iGEM 2023 — Team bioplus-china

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# Notes

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![](https://static.igem.wiki/teams/4804/wiki/ack.png)

© 2022 Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository for this website is available at <gitlab.igem.org/2023/bioplus-china>.

1. [HOME](https://2023.igem.wiki/bioplus-china/)

- PROJECT

[DESCRIPTION](description)
[CONTRIBUTION](contribution)
[ENGINEERING](engineering)
[MODEL](model)

- WETLAB

[EXPERIMENTS](experiments)
[NOTES](notebook)
[SAFETY](safety)
[PART](part)

- [HUMAN PRACTICES](human-practices)

[EDUCATION](education)
[IHP](human-practices)

- TEAM

[MEMBERS](team)
[ATTRIBUTIONS](attributions)

# Engineering

Plastic pollution is a global crisis that affects aquatic ecosystems, human health, and livelihoods. Polyethylene terephthalate (PET) is one of the most abundantly produced plastics and is accumulating in the environment at a staggering rate as discarded packaging and textiles. We aim to use the combination of PETase (PET-digesting enzyme) and spider silk protein (R, a repetitive region of pyriform silk gene PySp1) to degrade PET plastics more efficiently. We successfully constructed the prokaryotic expression system, and a series of tests shows that PETase-R recombinant proteins could improve the degradation efficiency of PET.

## Design

We designed a prokaryotic expression system to obtain the PETase-R recombinant proteins. The composition part (PETase-R, BBa\_K4804000) consists of PETase (BBa\_K4804002) and R (BBa\_K4804003) (Figure 1). The successfully constructed PETase-R vector was transformed into E. coli BL21 (DE3). Then we did the following tests, as shown in Figure 2:

* SDS-PAGE analysis of PETase-R.
* Using p-NP assay to analyze the activity of recombinant protein proteolytic substrates.
* Using HPLC to analyze the ability to degrade real PET plastic.

![](https://static.igem.wiki/teams/4804/wiki/engineering/figure1.png)

Figure 1:Constitution of T7 Promoter-RBS-PETase-R-T7 Terminator gene circuits

![](https://static.igem.wiki/teams/4804/wiki/engineering/figure2.png)

Figure 2: Work flow of our project

## Synthesizing

We designed the essential parts (PETase and R) by reading background literature and data, and then the sequences were synthesized by GenScript and cloned into the pET-21a (+) vector. The R sequence was subcloned into the C-terminal of PETase using seamless cloning techniques to construct the composition part (Figure 3). Ultimately, we successfully recombined the PETase-R\_pET-21a (+) plasmid (Figure 4) and transformed it into E. coli BL21 (DE3) strain.

![](https://static.igem.wiki/teams/4804/wiki/engineering/figure3.png)

Figure 3:Ligation reaction of PETase-R\_pET-21a (+) by seamless cloning.

![](https://static.igem.wiki/teams/4804/wiki/engineering/figure4.png)

Figure 4:Recombinant Plasmid Map of PETase-R\_ pET-21a (+). pET-21a (+) vector contains ampicillin resistant fragment for screening positive clones

The vector fragment containing PETase was cloned from the plasmids PETase \_pET-21a(+) by PCR, and the product was detected by 0.8% agarose gel electrophoresis. The vector fragment size is 6283bp (Figure 5). At the same time, we amplified the R fragment by PCR, and the product was examined by 0.8% agarose gel electrophoresis (Figure 6). We use the seamless cloning kit (Sango Biotech, Seamless cloning Master Mix) to recombine the target fragment with the vector. Transformation of E. coli and DH5α competent cells was performed after the recombination reaction, and colony PCR results (Figure 7-A) and sequencing (Figure 7-B) showed that R was successfully inserted into the C-terminal of PETase. Then, the correctly sequenced plasmid was transferred into E. coli BL21(DE3).

![](https://static.igem.wiki/teams/4804/wiki/engineering/figure5.png)

Figure 5: Electrophoresis of PCR products amplified from PETase\_pET-21a (+) plasmid. 1: DNA marker; 2: PCR amplification band of vector fragment (product size: 6283bp)

![](https://static.igem.wiki/teams/4804/wiki/engineering/figure6.png)

Figure 6: Electrophoresis of PCR products amplified from R\_ pET-21a (+) plasmid. 1: DNA marker; 2-5: PCR amplification bands of R (product size: 675bp)

![](https://static.igem.wiki/teams/4804/wiki/engineering/figure7.png)

Figure 7: Identified the correct clone. (A) Electrophoresis of colony PCR. 1: DNA marker; 2-13: colony PCR. (B) Sequencing peak diagram shows the results of sequencing.

## Test

* **1. Protein Expression**

We conducted SDS-PAGE to detect the PETase-R expression after IPTG induction. As shown in Figure 8, compared to the simple without IPTG induction (lane 2), PETase-R induction by IPTG (lanes 3-6) has protein bands near 45 kDa, and the results indicate that the protein has been successfully expressed in BL21(DE3).

![](https://static.igem.wiki/teams/4804/wiki/engineering/figure8.png)

Figure 8: SDS-PAGE analysis of PETase-R. 1: Marker; 2: Before IPTG induction simple; 3: With IPTG induction simple; 4: pellet of ultrasonic crushing with induction; 5: Flow-through solution after nickel column affinity; 6: 20mM imidazole eluent

* **2. Activity Test of PETase-R**

The enzyme activity of PETase was performed by p-NP assay which is a common way to quantify hydrolytic activity. We selected p-Nitrophenyl Butyrate (p-NPB) as the substrate, which can be hydrolyzed to p-nitrophenol (p-NP) (Figure 8-A).

pNP can be measured by microplate reader (BioTek Syner gyH1) by the characteristic absorption at 405 nm (Figure 9-B). The bacterial supernatant after ultrasonic crushing was mixed with p-NPB, and the absorbance was measured at a series of time points. As shown in Figure 9-C, with the extension of reaction time, the OD405 value increased, which indicates that the degradation activity of the PETase-R. In addition, the p-NP assay was used to further test the effects of the degradation activity of PETase only. Figure 9-D demonstrated that, when the substrate concentration is 2 mM, the OD405 value increased with time. The OD405 value of the PETase-R system is slightly higher than PETase alone after 40 minutes. In other words, with R, PETase still have degradation activity.

![](https://static.igem.wiki/teams/4804/wiki/engineering/figure9.png)

Figure 9: Activity Test of PETase-R. (A)The mechanism of pNPB degradation; (B) Loaded samples into 96-well plates for the detection by microplate reader. (C) OD405 of pNPB hydrolysis by overexpressed PETase-R. (D) OD405 of pNPB hydrolysis by overexpressed PETase and PETase-R.

* **3. Real Sample Degradation Test**

To verify the degradation effect of our system on the actual PET plastic, we cultured PET plastic fragments overnight in the ultrasonically broken supernatant, and the concentration of the PET degradation product-TPA (terephthalic acid) in the solution was measured by HPLC (Figure 10-A). Figure 10-B shows the standard curve of the TPA standard. The results show good linearity between TPA concentration and peak area with R^2 = 0.995. The results are shown in Figure 10-C. The relationship between peak area from HPLC results and TPA concentration was plotted to analyze the degradation efficiency further. Under the same experimental conditions, the engineered E. Coli overexpressed with PETase and PETase-R were performed to degrade the real PET microplastics. LB medium and R-only solutions were set as the control groups. The results shown in Figure 11 reveal that the peak area obtained by the PETase-R is higher than that of the PETase alone and a mixture of protein (PETase and R) group.

![](https://static.igem.wiki/teams/4804/wiki/engineering/figure10.png)

Figure 10: HPLC results of TPA standard and real PET fragment degradation.

![](https://static.igem.wiki/teams/4804/wiki/engineering/figure11.png)

Figure 11: HPLC results of TPA relative concentration after proteins normalized in real sample test.

## Summary

We amplified R and then using seamless cloning technology insert it into the C-terminal of PETase gene, and transformed the recombinant plasmid into the E. coli BL21(DE3) strain. We successfully constructed the prokaryotic expression system, and IPTG induction to obtain the recombinant proteins. p-NP assay results revealed that the fusion protein could degrade p-NPB. Besides, HPLC analysis of protein degradation of real PET plastics showed that fusion protein was indeed more efficient than PETase only for PET degradation. The expression of spider silk protein-R in the fusion protein did not affect the activity of PETase, and in the real PET plastic degradation experiment, the fusion expression of spider silk protein may improve the function of PETase to degrade plastics due to the adhesion of spider silk protein.

![](https://static.igem.wiki/teams/4804/wiki/ack.png)

© 2022 Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository for this website is available at <gitlab.igem.org/2023/bioplus-china>.

1. [HOME](https://2023.igem.wiki/bioplus-china/)

- PROJECT

[DESCRIPTION](description)
[CONTRIBUTION](contribution)
[ENGINEERING](engineering)
[MODEL](model)

- WETLAB

[EXPERIMENTS](experiments)
[NOTES](notebook)
[SAFETY](safety)
[PART](part)

- [HUMAN PRACTICES](human-practices)

[EDUCATION](education)
[IHP](human-practices)

- TEAM

[MEMBERS](team)
[ATTRIBUTIONS](attributions)

# Protocol

Your browser does not support PDFs. Please download the PDF [here](/protocol.pdf)

![](https://static.igem.wiki/teams/4804/wiki/ack.png)

© 2022 Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

The repository for this website is available at <gitlab.igem.org/2023/bioplus-china>.

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}