and the sample was impure.

#### Learn

In the original anchoring peptide vector, the TEV cleavage site was located after the 6xHis tag. TEV protease was used to cleave the TEV site on the fusion protein, leaving the His-tag on the anchoring peptide while GFP lacked the tag, allowing for separation and purification via a nickel column. However, since the TEV protease also contains a His tag, and the His-tagged anchoring peptide strongly adhered to the nickel column and was difficult to elute, it was discovered that TEV protease mixed with the purified anchoring peptide. Additionally, there was significant loss of the anchoring peptide during the process.

## **Cycle 2: Reversing the TEV Site and His-tag Order**

#### Design

The vector was redesigned by swapping the positions of the TEV site and the 6xHis tag **(Fig 1)**.

#### Build

Primers were redesigned for PCR to obtain the modified plasmid. The new plasmid was transformed into BL21(DE3), and after induction, the anchoring peptide-GFP fusion protein was purified using a nickel column. TEV protease was used to cleave the anchoring peptide-GFP fusion protein at 30°C for 4 hours.This yielded His-tagged GFP and anchoring peptide without the His-tag. The cleavage mixture was then passed through a His gravity column (Takara, Beijing, China) to collect the anchoring peptide.

#### Test

The newly constructed vector significantly increased the concentration of the anchoring peptide and eliminated TEV protease contamination.

#### Learn

The concentration of the anchoring peptide increased to meet the requirements of the subsequent experiments.

![engineer-f1.png](https://static.igem.wiki/teams/5487/engineer-f1.png)

Fig 1: Removal and Re-insertion of the TEV Site in Reverse Order Using Two Rounds of PCR

---

# **PET Biodegradation**

## **Cycle 1: Enhancement of GC linker and Reordering of Component Assembly**

#### Design

Taking into account the necessity for robust and efficient assembly, we have adopted a strategy that involves the separate assembly of functionally synthesized components. We have selected the pET-30a vector as the framework for this section. This vector offers well-documented advantages such as high expression levels, a strong promoter for efficient transcription, and a multiple cloning site that facilitates the insertion of multiple genes.

#### Construct

The plasmid BBa\_K5487005 encompasses key elements: the membrane protein Lpp-OmpA and the green fluorescent protein sfGFP, which serve as a test for the efficiency of membrane protein linkage. This plasmid was constructed using homologous recombination seamless cloning, a method that efficiently inserts multiple genes into a vector with minimal effort and time. The process involves the amplification of the target genes with specific primers, followed by an overlap extension PCR. The direct transformation into competent *E. coli* cells DH5α allows for the in vivo recombination and integration of the genes into the plasmid. The plasmid BBa\_K5487003 contains three critical proteins, connected in a sequential order: Lpp-OmpA, MHETase, and Z1-PETase, designed for the degradation of PET plastics. Subsequently, these plasmids were introduced into *E. coli* DH5α using the heat shock method, a widely used technique for transforming *E. coli* with foreign DNA. This optimization can lead to a higher yield of transformed cells, affecting the overall efficiency of the transformation process.

#### Testing

After transformation, *E. coli* DH5α was cultured overnight in LB medium containing kanamycin resistance, and plasmid extraction and sequencing revealed that the segments MHETase and Z1-PETase did not successfully integrate within the pET-30a framework.

#### Learning and Experimentation

Through literature review and consultation with peers, we discovered that several articles have incorporated multiple rigid GC linkers to enhance the efficiency of segment-to-segment connections. This insight has prompted us to reconsider our approach and explore the potential benefits of incorporating such linkers into our construct design.

## **Cycle 2: Chassis Organism Replacement**

#### Design

Building upon the lessons learned from the first cycle, we recognized the necessity to enhance the functionality and expression of our key components, Lpp-OmpA, MHETase, and Z1-PETase. To achieve this, we strategically incorporated multiple rigid GC linkers within these elements to reinforce their structural stability and improve their interaction dynamics. Furthermore, we experimented with the sequence of MHETase and Z1-PETase to optimize their arrangement for enhanced expression and functionality.

#### Construct

With the revised order of Lpp-OmpA, Z1-PETase, and MHETase, we re-engineered the plasmid. We then decided to introduce it into *E. coli* BL21, a chassis organism known for its robust protein expression capabilities. This choice was made after considering the organism's compatibility with our construct and its potential to improve the overall performance of the system.

#### Testing

The transformed *E. coli* BL21 was cultured under conditions optimized for protein expression. Both the growth rate and the expression levels of the target proteins were closely monitored. The results were analyzed to assess the impact of the chassis organism on the construct's performance.

#### Learning and Experimentation

Upon reviewing the data from the tests, we observed that the growth rate and protein expression efficiency in *E. coli* BL21 were significantly improved compared to our initial trials with *E. coli* MG1655. This led us to conclude that *E. coli* BL21 might be a more suitable chassis organism for our construct. The decision to use BL21 was further supported by its well-documented history of successful use in protein expression studies and its ability to handle the metabolic demands of our complex construct.

In light of these findings, we will continue to refine our construct within the *E. coli* BL21 chassis, focusing on further optimization of the rigid GC linkers and the component arrangement. We will also explore additional genetic modifications and environmental factors that could potentially enhance the expression and functionality of our system. This iterative process will involve a combination of computational modeling, laboratory experimentation, and data analysis to fine-tune our approach and achieve the desired outcomes.

## **Cycle 3: Optimization of Degradation Conditions**

#### Design

Following the successful expression of the target proteins in *E. coli* BL21 during the second cycle, we have now established a baseline for PET degradation under the initial conditions of 50°C in PBS buffer. With the detection of TPA, a key degradation product, we are poised to optimize the degradation process further. To enhance the efficiency and specificity of PET degradation, we will systematically vary the bacterial concentration (OD600), buffer pH, and IPTG induction concentration to identify the optimal conditions that maximize the degradation efficiency.

#### Construct

The plasmid containing the optimized sequence of Lpp-OmpA, Z1-PETase, and MHETase, along with the rigid GC linkers, remains unchanged. We will utilize this construct for all degradation experiments to ensure consistency and to isolate the effects of the varying conditions on the degradation process.

#### Testing

A series of degradation experiments will be conducted under the following conditions:

Bacterial Concentration (OD600): Varying the initial bacterial concentration to 1, 5, 20, and 50 to determine the optimal cell density for PET degradation.

Buffer pH: Buffers of different pH (7/8/9/10) were used to assess the impact of pH on the enzymatic activity and degradation rate.

IPTG Concentration: Testing different concentrations of IPTG to induce protein expression and identify the level that yields the highest degradation efficiency without causing undue stress to the bacterial cells.

Each condition will be tested in triplicate to ensure the reliability of the results. The degradation efficiency will be quantified by measuring the amount of TPA produced after 24 hours of incubation at 50°C.

## **Cycle 4: Enhancing MHETase Catalytic Efficiency**

#### Design

In pursuit of higher catalytic efficiency for MHETase, we are considering employing virtual mutagenesis technology to predict the structural impact of every possible mutation within each residue of MHETase. This approach will allow us to simulate and analyze the effects of these mutations on the enzyme's structure and function. Subsequently, we will conduct molecular docking of all single-point mutations with the MHET molecule to evaluate their binding affinity. Through this screening process, we aim to identify mutations that exhibit a higher affinity for MHET compared to the wild-type MHETase. The promising candidates will then be subjected to molecular dynamics simulations to further confirm their stability and improved binding characteristics.

#### Construct

Leveraging homologous recombination techniques, we will construct several variants of MHETase based on the identified mutation hotspots. Specifically, we will create the following mutants: K397A, R71N, R273A, S305T, and N89A. Each of these variants will be tested for their ability to produce TPA, a key intermediate in the degradation pathway of PET, to determine which mutation(s) result in the most significant enhancement of catalytic efficiency. The goal is to select the variant(s) that not only maintain the enzyme's structural integrity but also exhibit improved catalytic properties, thereby accelerating the PET degradation process.

---

# **Upcycling**

## **Cycle 1 Feasibility Assessment of Upcycling System**

#### Design

Under the catalysis of terephthalate 1,2-dioxygenase (TPADO) and 1,4-dicarboxylic acid dehydrogenase (DCDDH), terephthalic acid (TPA) can be converted into protocatechuic acid (PCA). We utilized the Escherichia coli strain MG1655 (DE3) for the expression of target proteins and employed ultrasonication to lyse the cell suspension, thereby obtaining a crude enzyme solution for substrate conversion. This approach aims to verify the feasibility of the proposed conversion pathway and to explore the optimal conditions regarding time, temperature, and the concentration of key components in the system.

#### Build

A 10 mL transformation system was established using 100 µL of crude enzyme solution prepared from a cell suspension with an optical density (OD600) of 80. A 5 mL solution of 2.4 mM TPA in dimethyl sulfoxide (DMSO) was added, and the final volume was adjusted to 10 mL with 50 mM glycine buffer (pH 8.5). The reaction mixture was incubated at 30°C for 24 hours to facilitate the degradation process.

#### Test

High-performance liquid chromatography (HPLC) analysis revealed that the target product, PCA, was not detected (Fig 2).

![engineer-f2.png](https://static.igem.wiki/teams/5487/engineer-f2.png)

Fig 2 : HPLC result (no target product PCA detected )

#### Learn

During the optimization process, it was determined that excessive DMSO exerts toxic effects on the cells, 30°C is not the optimal temperature for degradation, the current culture medium is insufficient to support the reaction, and a high concentration of enzymes is critical for effective transformation.

## **Cycle 2 Replacement of *E. coli* strain**

#### Design

Initially, the *E. coli* MG1655 (DE3) strain was utilized for protein expression; however, low expression levels were observed. Consequently, the *E. coli* BL21 (DE3) strain was utilized for enhanced protein expression. A novel transformation system was established, incorporating altered temperature conditions, transformation protocols, and bacterial suspension densities to upgrade the degradation of TPA.

#### Build

Following a 24-hour induction period, the *E. coli* BL21 (DE3) strain was resuspended in 3 mL of M9 medium to an OD600 of 65 and lysed using ultrasonication to obtain a crude enzyme solution. Subsequently, 100 µL of a 0.12 M TPA solution in DMSO was added, and the mixture was adjusted to a final volume of 10 mL with a pH 8.5, 50 mM glycine buffer. The degradation process was carried out at 20°C with orbital shaking for 24 hours.

#### Test

![engineer-f3.png](https://static.igem.wiki/teams/5487/engineer-f3.png)

Fig 3 : HPLC result (Target product PCA detected）

HPLC analysis results demonstrate the successful detection of the target PCA (Fig 3).

#### Learn

The *E. coli* BL21 (DE3) strain exhibits superior protein expression capabilities compared to the *E. coli* MG1655 (DE3) strain.

## **Cycle 3 Optimization of Cell Membrane Permeability System**

#### Design

To achieve whole-cell catalysis, we focused on the transmembrane transport of TPA. Enhancing cell membrane permeability can improve the overall yield of the upcycling pathway. Therefore, increasing membrane permeability has become our primary strategy.

#### Build

We decided to use n-butanol to facilitate transmembrane transport. Through experimental investigation, we aim to determine the optimal timing and concentration of n-butanol addition within the system. Initially, n-butanol was added to *E. coli* cultures in TB medium at an OD600 of 0.3 to a final concentration of 10 mM, which resulted in suboptimal enzyme expression. Consequently, the timing and concentration of n-butanol addition were adjusted.  *E. coli* was cultured in TB medium for 24 hours, and following cell harvest, 1% (v/v) n-butanol was introduced to the 10 mL upcycling system.

#### Test

It was confirmed that the BL21 (DE3) strain, after 24 hours of cultivation in TB medium, followed by cell harvest and the addition of 1% n-butanol to the 10 mL degradation system, could successfully mediate the transmembrane process of TPA without toxic effects on protein expression, leading to its degradation into PCA (Fig 4).

![engineer-f4.png](https://static.igem.wiki/teams/5487/engineer-f4.png)

Fig 4 : HPLC result (Target product PCA detected）

#### Learn

The presence of n-butanol in the degradation system can exert toxic effects on *E. coli* during the logarithmic growth phase. On the other hand, an appropriate concentration of n-butanol can enhance the permeability of the *E. coli* cell membrane, thereby facilitating the transmembrane transport of TPA. This achieves the effect of whole-cell catalysis and lays the foundation for the subsequent conversion of PCA into vanillin.

## **Cycle 4 Detection of Vanillin**

#### Design

In order to achieve whole-cell catalysis and facilitate the transmembrane transport of terephthalic acid without cell lysis, thereby obtaining the desired product, we opted to incorporate n-butanol into the degradation system to enhance the permeability of the cell membrane. This approach is based on the synergistic action of catechol O-methyltransferase, carboxylic acid reductase, and phosphopantetheinyl transferase, which can convert PCA into the high-value product vanillin. Building upon the validation from the first cycle, we further verified the feasibility of the second step in the multi-enzyme cascade.

#### Build

The *E. coli* BL21 (DE3) strain was resuspended in 3 mL of M9 medium to an OD600 of 65, and n-butanol was added to a final concentration of 1% (v/v) after the cells were fully induced for 24 hours. Subsequently, 100 µL of a 0.12 M TPA solution in DMSO was added, and the mixture was adjusted to a final volume of 10 mL with a pH 8.5, 50 mM glycine buffer. The degradation process was carried out at 20°C with orbital shaking for 24 hours.

#### Test

The successful detection of vanillin confirmed the complete feasibility of the pathway (Fig 5).

![engineer-f5.png](https://static.igem.wiki/teams/5487/engineer-f5.png)

Fig 5 : HPLC result (Target product Vanillin detected）

#### Learn

During our investigation, we identified the optimal transformation system.

---

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

Experiments

* [Culture](#Culture)
  + [1.Culture medium](#Culturemedium)
  + [2.Antibiotic](#Antibiotic)
  + [3.IPTG\L-arabinose](#IPTGLarabinose)
* [Breed Preservation](#BreedPreservation)
  + [1.Glycerol Stock](#GlycerolStock)
  + [2.Recovery](#Recovery)
* [Plasmid construction and Transformation](#PlasmidconstructionandTransformation)
  + [1.PCR](#PCR)
  + [2.NucleicAcidGelElectrophoresis](#NucleicAcidGelElectrophoresis)
  + [3.Gel Recovery](#GelRecovery)
  + [4.Homologous Recombination](#HomologousRecombination)
  + [5.Transformation](#Transformation)
* [Expression](#Expression)
  + [1.Bacterial Culture and Target Protein Expression](#BacterialCultureandTargetProteinExpression)
  + [2.Bacterial Culture Harvesting and Pellet Resuspension](#BacterialCultureHarvestingandPelletResuspension)
* [Verification](#Verification)
  + [1.SDS-PAGE](#SDS-PAGE)
  + [1.Preparation of polyacrylamide gels](#Preparationofpolyacrylamidegels)
  + [2.Anchor Peptide Purification](#AnchorPeptidePurification)
  + [3.Affinity validation of the anchored peptide](#Affinityvalidationoftheanchoredpeptide)
  + [4.Colorimetric identification of plastics](#Colorimetricidentificationofplastics)
  + [5.High-Performance Liquid Chromatography Analysis](#High-PerformanceLiquidChromatographyAnalysis)
  + [6.Degradation](#Degradation)
* [Gene editing](#Geneediting)
  + [1.Plasmid Construction Workflow for PHCY-yahK-KO(piGEM24\_09)](#PlasmidConstructionWorkflowforPHCY-yahK-KO(piGEM24_09))
  + [2.YahK Gene Knockout Procedure](#YahKGeneKnockoutProcedure)
  + [3.Plasmid Curing Procedure](#PlasmidCuringProcedure)

# **Culture**

## **1.Culture medium**

### **1.1 TB**

Cultivation of E. coli using TB medium while expressing proteins in experiments.

The TB medium was configured as follows.

| Ingredient | Quantity |
| --- | --- |
| YEAST EXTRACT | 24 g |
| TRYPTONE | 12 g |
| K2HPO4·3H2O | 12 g |
| KH2PO4 | 2.2 g |
| GLYCERINUM | 4 mL |
| ddH2O | To 1 L |

The preparation process is as follows.

Calculate → Weigh → Dissolve → Sterilize → Save
Detailed steps:

1. Calculation of substance requirements based on the volume of medium required.
2. Weigh the substances according to the calculation.
3. Add ddH2O to fix the volume and stir well to dissolve it.
4. Autoclave the conical vials after closing the mouth with a breathable parafilm and a leather band.
5. Store the liquid medium in a 4 ℃ refrigerator and use it as soon as possible. Close the solid medium with a parafilm and store it in a refrigerator at 4℃ and use it as soon as possible.

### **1.2 LB**

Cultivation of E.coli in experiments using LB medium (with appropriate antibiotics if needed).

The LB medium was configured as follows.

| Ingredient | Quantity |
| --- | --- |
| NaCl | 10g |
| TRYPTONE | 10g |
| YEAST EXTRACT | 5g |
| ddH2O | 2.2 g |

If formulated as a solid medium, add an additional 15 g of agar powder (1.5%) before sterilization, then autoclave the configured medium.

The preparation process is as follows.

Calculate → Weigh → Dissolve → Sterilize → Pour Plate (if needed) → Save

Detailed steps:

1. Calculation of substance requirements based on the volume of medium required.
2. Weigh the substances according to the calculation.
3. Add ddH2O to fix the volume and stir well to dissolve it.
4. Autoclave the conical vials after closing the mouth with a breathable parafilm and a leather band.
5. In case of LB agar the inverted plate operation is carried out when the medium is cooled to about 45 ℃. The petri dish lid can be tilted to minimize condensate contamination, and the inverted plate operation is carried out in an ultra-clean bench next to an alcohol lamp.
6. Store the liquid medium in a 4 ℃ refrigerator and use it as soon as possible. Close the solid medium with a parafilm and store it in a refrigerator at 4 ℃ and use it as soon as possible.

### **1.3 M9 Medium**

1. Configure of 1 mol/L MgSO4 : weigh 2.46g of MgSO4-7H2O and dissolve it in 10ml of ddH2O, autoclaved it.
2. Configure 1 mol/L CaCl2: weigh 2.191g of CaCl2-6H2O and dissolve it in 10ml of ddH2O, autoclave it.
3. Configure 5×M9 salt solution:

   | Ingredient | Quantity |
   | --- | --- |
   | Na2HPO4・H2O | 12.8g |
   | KH2PO4 | 3g |
   | NACL | 0.5g |
   | NH4CL | 1 g |
   | ddH2O | To 200ml |

   Autoclave standby.
4. Configure 20% glucose solution: weigh 4g glucose and dissolve it in 20ml of ddH2O, then filter with 0.22μm filter to remove bacteria.
5. Configure M9 culture medium

   | Ingredient | Quantity |
   | --- | --- |
   | 5×M9 | 200ml |
   | 1M MgSO4 | 2ml |
   | 20% Glucose solution | 20ml |
   | 1M CaCl2 | 0.1ml |
   | ddH2O | To 1L |

### **1.4Kana+Amp+Glucose LB:**

The LB medium was configured as follows. To this solution, glucose was added to achieve a final concentration of 20 mM in the medium. Add an additional 15 g of agar powder (1.5%) before sterilization, then autoclave the configured medium.

| Ingredient | Quantity |
| --- | --- |
| NaCl | 10g |
| TRYPTONE | 10g |
| YEAST EXTRACT | 5g |
| ddH2O | To 1L |

The preparation process is as follows.

Calculate → Weigh → Dissolve → Sterilize → Pour Plate → Save

Detailed steps:

* Calculation of substance requirements based on the volume of medium required.
* Weigh the substances according to the calculation.
* Add ddH2 O to fix the volume and stir well to dissolve it.
* Autoclave the conical vials after closing the mouth with a breathable parafilm and a leather band.
* Kanamycin and benzylpenicillin should be added to the medium at a concentration of 1:1000 at a temperature of approximately 65°C, and the solution should be thoroughly agitated.
* In case of LB agar the inverted plate operation is carried out when the medium is cooled to about 45 ℃. The petri dish lid can be tilted to minimize condensate contamination, and the inverted plate operation is carried out in an ultra-clean bench next to an alcohol lamp.
* Sign the signal and store the liquid medium in a 4 ℃ refrigerator and use it as soon as possible. Close the solid medium with a parafilm and store it in a refrigerator at 4 ℃ and use it as soon as possible.

### **1.5 Kana+Amp+L-arabinose LB:**

The LB medium was configured as follows. Add an additional 15 g of agar powder (1.5%) before sterilization, then autoclave the configured medium.

| Ingredient | Quantity |
| --- | --- |
| NaCl | 10g |
| TRYPTONE | 10g |
| YEAST EXTRACT | 5g |
| ddH2O | To 1L |

The preparation process is as follows.

Calculate → Weigh → Dissolve → Sterilize → Pour Plate → Save

Detailed steps:

1. 1. Calculation of substance requirements based on the volume of medium required.
2. 2. Weigh the substances according to the calculation.
3. 3. Add ddH2O to fix the volume and stir well to dissolve it.
4. 4. Autoclave the conical vials after closing the mouth with a breathable parafilm and a leather band.
5. 5. Kanamycin, benzylpenicillin and L-arabinose should be added to the medium at a concentration of 1:1000 at a temperature of approximately 65°C, and the solution should be thoroughly agitated.
6. 6. In case of LB agar the inverted plate operation is carried out when the medium is cooled to about 45 ℃. The petri dish lid can be tilted to minimize condensate contamination, and the inverted plate operation is carried out in an ultra-clean bench next to an alcohol lamp.
7. 7. Sign the signal and store the liquid medium in a 4 ℃ refrigerator and use it as soon as possible. Close the solid medium with a parafilm and store it in a refrigerator at 4 ℃ and use it as soon as possible.

All of the aforementioned operations must be conducted within a biological safety cabinet in order to prevent contamination.

## **2. Antibiotic**

The following table shows the concentration of antibiotics and working solution used, which should be diluted 1:1000 at the time of use. Note that solid media need to be added after sterilization when cooled to approximately 45 ℃ to avoid inactivation of antibiotics.

| Ingredient | Concentration |
| --- | --- |
| Kanamycin | 100 mg/mL |
| Ampicillin | 100 mg/mL |
| Chloramphenicol | 50 mg/mL |
| Streptomycin | 50 mg/mL |

The preparation process is as follows.

Calculation → Weighing → Dissolving → Dispense

Detailed steps:

1. Calculation: according to the concentration and the required amount to calculate the antibiotic mass and sterile water volume to be weighed.
2. Weighing: according to the calculation results.
3. Dissolution: use a pipette gun ( the gun’s head needs to be sterilized ) to suck sterile water (ddH2O is sterilized by high-pressure steam ) and dilute to the corresponding volume.
4. Sub-packaging: by using disposable syringe and 0.22 μM sterile syringe filter (bioshrap, catalog number: bs-pes-22 ) to filter and sub-pack the antibiotic into several 1.5/2 ml EP tubes and then store them in a - 20℃ refrigerator, and use it as soon as possible.

## **3. IPTG\L-arabinose**

### **3.1 Preparation of 0.5M IPTG Stock Solution**

1. **Personal Protective Equipment (PPE):**Put on appropriate PPE including lab coat, gloves, and safety goggles.
2. **Weighing IPTG:** In a fume hood, accurately weigh the required amount of IPTG powder to prepare a 0.5M solution. The amount needed can be calculated using the molecular weight of IPTG and the desired volume of the stock solution.
3. **Dissolution:**Add the weighed IPTG to a suitable volume of sterile water in a clean container. Stir gently until the IPTG is completely dissolved. Ensure that the solution is at room temperature to facilitate dissolution.
4. **Sterilization:**Filter the solution through a 0.22 µm sterile filtration system to remove any potential contaminants. This step should be done in a laminar flow hood to maintain sterility.
5. **Aliquoting:** Using aseptic technique, aliquot the filtered IPTG solution into 1 mL centrifuge tubes. Label each tube with the solution name, concentration, date of preparation, and any other relevant information.
6. **Storage:** Store the aliquoted IPTG stock solution at -20°C. Avoid repeated freeze-thaw cycles to maintain the integrity of the solution.

### **3.2 Preparation of L-arabinose**

1. **Personal Protective Equipment (PPE):**Put on appropriate PPE including lab coat, gloves, and safety goggles.
2. **Weighing L-arabinose:** In a fume hood, accurately weigh the required amount of L-arabinose powder to prepare a 1M solution. The amount needed can be calculated using the molecular weight of L-arabinose and the desired volume of the stock solution.
3. **Dissolution:**Add the weighed L-arabinose to a suitable volume of sterile water in a clean container. Stir gently until the L-arabinose is completely dissolved. Ensure that the solution is at room temperature to facilitate dissolution.
4. **Sterilization:** Filter the solution through a 0.22 µm sterile filtration system to remove any potential contaminants. This step should be done in a laminar flow hood to maintain sterility.
5. **Aliquoting:**Using aseptic technique, aliquot the filtered L-arabinose solution into 1.5 mL centrifuge tubes. Label each tube with the solution name, concentration, date of preparation, and any other relevant information.
6. **Storage:**Store the aliquoted L-arabinose stock solution at -20°C. Avoid repeated freeze-thaw cycles to maintain the integrity of the solution

All of the aforementioned operations must be conducted within a biological safety cabinet in order to prevent contamination

---

# **Breed Preservation**

## **1. Glycerol Stock**

1. **Prepare 50% Glycerol Solution:**Mix equal volumes of 100% glycerol and dH2O, then store at room temperature.
2. **Inoculate Glycerol Stock:** Transfer 500 μL of bacterial culture to a 2 mL tube, add 500 μL of 50% glycerol, and mix gently.
3. **Storage:** Use screw top tubes, label with strain and date, and store at -80°C to prevent unexpected opening.
4. **Stability:**Maintain stocks at -80°C for long-term stability; avoid repeated freeze-thaw cycles.

## **2. Recovery**

1. **Recovery of Bacteria:**Under sterile conditions, open the glycerol stock tube and use a sterile inoculation loop or pipette tip to scrape an appropriate amount of frozen bacteria, then streak onto an LB agar plate with or without the appropriate antibiotic.
2. **Overnight Cultivation:**Incubate the streaked LB agar plate at the optimal temperature for the bacterial strain to allow for growth.
3. **Isolation of Single Colonies:** After overnight incubation, pick individual colonies for further processing or analysis.

# **Plasmid construction and Transformation**

## **1. PCR**

Prepare a 25 µl reaction system using PCR tubes (ignite an alcohol lamp, no need to operate inside a laminar flow hood). 12.5 µl PCR MIX, 0.5 µl upstream primer, 0.5 µl downstream primer, 1 µl template, 10.5 µl ddH2O. Gently centrifuge to mix the reaction system.

Set up the PCR program: (Usually use high-fidelity enzymes from Shenggong, or set according to the instructions for different enzymes)

| Temperature | Time | Cycles |
| --- | --- | --- |
| 94℃ | 2：00 | 1x |
| 98℃ | 0：15 | 34x |
| X℃ | 0：15 | 34x |
| 72℃ | Y | 34x |
| 72℃ | 5: 00 |  |
| 4℃ | ∞ |  |

X should be set lower than the lowest Tm value of the two primers by 5-6. For example, if the Tm values of the two primers are 80 and 72, it should be set to 65.

Y is the extension time, determined by the length of the PCR fragment, usually 30s/1Kb. For example, a 6300 bp (6.3 Kb) fragment should be set to 3:30.

## **2. Nucleic Acid Gel Electrophoresis**

Prepare the nucleic acid gel: Remember the ratio 1:10:100 (0.8g agarose: 8 µl nucleic acid dye: 80 ml 1x TAE buffer).

After adding agarose and TAE buffer, mix well and heat in a microwave until the solution is clear and transparent without any agarose lumps (use a larger conical flask to prevent boiling over). After heating, add the nucleic acid dye (highly toxic, handle with care), mix well. Place the plastic plate in the groove, then pour the solution, insert the comb (remove it vertically without damaging the wells). Do not add all at once to prevent the gel from being too thick.

Add 5 µl marker, 25 µl PCR product. Electrophoresis at 150V for 20 minutes.

## **3. Gel Recovery** Remove the electrophoresed nucleic acid gel from the glass plate, place it under ultraviolet light, and remember to take protective measures. After taking a photo for preservation, cut off a small piece of the gel and place it into a 1.5 mL EP tube. 1. Cut the single DNA band from the agarose gel (try to remove excess parts) and place it into a clean centrifuge tube, weigh it. 2. Note: When using a gel cutter (OSE-GC) to cut the gel, align the cutter with the DNA band in the agarose gel and press down to cut. After cutting, push the center rod to push the gel piece into a clean centrifuge tube. Depending on the width of the gel well, you can perform single or continuous cutting. 3. Add 3 times the volume of gel dissolution liquid PE (if the gel weight is 0.1g, its volume can be considered as 100 µl, then add 300 µl gel dissolution liquid PE. When using a gel cutter to cut 1% agarose gel, the weight of a single piece is about 0.06 g, the actual weight of the gel piece is related to the gel concentration and thickness), dissolve at room temperature (15-25°C) for 5-10 minutes, during which gently invert the centrifuge tube up and down to ensure the gel piece is fully dissolved. (If the volume of the gel piece is too large, it can be cut into smaller pieces beforehand). 4. Note: For large fragments greater than 5 kb or gels with a concentration greater than 1.5%, it is recommended to heat dissolve at 50°C for 5-10 minutes; after the gel piece is completely dissolved, it is best to lower the solution temperature to room temperature before applying it to the column, as the adsorption column has a stronger DNA binding capacity at room temperature. 5. Add the solution from step 2 to an adsorption column CA5 (place the adsorption column into a collection tube), let it sit at room temperature for 2 minutes, centrifuge at 12,000 rpm (approximately 13,400 xg) for 30-60 seconds, discard the waste liquid in the collection tube, and place the adsorption column CA5 back into the collection tube. 6. Note: The volume of the adsorption column is 800 µl, if the sample volume is greater than 800 µl, it can be added in batches. 7. Add 600 µl wash solution PW (check if anhydrous ethanol has been added before use) to the adsorption column CA5, centrifuge at 12,000 rpm (approximately 13,400 xg) for 30-60 seconds, discard the waste liquid in the collection tube, and place the adsorption column CA5 back into the collection tube. Note: If the DNA being recovered is for salt-sensitive experiments, such as blunt-end ligation experiments or direct sequencing, it is recommended to let the PW sit for 2-5 minutes before centrifugation. 8. Repeat step 4. 9. Place the adsorption column CA5 back into the collection tube, centrifuge at 12,000 rpm (approximately 13,400 xg) for 2 minutes to remove as much wash solution as possible. Let the adsorption column CA5 sit at room temperature for a few minutes to thoroughly dry to prevent residual wash solution from affecting the next step of the experiment. 10. Note: Residual ethanol in the wash solution can affect subsequent enzymatic reactions (restriction enzyme digestion, PCR, etc.). 11. Place the adsorption column CA5 into a clean centrifuge tube, add an appropriate amount of elution buffer TB to the center of the adsorption membrane and let it sit at room temperature for 2 minutes. Centrifuge at 12,000 rpm (approximately 13,400 xg) for 2 minutes to collect the DNA solution. Note: The elution volume should not be less than 30 µl, as a smaller volume can affect the recovery efficiency. The pH value of the elution buffer has a significant impact on the elution efficiency. If sequencing is to be performed later, ddH2O should be used as the elution buffer, and its pH value should be maintained between 7.0-8.5. A pH value below 7.0 will reduce the elution efficiency; and the DNA product should be stored at -20°C to prevent DNA degradation. DNA can also be eluted with a buffer (10 mM Tris-Cl, pH 8.0). **4. Homologous Recombination** If it is simply to delete a certain site, add 5 µl gel recovery product: 5 µl homologous recombination enzyme. If it is to connect the target gene with the vector, adjust the system according to the concentration to make the concentrations of the two the same, for example, if the vector and gene concentrations are the same, prepare 2.5 µl vector gel recovery product: 2.5 µl target gene gel recovery product: 5 µl homologous recombination enzyme. Set the PCR program to react at 50°C for 30 minutes, then save at 4°C indefinitely. Note that this is a 10 µl system, remember to select the 10 µl system when setting up the PCR. **5. Transformation** For general verification of whether the vector is successfully constructed, use DH5α, and for induced expression, use BL21 competent cells. If commercial ones are not available, they can be prepared on-site: 1. Cultivate the original MC1061 bacterial liquid, use a toothpick to pick a single colony from the biosafety cabinet into 20 ml LB (without antibiotics), culture at 37°C at 180 rpm for 5 hours until it becomes turbid (measure the OD value to be 0.3-0.6). 2. Transfer 1 ml of the bacterial liquid into a 1.5 ml EP tube, centrifuge at 12,000 rpm for 1 minute at 4°C (use a refrigerated centrifuge), discard the supernatant (prepare as many centrifuged bacterial liquids as needed for the competent cells). 3. Add 1 ml of ice-bathed CaCl2 (calcium chloride concentration of 0.1 mol/l, i.e., dissolve 1.11 g of calcium chloride in 100 ml of sterile water) to each tube, gently mix, and ice bath for 30 minutes. (Be gentle, handle the bacterial liquid on ice) 4. Centrifuge at 12,000 rpm for 1 minute, invert to remove residual liquid, then add 100 µl of 0.1 mol/l calcium chloride, the competent cells are prepared, store at -80°C. 5. Add 1 µl of plasmid (constructed plasmid, such as the sample returned by the company) to the competent cells, if it is a PCR product after homologous recombination, add 10 µl, ice bath for 30 minutes (let the competent cells melt on ice, operate on ice throughout the process, the pipette tip should be below the liquid surface but not touch the wall). 6. (Preheat the water bath in advance) Heat shock at 42°C for 60-90 seconds. 7. Quickly ice bath for 2-3 minutes (must be fast and the ice bath time should not be too long, usually take 2 and a half minutes as a compromise). 8. Add 900 µl of LB medium, culture at 37°C at 180 rpm for 1 hour to recover. 9. Centrifuge at 12,000 rpm for 1 minute, discard the supernatant 900 µl, gently mix. 10. Take 20-30 µl and spread it on LB medium with the corresponding antibiotics, spread evenly with a glass rod, seal (to prevent water loss), and culture upside down for 12-16 hours. (If it is a plasmid obtained through the PCR process, spread it all). The entire transformation process must be carried out in a biosafety cabinet and an alcohol lamp must be ignited, the pipette tips, culture medium, and EP tubes must be sterilized. --- **Expression** **1. Bacterial Culture and Target Protein Expression** 1. **Colony Picking:** Pick a single colony from the transformation plate into 20 mL of liquid medium with kanamycin, incubate overnight at 37°C. 2. **Expression:**Add 400 µL kanamycin to 400 mL TB medium, then inoculate with 6-8 mL (2%) bacterial culture. Incubate for 3-4 hours until OD600 reaches 0.8. 3. **Induction:**Add 400 µL (1%) of 0.5 M IPTG and incubate at 16°C, 180-200 rpm for 24 hours. **2. Bacterial Culture Harvesting and Pellet Resuspension** 1. Take four 50 mL centrifuge tubes, and add 50 mL of bacterial culture to each tube, balancing the tubes appropriately. 2. Centrifuge at 4500 rpm for 15 minutes. 3. Discard the supernatant, and resuspend the bacterial pellet in each tube with 5-10 mL of PBS buffer. Combine the resuspended cultures into a single centrifuge tube, and store at -20°C. 4. Depending on the total volume of the bacterial culture (e.g., 400 mL), repeat the above steps as necessary. --- **Verification** **1. SDS-PAGE** Polyacrylamide Gel Electrophoresis (PAGE) is a method of electrophoresis in which a polyacrylamide gel is used as a support medium for the separation of proteins or nucleic acids. Polyacrylamide gel is made of acrylamide monomer (ACR) and cross-linking agent N,N-methylene bisacrylsmide (N,N-methylene bisacrylsmide abbreviated as BIS) in the role of catalyst polymerization and cross-linking of three-dimensional mesh structure of the gel. By varying the ratio of monomer concentration to cross-linking agent, gels with different pore sizes can be obtained, which can be used to separate substances with different molecular weights. We used SDS-PAGE to verify the expression of the imported proteins. **2. Preparation of polyacrylamide gels** 1. Installation of the gel board model: Wash the glass board, dry it and set it aside. When making glue, choose the right glass plate, assemble the glue plate model, put the glass plate into the rack after alignment, and the clamps on both sides are tightened, so that the short glass is leaning out and the long glass is leaning in. 2. According to the following table formula configuration of the separation of the glue solution, mixing with a light hand shaking, carefully inject the mixture into the prepared glass plate gap, leaving enough space for the concentration of the glue (~ 2.5cm), gently in the top layer of the addition of 0.5ml of deionized water to cover the (liquid sealing should be slow, to avoid deformation of the glue is washed out), in order to stop the inhibition of the air oxygen on the coagulation. Just add water can be seen between the water and the gel liquid interface, and then gradually disappear, soon appeared again interface, which indicates that the gel has been polymerized. And then leave a few moments to make the polymerization complete, the whole process takes about 30 minutes (25 ℃ room temperature). Separation gel concentration | Gel volume | Volume of each component required(in ml) | | | | || | | H2O | 30%Acr-Bis（29：1） | SDS-PAGE Separating Gel Buffer(4×） | 10%APS | TEMED | | 6% | 5ml | 2.75 | 1.0 | 1.25 | 0.05 | 0.004 | | 8% | 5ml | 2.42 | 1.33 | 1.25 | 0.05 | 0.003 | | 10% | 5ml | 2.08 | 1.67 | 1.25 | 0.05 | 0.004 | | 12% | 5ml | 1.75 | 2.0 | 1.25 | 0.05 | 0.002 | | 15% | 5ml | 1.25 | 2.5 | 1.25 | 0.05 | 0.002 | 3. The preparation of concentrated gel: the first has been polymerized to separate the upper layer of the gel water suction, and then use filter paper to absorb the residual water. Prepare the concentrated gel solution according to the following formula. Mix it and inject it into the upper end of the separation gel, insert the comb and be careful to avoid air bubbles. 4. While the gel concentrate is being polymerized, mix the protein sample with an equal volume of 4X sample buffer, denature in a water or metal bath at 95°C for 10 min, and cool to room temperature for use. 5. After the polymerization of concentrated gel is complete, put the gel template into the electrophoresis tank and fix it, with the small glass plate facing inward and the large glass plate facing outward, add 1X electrophoresis buffer to both the upper and lower tanks, and carefully pull out the comb, check for leakage, and remove away air bubbles at the bottom of the gel between the two glass plates. **3. Anchor Peptide Purification** **3.1 Bacterial Lysis** 1. Transfer 20-50 mL bacterial culture to a 50 mL beaker. 2. Place the beaker in a larger beaker filled with ice, keeping the ice 1-2 cm below the small beaker's rim to prevent water backflow. 3. Sonicate at 170-190W, 1s on/3s off, for 40 minutes (avoid excessive agitation or foaming). 4. Stop when the liquid turns clear and less viscous; if not, extend by 5-10 minutes. **3.2 Obtaining GFP-Anchor Peptide** 1. Centrifuge lysate at 12,000 rpm for 1.5 hours in 2 mL tubes (ensure balance). 2. Collect supernatant into a 50 mL centrifuge tube. **Nickel Column Purification:** 1. Remove nickel column from 4°C, drain ethanol solution. 2. Equilibrate column with 1x PB buffer three times. 3. Equilibrate with 10 mM imidazole solution. 4. Add 10-15 mL supernatant to column, invert to mix, incubate on ice for 45 minutes. 5. Attach tubing, secure column, and collect flow-through. 6. Wash with 30 mM imidazole buffer at 2-3 drops/sec. 7. Test impurities with Coomassie Brilliant Blue; continue washing if blue. 8. Elute protein with 300 mM imidazole, incubate 5 minutes, and collect in 2 mL tubes. 9. Repeat elution if strong fluorescence; collect 15-20 mL, store at 4°C. 10. Wash column with 1x PBS, store in 20% ethanol at 4°C. **3.3 Anchoring peptide aquisition** **Enzymatic Cleavage with TEV Protease:** Use the TEV protease kit (P2308, Beyotime Inc.) and prepare the cleavage system according to the specified ratios. | Component | Volume(μl) | | --- | --- | | H2O | X | | 10X TEV Buffer | 5 | | Target Protein (8 μg) | Y | | TEV Protease (His-tag) (1U/μl) | 0,1.5or2.5 | | Total Volume | 50 | - Place the reaction system in a 4°C refrigerator for 24 hours. **Ni2+-NTA Column Purification:** 1. Remove Ni2+-NTA Column from 4°C, secure vertically, and drain ethanol. 2. Wash column with 1x PB buffer three times to equilibrate. 3. Fill with 10 mM imidazole solution, drain to remove non-specific bindings. 4. Close ports, add cleavage solution, invert to mix, incubate on ice for 10 minutes. 5. Open ports, collect eluted target protein. 6. Add 15 mL of 1 M imidazole, invert to dissociate residual proteins, then drain. **Ni2+-NTA Column Storage:** - Wash with 1x PB buffer, add 20% ethanol, and store at 4°C. **3.4 Coomassie Brilliant Blue Assay for Protein Concentration:** **Concentration:** 1. **Standard Curve Preparation:**Mix 1 mL Coomassie Brilliant Blue solution with 20 µL protein standard. After 1.5 minutes, measure absorbance at OD595 to create the standard curve. 2. **Sample Measurement:**Mix 1 mL Coomassie Brilliant Blue solution with 20 µL protein sample. After 1.5 minutes, measure OD595, and use the standard curve to determine protein concentration. **3.5 Determination of Protein Concentration Using BCA Assay Kit** **Dilution of Standards:** Dilute the BSA standards with the same buffer system as the samples according to the following table: | Tube Number | Volume of Dilutant | BSA Volume (Source) | Final BSA Concentration (μg/ml) | | --- | --- | --- | --- | | A | 0 μlL | 300 μl (Stock) | 2000 | | B | 125 μl | 375 μl (Stock) | 1500 | | C | 325 μl | 325 μl (Stock) | 1000 | | D | 175 μl | 175 μl (Tube B) | 750 | | E | 325 μl | 325 μl (Tube C) | 500 | | F | 325 μl | 325 μl (Tube E) | 250 | | G | 325 μl | 325 μl (Tube F) | 125 | | H | 400 μl | 100 μl (Tube F) | 25 | | I | 400 μl | 0 | 0 | **Preparation of BCA Working Solution** Based on the number of samples, prepare an appropriate amount of BCA working solution by mixing reagent A and reagent B in a volume ratio of 50:1, and mix well. *Note: Before preparing the BCA working solution, please shake reagent A well to mix.* **Standard Colorimetric Cup Method:** 1. Take 0.1 ml of each standard and sample in appropriate tubes. 2. Add 2.0 ml of BCA working solution, mix thoroughly. 3. Cap and incubate at 37°C for 30 minutes, then cool to room temperature or place at room temperature for 2 hours. 4. Measure the absorbance at 562 nm using a UV spectrophotometer. 5. Calculate the protein concentration in the samples based on the standard curve. **Microplate Method:** 1. Take 25 μl of freshly prepared BSA standard solution and sample from the table and add to a 96-well plate. 2. Add 200 μl of BCA working solution to each well and mix well. 3. Cap and incubate at 37°C for 30 minutes, then cool to room temperature or place at room temperature for 2 hours. 4. Measure the absorbance at 562 nm using a UV spectrophotometer. 5. Calculate the protein concentration in the samples based on the standard curve **4. Affinity validation of the anchored peptide** 1. Measure the concentration of the four purified EGFP-anchor peptides using the Coomassie Brilliant Blue method and dilute them to the same level. 2. Mix 200 µL of EGFP-anchor peptide solution with 200 µL of 100 µg/mL PS/PLA/PET/PP solution (plastic particle size 8-13 µm, solvent is pure water). 3. Incubate at 4°C for 15 minutes. 4. Centrifuge at 12,000g for 2 minutes and discard the supernatant to remove unreacted anchor peptides. 5. Resuspend the pellet in 100 µL of PB buffer. 6. Prepare slides and observe under an inverted fluorescence microscope, capturing both fluorescence and bright-field images. **5. Colorimetric identification of plastics****5.1 Preparation of Gold Nanoparticles** 1. Mix 1 mL of chloroauric acid with 49 mL water, then heat to boiling or until bubbles form. 2. Add 2 mL of 1% trisodium citrate (pre-prepared) all at once. Insert a stirring bar and heat for 30 minutes. 3. Turn off heat and stir for 15 minutes. 4. Aliquot into 50 mL tubes, centrifuge at 10,000 rpm for 1 hour, discard supernatant, and resuspend gold nanoparticles in glycine buffer (pH 8.5). 5. Store at 4°C. **5.2 Gold Nanoparticle Modification** 1. Mix 1 mL of gold nanoparticles with 30 µL of anchor peptide and stir. Incubate at room temperature for 10 minutes to prepare the anchor peptide-gold nanoparticle solution. *Note: For larger volumes, adjust proportions accordingly. Prepare fresh and use immediately.* **5.3 Microplastic Detection** 1. Prepare a plastic gradient solution (2, 4, 6, 8, 9, 10 µg/mL) using glass test tubes for each concentration. 2. Mix 100 µL of the prepared plastic gradient solution with 1 mL of anchor peptide-gold nanoparticle solution. Incubate at room temperature for 10 minutes. 3. Record the color and spectral data using an RGB sensor and UV-Vis spectrophotometer. **6. High-Performance Liquid Chromatography Analysis****6.1 HPLC analysis** A Waters E2695 chromatographic system, equipped with a Shim-pack GIS column, was employed in this study for HPLC analysis. The mobile phase was comprised of a mixture of methanol and acetonitrile, flowing at a rate of 0.5 mL/min, with detection at a wavelength of 254 nm. The elution protocol commenced with a gradient elution using 95% formic acid and 5% from 0 to 2 minutes. This was followed by a linear gradient elution, increasing the acetonitrile concentration to 20% while maintaining 80% formic acid from 2 to 14 minutes. The system was then re-equilibrated to the initial conditions of 95% formic acid and 5% acetonitrile over the 14 to 15-minute interval, employing a gradient pump mode throughout the entire chromatographic run. The total peak areas of MHET, terephthalic acid (TPA), PCA and Vanillin were utilized in determining the quantity of product from each PET hydrolysis reaction. **6.2 Standard Curve of HPLC analysis** Prepare stock solutions of tetradecanoylphorbol acetate (TPA), p-coumaric acid (PCA), vanillin and methyl 4-hydroxy-3-methoxyphenyl ether (MHET) at a concentration of 10 mg/mL, with TPA specifically dissolved in dimethyl sulfoxide (DMSO). For the construction of a standard curve, dilute these stock solutions to prepare a series of mixed solutions with final concentrations of 0.05, 0.1, 0.25, 0.5, 1, and 2 mM. Subject the mixed solutions to thermal treatment at 90°C for 15 minutes to facilitate compound solubilization and reaction. Post-thermal treatment, centrifuge the solutions at 12,000 rpm for 20 minutes to pellet any insoluble material. Carefully collect the supernatant and proceed with high-performance liquid chromatography (HPLC) analysis using a Waters E2695 system to quantify the compounds and establish the standard curve. **Degradation****6.3 PET Degradation:** PET powder (1000 mesh, granular form) was sourced from a commercial supplier. Precisely 0.4 grams of this PET powder was measured and transferred into a 50 mL conical flask. Thereafter, 10 mL of bacterial culture was inoculated into the flask. The culture was derived from a homogenous batch of bacteria that had been expressed and subjected to centrifugation at 4500 rpm for a period of 20 minutes to pellet the cells. Post-centrifugation, the bacterial pellet was resuspended in a series of buffer solutions with varying pH levels (pH = 7 - 10) to adjust the optical density (OD600) to the desired values. The flask containing the mixture was then sealed with a sterile membrane to maintain sterility and integrity of the reaction environment. The sealed flask was placed on a constant temperature shaker, which was set to oscillate at a speed of 180 rpm for a continuous period of 24 hours. The enzymatic reaction was terminated by subjecting the flask to a thermal treatment at 95°C for a duration of 15 minutes. **6.4 TPA Degradation** Resuspend the E. coli BL21 (DE3) strain, which has undergone triple plasmid co-transformation and is induced with 0.4 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 24 hours, in 3 mL of M9 medium to achieve an optical density at 600 nm (OD600) of 65. Then, incorporate 100 µL of n-butanol and 100 µL of a 0.12 mol/L terephthalic acid (TPA) standard solution in dimethyl sulfoxide (DMSO). Subsequently, adjust the volume to 10 mL with a 50 mM glycine buffer prepared at a pH of 8.5. Cultivate the assay mixture at a temperature of 20°C by shaking at a speed of 180 revolutions per minute (rpm) for a continuous period of 24 hours. Finally, terminate the enzymatic reaction by thermal inactivation at 95°C for 15 minutes. --- **Gene editing** **1. Plasmid Construction Workflow for PHCY-yahK-KO(piGEM24\_09)** The sgRNA sequence designed on the Benchling platform was introduced into the N20 site of the PHCY-26D plasmid through PCR and seamless ligation. The constructed plasmid was subsequently verified by sequencing. The verified plasmid was then linearized and seamlessly ligated with the HA1/2 fragments, which were obtained via colony PCR and overlap PCR, to generate the PHCY-yahK-KO(piGEM24\_09) plasmid. The successful construction was confirmed by sequencing. **2. YahK Gene Knockout Procedure** For the YahK gene knockout, PHCY-25A and piGEM24\_09 plasmids are used, and the strain employed for editing is Escherichia coli BL21(DE3). 1. **Preparation of Competent Cells and Transformation with PHCY-25A Plasmid:**Prepare chemically competent cells of *E. coli* BL21(DE3) and transform them with the PHCY-25A plasmid. The resulting strain is *E. coli* BL21(DE3)/PHCY-25A. 2. **Preparation of Competent Cells of the Transformed Strain:**The *E. coli* BL21(DE3)/PHCY-25A strain is prepared as chemically competent cells. The medium should contain kanamycin (Kan) and glucose. 3. **Transformation with piGEM24\_09 Plasmid:**Transform the piGEM24\_09 plasmid into the *E. coli* BL21(DE3)/PHCY-25A competent cells. Plate the transformants on LB agar plates containing ampicillin (Amp), kanamycin (Kan), and glucose, and incubate at 30°C. 4. **Colony Growth and Induction:**Once colonies have grown, select 3-4 colonies from the plate and inoculate them into 2 ml of LB medium. Add 2 μl of ampicillin (100 mg/ml) and 2 μl of kanamycin (50 mg/ml), and incubate at 30°C for 2 hours. Then, induce with 20 μl of IPTG (100 mM) and incubate at 30°C for 1 hour, followed by 40 μl of L-arabinose (1 M), incubating for an additional 3 hours at 30°C. 5. **Dilution and Plating for Colony Screening:**Dilute the culture 100-fold and 1000-fold. Plate 100 μl of the diluted cultures on LB agar plates containing ampicillin (Amp), kanamycin (Kan), and L-arabinose. Incubate at 30°C. After colonies grow, confirm successful knockout by PCR verification. ![experiment-f1.png](https://static.igem.wiki/teams/5487/experiment-f3.png) Fig.1. WT(B) and KO(A) group expected consequence of E.coli gene editing test. PCR validation was performed using the primers Test-KO-F and Test-KO-R, as shown in Figures 1 and 2. In the wild-type (WT) group, the PCR amplification product before knockout was 2219 bp, while the knockout (KO) group showed a PCR product of 1169 bp after successful gene deletion. Therefore, the success of the knockout can be determined based on the length of the PCR products. **3. Plasmid Curing Procedure** Inoculate a colony confirmed to have a successful knockout into 2 ml of LB medium without antibiotics, and incubate overnight at 37°C. Then, streak 2 μl of the culture onto an LB plate containing 1% sucrose, and incubate at 37°C. Most of the colonies that grow on the plate will have lost the pHCY-25A and piGEM24\_09 plasmids. To verify plasmid curing, select a colony and inoculate it into three different LB media: one without antibiotics, one with ampicillin (Amp+), and one with kanamycin (Kan+). Incubate all three tubes at 37°C. If the colony grows in the antibiotic-free medium but not in the Amp+ or Kan+ media, it confirms the loss of both the pHCY-25A and piGEM24\_09 plasmids. The culture in the antibiotic-free medium can then be used for subsequent experiments.

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Results

* [Colorimetric detection of microplastics based on anchoring peptide-modified Au colloid](#Colorimetricdetection)
  + [1. Expression and purification of anchoring peptides](#Expression)
  + [2. Affinity Assay of anchoring pepetide against plastics](#Affinity)
  + [3. Conclusion and Outlook](#ConclusionandOutlook)
* [PET Biodegradation](#PETBiodegradation)
* [Upcycling of TPA to vanillin](#Upcycling)
  + [1. Plasmid Construction for TPA Upcycling Enzymes](#Constructon)
  + [2.Protein expression](#Protein expression)
  + [3.HPLC standard curves](#HPLCanalysisresults)
* [Gene editing to downregulate vanillin metabolism](#Geneediting)

# **Colorimetric detection of microplastics based on anchoring peptide-modified Au colloid**

## **1. Expression and purification of anchoring peptides**

In order to specially recognize microplastics, we utilized genetic engineering strategies to express anchoring peptides Cg-Def, TA2, and LCI. Many experiements have been done to optimize the design of recombinant plasmids, protein expression and purification of these three anchoring peptides. Fig.1A,1B and 1C gave the finally designed plasmid maps in which the target protein gene LCI, TA2 and Cg-Def was follwed by TEV site, His Tag and sfGFP (superfolder Green Fluorescent Protein), respectively. PCR results in Fig. 1D showed that both the target protein and sfGFP were successfully inserted into pET-30(a). SDS-PAGE results indicated that the fusion protein was successfully expressed and purified, as shown in Fig. 1E.

![result-f1.png](https://static.igem.wiki/teams/5487/result-f1.png)

Fig.1: Plasmid profile of the recombinant pET-30(a) vector containing anchoring peptides LCI (A), TA2(B) and Cg-Def(C),

## **2. Affinity Assay of anchoring pepetide against plastics**

To check the affinity capability of the expressed target peptide against various microplstics, we utilized fluorescence microscopy to measure the fluorescence emitted from sfGFP in the fusion protein. Meanwhile optical image was also done to show the plastics appearance. It was found from Fig.2A that when PS, PLA or PP was incubated with TA2 anchoring peptides, there occurred strong fluorescence emission on the plastic surface. Similar phenomenon was also observed when we used LCI and CG target peptides to mix various plastics (Fig.2B and 2C). In contrast, no fluorescence was detected in the case of PET, indicating that these anchoring peptides showed rather weak affinity to recognize PET. Here it should be noted that we also used sfGFP containing no anchor peptides as control, and no fluorescence emission was detected. These results suggest that we have successfully obtained anchoring peptides.

![result-f2.png](https://static.igem.wiki/teams/5487/result-f2.png)

Fig.2 Fluorescence microscopy and optical imaging results of the mixture system containing the expressed anchroing peptide and PS, PLA, PET, PP plastics

## **3. Detection of Microplastics based on anchoring peptide modified Au colloid**

After we synthesized Au colloid by the reaction of HAuCl4 and sodium citrate, we added the anchoring peptide TA2 into Au colloid to make them to combine together. We optimized pH value and the ratio of Au colloid versus anchoring peptide to finally get the modified Au colloid for the next experiment.

### 3.1 RGB sensor-based detection results

Fig.3A gave the color changes induced by the addition of microplastics to the anchoring peptide modified Au nanoparticles solution. It was observed that when the concentration of microplastics was less than 2 ug/mL, Au nanoparticle remained red colour. As the concentration of microplastics increased, Au nanoparticle gradually aggregated in solution and displayed purple color. Here we used RGB sensors to measure the values of red light channel, green light channel and blue light channel. They are denoted as R-value, G-value and B-value, respectively. Next we used multiple linear regression model, and successfully find the linear relationship between the concentration of microplastics and RGB fitted data (Fitted\_Data = -0.1172\*R\_values + 0.5765\*G\_values - 0.5871\*B\_values+29.0269). As seen from Fig.3B, the standard deviation is ~ 0.99, indicating that RGB method we designed here was accurate and feasible for the detection of microplastics.

![result-f3.png](https://static.igem.wiki/teams/5487/result-f3.png)

Fig.3 Colour changes of TA2-anchored peptide-modified Au colloid in the presence of microplastics with the concentration of 0, 2, 4, 6, 8, 10μg/mL from left to right (A) and the linear relationship between the concentration of microplastics and the fitted RGB value

### 3.2 UV-Vis Spectral measurement results

In addition to RGB sensor, we also used UV-Vis absorption spectrometer to detect the spectral changes. As seen in Fig.4A, the introduction of microplastics gave rise to the appearance of one new wide peak at ~ 700 nm. This phenomenon inferred that Au colloid aggregated to larger particle in the presence of microplastics. In order to avoid the spectral overlapping between 525 nm and ~ 700 nm, We selected the absorbance ratio of A750/A525 to describe the extent of gold nanoparticle aggregation. As shown in the Fig.4B, there was a linear relationship between A750/A525 value and PS concentration in the range of 0-10 μg/mL, with a linear regression correlation coefficient of 0.99. This result indicated that UV-Vis absorption method is promising for the quantification analysis of PS plastic concentration.

![result-f4.png](https://static.igem.wiki/teams/5487/result-f4.png)

Fig.4 UV-Vis absorption spectra of TA2-modified Au colloid in the presence of differnt concentration of PS microplastics.

### Conclusion and Outlook

In our project, we have successfully developed a simple, convenient and specific detection methods for microplastics. Instead of expensive Raman or FTIR instruments, we can use much cheaper UV-Vis absorption spectra or even RGB sensors to carry out quantitative analysis of microplastics. We firmly believe that in the near future, we can use our mobile phone to achieve the detection of microplastics in the enviroment. It will help the public to realize the occurance and damage of microplastics and then take actions to protect our earth.

---

# **PET Biodegradation**

Taking into account that PET could hardly enter the *E. coli* cells, we hoped that PET-degrading enzymes could be displayed on the bacterial surface. To achieve this aim, here we used the membrane protein Lpp-OmpA. In order to ensure this protein could be expressed on the *E. coli* surface, we firstly constructed one plasmid pET-30(a)-Lpp-OmpA-sfGFP. Fig.5B and 5C showed the PCR results and fluorescence microscopic results. It was observed that sfGFP has been expressed on the surface of *E. coli*cell. This result indicated that Lpp-OmpA could work in our system. Next, we constructed the degrading recombinant pET-30(a) Lpp-OmpA-Z1-PETase-MHETase plasmid. As demonstrated in Fig.6B, the PCR amplified bands appeared at the expected position, suggesting that this plasmid has been successfully constructed. Although we had tried to use 15% SDS-PAGE to detect the expression of both Z1-PETase and MHETase, there were no significant bands at the expected molecular weight. It might be because the targets protein was mainly expresed in the form of membrane protein. It is not easy to extract enough amount of membrane protein. Next we conducted HPLC detection to make sure if our engineered *E. coli* cells could degrade PET or not.

![result-f5.png](https://static.igem.wiki/teams/5487/result-f5.png)

Fig.5 Schematic map of pET-30(a)-Lpp-OmpA-sfGFP plasmid (A) and 1% agarose gel electrophoresis results (B) of the PCR amplified bands. M: Trans 5K DNA Marker; Lanes 1-2: pET-30(a)-Lpp-OmpA; Lanes 3-4: sfGFP. (C) Fluorescence microscopic results of empty vector (control) and *E. coli* cells expresssing Lpp-OmpA-sfGFP (surface display).

![result-f6.png](https://static.igem.wiki/teams/5487/result-f6.png)

Fig.6 Schematic map of pET-30(a)-Lpp-OmpA-Z1-PETase-MHETase plasmid (A) and 1% agarose gel electrophoresis results of the PCR amplified bands (B). M: Trans 5K DNA Marker; Lanes 1-4: the pET30a vector backbone; Lanes 5-6: the amplified produce for Lpp-OmpA fusion protein; Lanes 7-8: the amplificed product for Z1-PETase enzyme; Lanes 9-10: the amplified product for MHETase enzyme.

In our project, we used HPLC to detect the concentation of TPA product. Fig.7A showed the standard curve of TPA in PBS buffer (pH 7.45) . Next we measured if TPA was released following the 24h treatment of PET using the engineered whole-cell *E. coli* harbouring Z1-PETase and MHETase. As demonstrated in Fig.7C, TPA was successfully produced, indicating that Z1-PETase and MHETase had been successfully expressed and they could work together. We also carried out some optimization experiments (seen in Fig.7B), and finally found that the released TPA could reach 0.579 mM when pH and OD was adjusted to 9 and 20 respectively (seen in Fig.7C).

![result-f7.png](https://static.igem.wiki/teams/5487/result-f7.png)

Fig.7 HPLC analysis of TPA and TPA released from PET following the treatment of enzyme expressed from the engineered *E. coli* harbouring Z1-PETase and MHETase. (A) Standard curve for terephthalic acid (TPA) obtained from HPLC profile using C18 column detected at 254 nm with the mobile phase 0.1% formic acid and acetonitrile solution. (B) Influence of initial pH and bacterial optical density (OD600) on the enzymatic degradation of PET using whole-cell expressing Z1-PETase and MHETase. (A: OD600 = 50, B: OD600 = 20, C: OD600 = 5, D: OD600 = 1) (C) Comparison of TPA concentration obtained from HPLC profile under different pH and OD600.

### Conclusion and Outlook

In this work, we used one membrane protein Lpp-OmpA Tag to achieve the surface display of both Z1-PETase and MHETase fusion protein. Under this condition, these dual enzymes could effectively catalyze PET into TPA. The yield of TPA varied with pH and bacterial density. In the further work, we could optimize the ratio of these two enzymes or adjust the linker between them to achieve higher yield or higher stability.

---

# **Upcycling of TPA to vanillin**

## **1. Plasmid Construction for TPA Upcycling Enzymes**

In order to convert terephthalic acid (TPA) into the high-value compound vanillin, we first constructed one recombinant plasmid containing 4 genes, i.e. *TphA1, TphA2, TphA3* and *TphB*. The former three genes encode the three subunits of terephthalate 1,2-dioxygenase (TPADO), and *TphB* encoded 1,4-dicarboxylic acid dehydrogenase (DCDDH). These two enzymes could catalyze TPA into protocatechuic acid (PCA). Next we constructed the second plasmid harbouring *NiCAR* gene and *S-COMT* gene, which encodes carboxylate acid reductase and catechol O-methyltranferase. The third plasmid was also successfully constructed, in which *Sfp* gene encoding phosphopantetheinyl transferase was incorporated into PACYDuet-1 vector. The enzymes are essential for the conversion of PCA to vanillin.

![result-f8.png](https://static.igem.wiki/teams/5487/result-f8.png)

Fig.8 Plasmid profile of TphA2-TphA1-TphA3-TphB fusion expression system (A),
NiCAR-SCOMT fusion protein system(B) and Sfp fusion protein expression system (C) involved in the coversion from TAP to vanillin.

## **2.Protein expression**

SDS-PAGE analysis revealed that several target proteins were successfully expressed in soluble form using *E. coli* BL21(DE3) as host cell. (Fig.9).

![result-f9.png](https://static.igem.wiki/teams/5487/result-f9.png)

Fig.9 15% SDS-PAGE results of the pellets and supernant of empty vector (panel A: lane 1 and lane 2, panel B: lane 3 and lane 4) and the lysed *E. coli* cell harbouring piGEM24\_06 plasmid (panel A: lane 3 and lane 4 ) , piGEM24\_08 plasmid (panel A: lane 5 and lane 6) and piGEM24\_07 plasmid (panel B : lane 1 and lane 2).

## **3.HPLC analysis results**

Fig.10A showed the HPLC eluting profile of the supernatant collected following the addition of TPA to the *E. coli* intact cells co-expressing TPADO and DCDDH enzymes. HPLC comparison was also made using the standard sample of TPA and PCA under the same condition. It was found that PCA was produced successfully with the elution time of 7.068 min. The concentration of PCA was also calculated to be 0.078 mM.

Based on the standard curve we had done (data not shown). This result suggested that the first plasmid can work well. Next we detected if the second and the third plasmid could also work or not using HPLC method. As seen in Fig.10B, there appearred one small eluting peak at 14.279 min after the incubation of TPA with the intact *E. coli* celles which was co-transformed by plasmids piGEM24\_06, piGEM24\_07, and piGEM24\_08. The injection of standard vanillin sample into HPLC gave rise to the appearance of the same eluting peak, indicating that we have successfully converted the reactant TPA to the product vanillin using the engineered whole cell. In this case, the concentration of vanillin was almost 0.031 mM by calculating using standard curves of vanillin.

![result-f11.png](https://static.igem.wiki/teams/5487/result-f11.png)

Fig.10 HPLC profile of the supernetant collected following the 24h treatment of TPA using the intact *E. coli* cell expressing TPADO and DCDDH (A) and coexpressing multiply enzymes (B) in 50 mM Glycine buffer (pH 8.5)

### Conclusion and Outlook

For the time being, we have successfully achieved the upcycling of TPA into vanillin using engineered *E. coli* coexpressing multiple enzymes. In further work, we need to continue optimizing various conditions, particularly focusing on enhancing the transmembrane transport of TPA, which aims to improve the yield of vanillin.

# Gene editing to downregulate vanillin metabolism

In order to enhance the yield of vanillin, we carried out gene editing experiment to knock out one key enzyme YahK in the side pathway. Electrophoresis results showed that the target gene has been successfully knocked out, as seen from Fig.11C. More work needs to be done to check the effect of knoted-out gene on the yield of vanillin.

![result-11.png](https://static.igem.wiki/teams/5487/result-f16.png)

Fig.11 KO (A) and WT (B) partial gene sequence and 1% agarose gel electrophoresis results of PCR products using Test-KO-F and Test-KO-R as primers. Comparison showed that the target gene had been knocked out.

### Conclusion and Outlook

In this part, we have successfully utilized a Cas9-homologous reorganization fusion system to achieve gene editing of *E. coli* BL21(DE3). In further work, more key genes relating to *E. coli* BL21(DE3) vanillin metabolism pathways need to be knocked out, downregulated or upregulated so as to significantly enhance the yield of vanillin.

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