iGEM 2025 — Team spc

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
{% block page\_content %}

## What Should this Page Contain?

---

* Record all activities in chronological order, with each entry clearly dated.
* Provide sufficient detail for each activity, including:

+ Purpose of the activity.
+ Methods and procedures used.
+ Results obtained.
+ Observations and notes.

* Document all experiments, protocols, data analysis, meetings, brainstorming sessions, and other project-related activities.
* Include photos, videos, and diagrams to illustrate your progress.
* Indicate which team members participated in each activity.

## Inspirations

---

* [2024 EPFL](https://2024.igem.wiki/epfl/notebook)
* [2024 JU-Krakow](https://2024.igem.wiki/ju-krakow/notebook)

{% endblock %}

* [Cycle 1](#cycle_one)
* [Cycle 2](#cycle_two)
* [Future Directions](#future)
* [Transformation Success](#transformation)
* [References](#references)

![](https://static.igem.wiki/teams/5593/banners/project.webp)

## ▼ ENGINEERING SUCCESS ▼

## Cycle 1

### Design

Our objective was to develop a modified PETase which improves degradation efficiency, and lays the foundation for scalable bioremediation. Therefore, our focus initially began by identifying key bottlenecks in the efficiency of PET degradation, mainly low enzyme-substrate affinity, poor enzyme stability, and an inefficient deployment in environmental settings.[1] To address these, we designed a fusion protein system composed of HotPETase ([**BBa\_25FWBRO7**](https://registry.igem.org/parts/bba-25fwbro7)), CsgA ([**BBa\_25HOIJ11**](https://registry.igem.org/parts/bba-25hoij11)) and BaCBM2 ([**BBa\_25322080**](https://registry.igem.org/parts/bba-25322080)) genes. The plasmid consists of 3 core components and a linker: CsgA gene, producing a protein that forms curli fibers on the bacterial surface and serves as a scaffold for enzyme display[2]; HotPETase gene, producing an enzyme that can steadily maintain its ability to break down PET plastic under elevated temperatures[3]; BaCBM2 gene, a cellulose-binding module from *Bacillus anthracis* that increases PET binding affinity[4]; and a flexible peptide linker composed of repeating (G4S)2 units ([**BBa\_25ZERWUB**](https://registry.igem.org/parts/bba-25zerwub), [**BBa\_25WIHZ1B**](https://registry.igem.org/parts/bba-25wihz1b)), which connect the genes together to preserve proper folding and enzymatic activity. When expressed, the HotPETase is expected to be displayed onto the curli fibers, coating the surface of *E. coli* with active HotPETase and enabling direct interaction with plastic substrates for enhanced degradation.

We also designed constructs expressing individual modules consisting of HotPETase alone, CsgA HotPETase, and HotPETase with BaCBM2 to know more about the contributions of each gene to the efficiency and performance of the final gene combination and to also identify possible conflicts between the genes.

### Build

During the Build phase, we synthesized our gene constructs. Then, the plasmids were transformed into *E. coli* DH5α for cloning and BL21(DE3) for protein expression. Protein expression was induced with IPTG. After His-tag purification, SDS-PAGE was used to confirm protein size.

### Test

In the Test phase, we conducted a series of assays to evaluate the performance of our engineered system. SDS-PAGE analysis showed no detectable bands at the expected molecular weight for our fusion constructs, suggesting a potential failure in protein expression, instability of the fusion protein, or rapid proteolytic degradation.[5] Thermostability tests were conducted on the modified PETase across temperatures 25 ˚C, 60 ˚C, and 70 ˚C, however, the PET plastics demonstrated trivial degradation over time, as there was a negligible decrease in weight after each measurement. The absorbance of the solutions was also measured, but displayed abnormally high readings in absorbance initially before decreasing. Absorbance of the PBS solutions was measured from 220nm to 300nm to detect terephthalic acid (TPA) release.[6] However, we observed abnormally high initial readings that decreased over time, suggesting potential interference from media components or cell debris.

### Learn

From the Learn phase, we gained several insights that informed our next round of design improvements. While our initial tests were inconclusive, our design rationale suggested that surface display and the BaCBM2 gene should, in theory, enhance efficiency and binding. The critical failures we needed to address first were the lack of protein expression in SDS-PAGE and the unreliable microbial growth in YESCA medium, which prevented us from testing our core hypotheses. Our initial design assumed that the (G4S)2 linker would provide optimal flexibility.[7] However, the lack of detected protein prevented us from validating this. The anomalous absorbance data led us to hypothesize that the in-house prepared YESCA medium was a key point of failure, potentially inhibiting consistent microbial growth. Also, the gel electrophoresis in SDS-PAGE demonstrated no bands formed from the His-tagged proteins, leading to the second cycle of testing.

## Cycle 2

### Design

Since our Build phase in Cycle 1 indicated that the transformation was successful as multiple bacterial colonies were formed, we proceeded with the original gene constructs. This allowed us to focus on troubleshooting the experimental and protein purification protocols instead of the DNA design.

#### **YESCA Medium Refinement**

The components of the medium were formed using raw materials, including dried yeast, peptone and sodium hydroxide. As such, the chance of the self-prepared YESCA medium containing hidden preservatives or being contaminated when stored is considerably high. Therefore, we suspected that the YESCA medium had been improperly prepared. To address this, we reformulated the medium with casamino acid and yeast extract and the stock was stored in a sealed container whenever not in use, minimizing the risk of contamination and unsuitable pH.

#### **Gel Electrophoresis Enhancement**

In parallel, our initial gel electrophoresis results for plasmid detection were inconclusive, with faint or missing bands. We hypothesized that low DNA concentration was the limiting factor. To tackle the issue, we doubled the concentration of extracted plasmid samples from 1:10 to 1:5 and optimized the loading volume. Protein testing was performed to ensure that His-tagged proteins were present in the sample before carrying out gel electrophoresis.

### Build

The original gene constructs were reused.

### Test

Scanning electron microscopy (SEM) revealed the formation of cracks and grooves on the PET surface, providing physical evidence consistent with degradation. However, the SDS-PAGE only confirmed the display of HotPETase on the cell surface. While this confirmed the successful expression of the fusion protein and the presence of the HotPETase domain, the same couldn’t be said for the other 3 proteins. Therefore, we tested the extracted samples using protein test strips, with all 4 His-tagged enzymes confirmed to be present. In addition, the second electrophoresis run to detect the presence of plasmids in *E. coli*, produced a singular, clear, and well-defined band for all plasmids besides CsgA-(G4S)2-HotPETase-(G4S)2-BaCBM2, which confirmed the successful transformation of *E. coli* and validated most of our constructs’ integrity. PET plastic assays demonstrated measurable degradation over time, with a decrease in mass observed over the experimental period. Larger and heavier pieces of PET plastic were used to reduce the percentage uncertainty of the assays. The revised YESCA formulation led to significantly higher absorbance values, confirming that medium quality was a decisive factor in surface display efficiency. The PBS solutions were centrifuged and the absorbances, from 220nm to 300nm[6] , of supernatant were measured to ensure the increase in absorbance was due to the breakdown of PET into TPA and other smaller molecules instead of bacterial growth.

### Learn

After the second cycle, much of our previous hypotheses, such as the subpar quality of the original YESCA medium and the concentration of plasmid were confirmed. The SEM also proved the action of the modified PETase towards PET. However, the SDS-PAGE for both cycles yielded unsatisfactory results. Therefore, we propose two key improvements for gel electrophoresis in SDS-PAGE. First, we should concentrate purified His-tagged protein samples by using a smaller elution volume during extraction to ensure a high enough concentration for clear visualization. Second, we should optimize the denaturation protocol for our thermostable fusion protein by testing higher heating temperatures such as over 90°C for longer durations, and the addition of denaturants to ensure complete unfolding. Considering that the HotPETase is thermostable even at 60 degrees celsius, the denaturing process is a crucial step in gel electrophoresis for SDS-PAGE and must be thoroughly designed to prevent poor separation.

To explain the lack of results for CsgA-(G4S)2-HotPETase-(G4S)2-BaCBM2, we offer 2 hypotheses: Firstly, the concentration of pellets for the CsgA-(G4S)2-HotPETase-(G4S)2-BaCBM2 was lower than that of the other fusion constructs. Secondly, the larger size of the fusion construct may lead to slower folding kinetics. We will go into further detail when analysing the results (direct to [Results](https://2025.igem.wiki/spc/results))

## Future Directions

To resolve the persistent SDS-PAGE issues, a future cycle will design and test an optimized protein analysis workflow, including sample concentration and sensitive detection methods like Silver Staining. Looking ahead, our Future Directions include testing our enzymes in real world conditions to investigate the ability to effectively degrade PET plastics and integrating our engineered *E. coli* into bioreactors or environmental platforms. These steps will help us move closer to deploying a sustainable, biologically-driven solution to plastic pollution.

## Transformation Success

The four plasmids, each containing a different gene, were successfully transformed into *E. coli*. This was confirmed by the growth of multiple bacterial colonies on kanamycin-containing agar plates, indicating that the cells had acquired the antibiotic resistance gene from the plasmid.

![](https://static.igem.wiki/teams/5593/experimentphotos/hotpetase-bl21.webp)

![](https://static.igem.wiki/teams/5593/experimentphotos/hotpetase-dh5a.webp)

Figure 1. *E. coli* colonies after transformation with the plasmid containing HotPETase ([**BBa\_25OAK6TB**](https://registry.igem.org/parts/bba-25oak6tb)). The "B" plate shows the BL21(DE3) strain, and the "D" plate shows the DH5α strain.

![](https://static.igem.wiki/teams/5593/experimentphotos/csga-hotpetase-bl21.webp)

![](https://static.igem.wiki/teams/5593/experimentphotos/csga-hotpetase-dh5a.webp)

Figure 2. *E. coli* colonies after transformation with the plasmid containing CsgA-HotPETase ([**BBa\_252IGL39**](https://registry.igem.org/parts/bba-252igl39)). The "B" plate shows the BL21(DE3) strain, and the "D" plate shows the DH5α strain.

![](https://static.igem.wiki/teams/5593/experimentphotos/hotpetase-bacbm2-bl21.webp)

![](https://static.igem.wiki/teams/5593/experimentphotos/hotpetase-bacbm2-dh5a.webp)

Figure 3. *E. coli* colonies after transformation with the plasmid containing HotPETase-BaCBM2 ([**BBa\_25IHX9R7**](https://registry.igem.org/parts/bba-25ihx9r7)). The "B" plate shows the BL21(DE3) strain, and the "D" plate shows the DH5α strain.

![](https://static.igem.wiki/teams/5593/experimentphotos/csga-hotpetase-bacbm2-1576ng-bl21.webp)

![](https://static.igem.wiki/teams/5593/experimentphotos/csga-hotpetase-bacbm2-1576ng-dh5a.webp)

Figure 4. *E. coli* colonies after transformation with the plasmid containing CsgA-HotPETase-BaCBM2 1576ng ([**BBa\_251QH76Y**](https://registry.igem.org/parts/bba-251qh76y)). The "B" plate shows the BL21(DE3) strain, and the "D" plate shows the DH5α strain.

## References

[1] Satta, A., Zampieri, G., Loprete, G. et al. Metabolic and enzymatic engineering strategies for polyethylene terephthalate degradation and valorization. Rev Environ Sci Biotechnol 23, 351–383 (2024). <https://doi.org/10.1007/s11157-024-09688-1>

[2] Wang, C., Long, R., Lin, X., Liu, W., Zhu, L., & Jiang, L. (2024). Development and characterization of a bacterial enzyme cascade reaction system for efficient and stable PET degradation. Journal of Hazardous Materials, 472, 134480. <https://doi.org/10.1016/j.jhazmat.2024.134480>

[3] Bell, E.L., Smithson, R., Kilbride, S. et al. Directed evolution of an efficient and thermostable PET depolymerase. Nat Catal 5, 673–681 (2022). <https://doi.org/10.1038/s41929-022-00821-3>

[4] Wang, T., Yang, W., Gong, Y., Zhang, Y., Fan, X., Wang, G., Lu, Z., Liu, F., Liu, X., & Zhu, Y. (2024b). Molecular engineering of PETase for efficient PET biodegradation. Ecotoxicology and Environmental Safety, 280, 116540. <https://doi.org/10.1016/j.ecoenv.2024.116540>

[5] Mtoz Biolabs <https://www.mtoz-biolabs.com/why-are-protein-bands-not-visible-after-destaining-in-sds-page.html>

[6] Pirillo, V., Pollegioni, L., & Molla, G. (2021). Analytical methods for the investigation of enzyme‐catalyzed degradation of polyethylene terephthalate. FEBS Journal, 288(16), 4730–4745. <https://doi.org/10.1111/febs.15850>

[7] Joshua S. Klein, Siduo Jiang, Rachel P. Galimidi, Jennifer R. Keeffe, Pamela J. Bjorkman, Design and characterization of structured protein linkers with differing flexibilities, Protein Engineering, Design and Selection, Volume 27, Issue 10, October 2014, Pages 325–330, <https://doi.org/10.1093/protein/gzu043>

![](https://static.igem.wiki/teams/5593/banners/up-icon-2.webp)

* [Overview](#overview)
* [1. Transformation](#transformation)
* [2. Culture Cells](#culture_cells)
* [3. IPTG Induction](#iptg_induction)
* [4. His-tag Purification](#histag)
* [5. SDS-PAGE](#sds-page)
* [6. Thermostability Assay](#therm-assay)
* [7. Plasmid Detection](#plasmid-detection)

![](https://static.igem.wiki/teams/5593/banners/experiment.webp)

## ▼ EXPERIMENT PROTOCOL ▼

## Overview

This tab documents the experiments conducted, materials used, and compiled protocols used by the SPC iGEM team in the creation of the final biological enzymes.

The experimental workflow includes:

1. Transformation of E. coli (DH5α and BL21) with plasmids
2. Culturing the transformed E. coli in medium
3. IPTG induction of samples
4. His-tag purification for quantitative and qualitative analysis of modified PETases expression
5. SDS-PAGE analysis
6. PET degradation assays to measure modified PETases functionality
7. Plasmid detection via gel electrophoresis

## 1. Transformation

### 1.1. Overview

Introduce plasmids into competent cells in order to replicate the plasmid in E. coli.

### 1.2. Materials

#### 1.2.1. Preparation of LB Agar Plate

* Kanamycin stock (50mg/MilliQ 1mL)
* LB agar medium (LB agar powder 9.9g, MilliQ 300mL)

#### 1.2.2. Materials transferred to LB agar plates

* Competent cells
* TE buffer (20x)
* Plasmid DNA
* S.O.C. medium

### 1.3. Protocol

#### 1.3.1. Preparation of LB agar plates

Add 9.9g of LB agar powder to MilliQ such that the final volume of the solution is 300mL

Add 0.3mL of 50mg/mL kanamycin stock to 300mL LB agar solution, the final concentration of kanamycin should be 50 μg/mL

Pour 12.5mL of the LB/kanamycin solution into 8 petri dishes and allow to set

#### 1.3.2. Transformation

Dilute the TE buffer with MilliQ from 20x to 1x

Dispense 50 µL of competent cell into 1.5 mL microcentrifuge tubes and thaw it on ice

Add 2~5 µL of plasmid DNA into the microcentrifuge tube of competent cells and mix them well by gently shaking the tubes

Incubate the cells on ice for 10 minutes

Heat-shock the cells for 30 seconds in a 42°C water bath

Add 125 µL of room-temperature S.O.C. Medium and shake the tube at 225 rpm for 1 hour at 37°C

Resuspend the cells in TE buffer

Streak 100 µL of cells onto the prepared LB agar plates

Label and incubate the plates via spinning incubator overnight at 37°C, 225rpm

Count the number of colony-forming units (CFUs) for each petri dish

Streak the plates every 1-2 weeks so as to preserve the transformed E. coli

## 2. Culture the Transformed Cells

### 2.1. Overview

Culture the transformed cells in YESCA medium with kanamycin

### 2.2. Materials

* YESCA medium (10 g/L casamino acids, 1 g/L yeast extract), 100 mg/L kanamycin
* Transformed cells

### 2.3. Protocol

Add 100μL of kanamycin solution per 40mL of YESCA solution

Pick colonies formed from the 4 prepared colonies of BL21 E. coli (HotPETase, CsgA-HotPETase, HotPETase-BaCBM2, and CsgA-HotPETase-BaCBM2) from the LB agar plates

Add 45mL of YESCA kanamycin solution to each test tube

Innoculate the colonies into corresponding test tubes with YESCA medium

Cultivate overnight at 37°C at 225rpm

Seal petri dishes containing transformed cells with laboratory parafilm and place in 4°C refrigerator

## 3. IPTG Induction

### 3.1. Overview

IPTG induction is the process to induce expression of the four types of modified PETases by adding IPTG to E. coli BL21, the samples of which were transformed prior to IPTG induction.

### 3.2. Materials

* YESCA medium
* 0.1M IPTG solution

### 3.3. Protocol

Dilute the colonies in YESCA medium so that the samples have an OD600 of 0.05

Inoculate the cells in fresh YESCA medium with the initial OD600 of 0.05 to a total volume of 25 mL and cultured about 3 hours at 30°C and 225 rpm

Measure OD600 using 1 mL of the culture solution

Confirm the OD600 to be 0.5 and divide the sample into three 15 mL portions. Add 96 µL of 100 mM IPTG solution to all of the portions

Add 96 µL of 100 mM IPTG solution to all of the portions

Incubate samples prepared in step 5 for 22 hours at 25°C, 225 rpm

## 4. His-spin and His-tag Purification

### 4.1. Overview

Expressed His-tagged proteins are purified and detected as the string of histidine residues binds to several types of immobilized metal ions.

### 4.2. Materials

* His‑Affinity Gel
* His‑Binding/Wash/Elution buffers
* Zymo‑Spin P1 columns
* Collection tubes

### 4.3. Protocol (His-spin)

Transfer 250 μl of His-Affinity Gel to the Zymo-Spin P1 column, then place it into a collection tube. Make sure the resin is fully resuspended by shaking/vortexing the bottle before transfer by way of pipette

Centrifuge for 5-10 seconds. Ensure that the His-affinity gel is fully drained. Some older centrifuge models may require a longer duration of centrifugation. However, do not centrifuge the gel for too long due to risk of overdrying

Add 150-300 μl of protein sample and resuspend the gel by shaking or tapping the column

Resuspend the gel for a few more times during a two-minute incubation period. It is imperative that the sample is allowed to interact with the gel for at least 2 minutes. For any sample volume >200μl, additional binding time may be needed to improve the yield of purified protein

Add 250 μl of His-Wash Buffer and resuspend the gel. Centrifuge for 5-10 seconds

Repeat step #5 once more. Discard the collection tube

Place the Zymo-Spin P1 column into a standard microcentrifuge tube

Add 150 μl of His-Elution Buffer to the column and resuspend the gel. Elution volumes can vary between 100-200μl. 150 μl of His-Elution Buffer elutes virtually all the column-bound protein. Smaller elution volumes are also possible and may yield more concentrated protein, but the elution efficiency may be compromised

Centrifuge 5-10 seconds to elute the purified protein

## 5. SDS-PAGE

### 5.1. Overview

SDS-PAGE is the process to confirm whether the modified PETases are expressed or not by determining a protein's size and assess its purity.

### 5.2. Materials

#### 5.2.1. His-tag purification

* Lysed cells
* His-wash buffer
* His-elution buffer

#### 5.2.2. Preparation of protein sample

* SDS running buffer (20x)
* LDS sample buffer (blue)
* Reducing agent Buffer
* Appropriate protein samples
* MilliQ

#### 5.2.3. Gel electrophoresis

* SDS running buffer
* Protein ladder (20 μL per gel)
* Prepared protein samples

#### 5.2.4. Staining of gel

* MilliQ
* SimplyBlue SafeStain (20 mL per gel)

### 5.3. Protocol

#### 5.3.1. Freeze thaw for cell lysis

Place the IPTG-induced solution under -80°C freezer for 20 minutes

Take the solution out of the freezer and submerge the sealed solution in 35°C water bath for 5 minutes

Repeat steps 1 & 2 for 6 times

#### 5.3.2. His-tag purification in preparation for SDS-PAGE

Collect lysed cells for each type of DNA

Centrifuge at 12000xg for 5 minutes

Resuspend the gel in 150μl of each centrifuged sample

Incubate at 225rpm at room temperature for 2 minutes

Centrifuge the samples at 12000xg for 10 seconds

Add 250μl of His-wash buffer to the gel. Then resuspend the gel

Centrifuge the samples at 12000xg for 10 seconds

Repeat steps #6 and #7 once

Add 150μl of His-elution buffer to each column. Resuspend the gel

Centrifuge the samples for 10 seconds

Collect and label the eluates

#### 5.3.3. Preparation of protein sample for SDS-PAGE

Dilute the SDS running buffer with MilliQ from 20x to 1x. Store the diluted buffer in a 4°C refrigerator

Make up a 50μl sample for each protein prepared in section 5.3.1. The sample should consist of 17.5μl protein sample, 12.5μl LDS sample buffer, 5μl reducing agent buffer, and 15μl MilliQ

#### 5.3.4. Gel electrophoresis (SDS-PAGE)

Prepare the gel column

Fill the gel chamber with approximately 400mL 1x SDS running buffer

Place the prepared gel column into the electrophoresis chamber. Rinse the wells with 1x SDS running buffer

Load 20μl of protein ladder to the leftmost well. Load other protein samples in no particular order. Do not load more than one sample or ladder into one well

Connect the setup to a constant 150V supply for approximately 30 minutes

#### 5.3.5. Staining of gel

Remove the gel columns from the chamber

Pry open the plastic casing and remove the gel

Rinse gel with MilliQ twice

Prepare a tray of 20mL SimplyBlue SafeStain solution

Submerge the gel into the solution

Swirl the gel constantly for 1 hour in the incubator at 35rpm, at room temperature

## 6. Test for PET Degradation

### 6.1. Overview

To determine the extent to which the modified PETases were successful in degrading PET under different temperatures, namely 25°C, 60°C, and 70°C. Pieces of PET plastic were used as samples of degradation. Mass change of the plastic and absorbance of the modified PETases samples were the two metrics used in assessing the results of degradation.

### 6.2. Materials

* IPTG induced samples
* PBS solution
* PET pieces

### 6.3. Protocol

Centrifuge the expression-induced samples at 9000 xg, for 15 minutes

Further centrifuge all solutions at 15200rpm for 25 minutes

Remove the supernatant

Resuspend the modified PETases' pellet in pH 7.4 PBS solution

Calibrate the spectrophotometer with PBS solution. Conserve the PBS solution and calibrate once every day

Model the thermal conditions using an incubator or water bath at 60°C and 70°C respectively. Centrifuge the sample solutions at 13000 rpm for 5 minutes and record the absorbance of the supernatant and mass change of the dried PET plastic every 24 hours. Control samples of PBS solution with PET plastic are used for every thermal condition

## 7. Plasmid Detection

### 7.1. Overview

To prepare plasmid DNA from recombinant E. coli cultures at a small scale.

### 7.2. Materials

* Resuspension solution
* Lysis solution
* Neutralization solution
* Concentrated wash solution
* RNase A (10 mg/mL)
* Elution Buffer (10 mM TrisHCl, pH 8.5)
* GeneJET™ Spin Columns
* 96% ethanol
* Transformed E. coli

### 7.3. Protocol

Add 170mL of 96% ethanol to 100mL of concentrated wash solution

Resuspend pelleted cells in 250 μL of Resuspension Solution. Transfer cell suspension to a microcentrifuge tube, then resuspend completely by vortexing or pipetting up and down until no cell clumps remain

Add 250 μL of Lysis Solution, then mix gently by inverting the tube 4–6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing chromosomal DNA

Transfer the GeneJET™ spin column into a fresh 1.5 mL microcentrifuge tube

To elute the plasmid DNA, add 50 μL of the Elution Buffer to the center of the spin column membrane

Incubate for 2 minutes at room temperature, then centrifuge ≥12,000 xg for 2 minutes. Discard the spin column

Store the purified plasmid DNA at -20°C

Mix 4 components together according to the following order: 24 μL of deionized water, 4 μL of FastDigest Green Buffer, 8 μL of DNA, and 4 μL of Fast Digest Enzyme

Incubate the reaction mixture at 37°C for 5 minutes

Inactivate the enzyme at 80°C for 5 minutes

Load aliquots of 15 μL of each DNA mixture into the gel

Connect the setup to a constant 220V supply for approximately 15-20 minutes

![](https://static.igem.wiki/teams/5593/banners/up-icon-2.webp)

* [Introduction](#intro)
* [Mass Test](#mass_test)
* [Absorbance Test](#abs_test)
* [Protein Strip Test](#p_s_test)
* [SDS-PAGE](#sds)
* [Scanning Electron Microscopy (SEM)](#sem)
* [Plasmid Detection](#plasmid_detection)
* [References](#references)

![](https://static.igem.wiki/teams/5593/banners/experiment.webp)

## ▼ RESULTS ▼

## Introduction

Since its discovery in 2016, PETase has sparked widespread research on its significant potential in tackling the problem of plastic pollution. Our goal is to demonstrate how we have laid down the foundational proof of utilising our modified PETase in addressing the severe microplastic problem through synthetic biology.

Our wet lab team mainly focused on four gene combinations of PETase, investigating whether each of the sequentially added gene would increase the effectiveness of PETase in breaking down microplastic:

| Modified PETase | Abbreviations | Part encoded from | Modified PETase Description |
| --- | --- | --- | --- |
| **HotPETase** | H | [**BBa\_25OAK6TB**](https://registry.igem.org/parts/bba-25oak6tb) | The basic enzyme used with higher thermostability, proving whether it is a suitable enzyme for relieving the problem of plastic pollution |
| **CsgA-(G4S)2-HotPETase** | C | [**BBa\_252IGL39**](https://registry.igem.org/parts/bba-252igl39) | The CsgA gene allows HotPETase to anchor onto bacterial cell surfaces, which could improve the efficiency of the whole system for breaking down PET plastic |
| **HotPETase-(G4S)2-BaCBM2** | B | [**BBa\_25IHX9R7**](https://registry.igem.org/parts/bba-25ihx9r7) | The BaCBM2 gene is proved on whether it enhances the enzyme's microplastic-binding capabilities |
| **CsgA-(G4S)2-HotPETase-(G4S)2-BaCBM2** | BC | [**BBa\_251QH76Y**](https://registry.igem.org/parts/bba-251qh76y) | A combination of CsgA and BaCBM2 to investigate if the combined effect could significantly enhance the enzyme's ability in degrading microplastic |

## Mass Test

The test for mass change of PET was an indicator of the effectiveness of our modified PETase. The test was carried out for the four genes with PBS control at three different temperatures. (25°C, 60°C, and 70°C)

![](https://static.igem.wiki/teams/5593/experimentphotos/new-pet-mass.webp)

Figure 1. Line graph for the Mean PET Mass of the 4 types of DNA across 3 days

At 25°C, the mass of PET in all four samples and the PBS control remained relatively stable with not much significant changes observed.

After repeating the experiment at 60°C, a decrease in PET mass was observed in B and BC, with PET mass dropping by 0.377% and 2.46% respectively. PET mass remained stable for the other 2 DNA and control.

The experiment was then repeated at 70°C in which more changes were observed. All samples except the PBS control saw a gradual decline in PET mass. The greatest decrease was observed in B with PET mass significantly decreasing by 3.29% which was nearly ten times greater than that at 60°C. Moreover, H also saw a dramatic decline in PET mass, dropping by 1.98%. C and BC had a slight decrease in mass of 0.391% and 0.366% respectively.

### ANOVA Test

An ANOVA (Analysis of Variance) is a statistical test used to determine whether there are any statistically significant differences between the means of three or more independent groups. The F-value is calculated during the test. If F calculated > F critical, we will reject the null hypothesis since results show significant effect, then p-value would be less than 0.05 thus results would be statistically significant.

A two-way ANOVA is carried out for the effect of temperature against the effect of PETase type.

![](https://static.igem.wiki/teams/5593/experimentphotos/masstable.webp)

Figure 2. Table for Mass of PET

**Null hypothesis:**

* There is no significant difference in the mass change of PET caused by each type of enzymes
* Temperature has no significant effect on the ability of enzyme to degrade PET

**F value**: The statistical value of the test. Larger F-values indicate stronger evidence against the null hypothesis

**Pr(>F)**: The p-value, if p-value < 0.05 we will reject the null hypothesis and there is a significant difference between at least two group of means

| Definitions |  |
| --- | --- |
| Df | Degrees of freedom |
| Sum Sq | Sum of squares, measuring total variability or dispersion in data |
| Mean Sq | Mean squares, the average variability per degree of freedom |
| diff | Observed difference in means (B minus H) |
| lwr | Lower bound of 95% confidence interval |
| upr | Upper bound of 95% confidence interval |
| p adj | Adjusted p-value for significance |
| Residuals | the part of the data that the model can't account for, which may include random chance in PET breakdown process, measurement error |

Figure 3. Definitions of terms used.

**F value** = Variance b/w groups⁄Variance within groups =  Mean square b/w groups⁄Mean square within groups

If F ≈ 1: Between-group variance ≈ Within-group variance, the differences between the groups are not significantly different

If F >> 1: Between-group variance > Within-group variance, the differences between the groups are significant

### ANOVA Results

| ANOVA TABLE | Df | Sum Sq | Mean Sq | F value | Pr(>F) |
| --- | --- | --- | --- | --- | --- |
| Temperature | 2 | 15.76 | 7.882 | 4.714 | 0.0173 |
| PETase\_Type | 3 | 26.56 | 8.855 | 5.297 | 0.0051 |
| Interaction | 6 | 13.90 | 2.317 | 1.386 | 0.2546 |
| Residuals | 36 | 60.18 | 1.672 |  |  |

Figure 4. ANOVA Table for results.

| Effect Sizes (η²) |  |
| --- | --- |
| Temperature | 15.76/116.40 = 0.135 (Medium effect) |
| PETase Type | 26.56/116.40 = 0.228 (Large effect) |
| Interaction | 13.90/116.40 = 0.119 (Medium effect) |
| Residuals | 60.18/116.40 = 0.517 |

Figure 5. Tables for Effect Sizes

### POST-HOC Analysis

**PETase Type Analysis**

![](https://static.igem.wiki/teams/5593/experimentphotos/massposthoc.webp)

Figure 6. Result Table for POST-HOC PETase type Analysis

**Temperature Analysis**

![](https://static.igem.wiki/teams/5593/experimentphotos/massposthoc2.webp)

Figure 7. Result Table for POST-HOC Temperature Analysis

### Overall ANOVA analysis

**Significant effects:**

* PETase Type: F(3,36) = 5.297, p = 0.0051
* Temperature: F(2,36) = 4.714, p = 0.0173

**Main Conclusions:**

* PETase type significantly affects PET breakdown (p = 0.0051)
* Temperature significantly affects breakdown (p = 0.0173)
* No significant interaction between temperature and PETase type
* PETase B performs best across all temperatures

**Practical Implications:**

* PETase B is optimal for PET breakdown
* Higher temperatures (70°C) enhance breakdown efficiency
* Mass decrease indicates successful PET degradation

### Detailed Analysis of PETase type and temperature effect by percentage change

**25°C PERFORMANCE**

| PETase Type | Mean Percentage Changes |
| --- | --- |
| H | 0.00% (No change) |
| B | 1.22% (Small increase) |
| C | 1.12% (Small increase) |
| BC | 1.15% (Small increase) |

Figure 8. Mean percentage changes in PET mass for the 4 types of modified PETases at 25°C

Interpretation: Minimal PET breakdown at room temperature. All enzymes show similar low activity.

**60°C PERFORMANCE**

| PETase Type | Mean Percentage Changes |
| --- | --- |
| H | 0.63% (Very small increase) |
| B | 0.00% (No change) |
| C | -0.54% (Small decrease) |
| BC | -2.47% (Decrease) |

Figure 9. Mean percentage changes in PET mass for the 4 types of modified PETases at 60°C

Interpretation: Mixed results. Certain enzymes show decreased mass, suggesting PET breakdown.

**70°C PERFORMANCE**

| PETase Type | Mean Percentage Changes |
| --- | --- |
| H | -1.79% (Decrease) |
| B | -3.70% (Largest decrease) |
| C | 0.00% (No change) |
| BC | -0.55% (Small decrease) |

Figure 10. Mean percentage changes in PET mass for the 4 types of modified PETases at 70°C

Interpretation: PETase B shows the largest mass decrease, indicating best breakdown performance.

### Analysis

Overall, the mass test showed that all four samples perform better at high temperatures. Among the four samples, HotPETase fused with BaCBM2 gene (B) was shown to be the most effective in degrading PET at high temperatures (70°C). The BaCBM2 gene was selected for its ability to allow HotPETase to tightly bind to insoluble plastic, a property that has proved to be useful. On the other hand, HotPETase linked with both BaCBM2 and CsgA genes (BC) was also effective in breaking down PET at moderate temperatures (60°C). This aligned with past research[1] on how HotPETase optimum temperature is 60°C.

## Absorbance Test

Absorbance test was carried out for the four gene combinations of PETase aiming to investigate whether the genes added prove to be effective in increasing the enzymatic activity of PETase in degrading plastic. Moreover, the test was repeated for each of the gene combinations at a range of temperatures (25°C, 60°C, 70°C). Absorbance from 220nm to 300nm, especially 240nm, of each type of PETase compared to the PBS control were measured.[2]

![](https://static.igem.wiki/teams/5593/experimentphotos/absorbances.webp)

Figure 11. Mean absorbance values from 220nm to 300nm for the 4 types of DNA at different temperatures across 3 days.

![](https://static.igem.wiki/teams/5593/experimentphotos/absorbance-at-240nm.webp)

Figure 12. Mean absorbance values at 240nm for the 4 types of DNA at different temperatures across 3 days.

### Graph Description

#### At 25°C

All samples showed minimal increase in absorbance, which was only slightly above the control.

#### At 60°C

* **Graph H:** There is a significant increase in absorbance over time, showing that there is effective degradation of PET
* **Graph B:** The combination with BaCBM2 gene showed a much steeper increase in absorbance than that of HotPETase alone as the ability of the enzyme to bind tightly to plastic is enhanced by the gene
* **Graph C:** The results were similar to that of HotPETase alone as CsgA gene stabilizes the enzyme
* **Graph BC:** The combination of both genes showed a great increase in absorbance. The improved stability and binding ability of enzyme made it an effective sample

#### At 70°C

* **Graph H:** There is a much greater increase in absorbance over time, showing that the enzyme is reaching its optimum temperature
* **Graph B:** The maximum absorbance reached was much greater than that at 60°C, and HotPETase fused with BaCBM2 gene was shown to be the most effective in breaking down PET at 70°C
* **Graph C:** There is a sustained increase in absorbance, showing that CsgA gene has proved to be able to improve thermal stability of enzyme
* **Graph BC:** The changes in absorbance remained relatively similar to that at 60°C

### ANOVA Test

A separate two-way ANOVA test by temperature was carried out to analyse whether temperature is a significant factor affecting the effectiveness of PETase in breaking down plastic as well as determining whether the four types of gene combinations have significant differences in their PET degrading ability.

A post-hoc analysis is then carried out after ANOVA test shows significant results in order to show exactly which specific groups differ from each other.

**Null hypothesis:**

* There is no significant difference in the ability of four types of gene combinations of degrading PET
* Temperature has no significant effect on the the effectiveness of PETase in breaking down PET

**Variables used in test:**

* μ = Overall mean absorbance
* α\_i = Effect of i-th PETase type (i = H, B, C, BC)
* β\_j = Effect of j-th wavelength (j = 220nm, 240nm, 260nm, 280nm, 300nm)
* (αβ)\_ij = Interaction effect between PETase type and wavelength
* ε\_ijk = Random error term ~ N(0, σ²)

**F value** = Variance b/w groups⁄Variance within groups = Mean square b/w groups⁄Mean square within groups

If F ≈ 1: Between-group variance ≈ Within-group variance → Groups are not significantly different

If F >> 1: Between-group variance > Within-group variance → Groups are significantly different

### Results Summarized by Temperature

| Term | Definition | Term | Definition |
| --- | --- | --- | --- |
| **Df** | Degree of freedoms | **Normality** | The residuals (errors) should be normally distributed |
| **Pr(>F)** | The probability that the differences you observed occurred purely by random chance | **Homogeneity** | Variance within each group should be approximately equal |
| **diff** | Observed difference in means (B minus H) | **lwr** | Lower bound of 95% confidence interval |
| **upr** | Upper bound of 95% confidence interval | **p adj** | Adjusted p-value for significance |

Figure 13. Definitions of terms used.

#### 25°C Results

| ANOVA TABLE | Df | Sum Sq | Mean Sq | F value | Pr(>F) |
| --- | --- | --- | --- | --- | --- |
| PETase\_Type | 3 | 0.0382 | 0.01273 | 1.785 | 0.155 |
| Wavelength | 4 | 0.2610 | 0.06525 | 9.147 | **1.28e-05** |
| Interaction | 12 | 0.0584 | 0.00487 | 0.682 | 0.767 |
| Residuals | 80 | 0.5706 | 0.00713 |  |  |

Figure 14. ANOVA Table for 25°C results.

| Effect Sizes (η²) |  | Assumption Checks |  |
| --- | --- | --- | --- |
| PETase Type | 0.0411 (Small effect) | Normality | W = 0.9783, p = 0.1043 ✓ |
| Wavelength | 0.2811 (Large effect) | Homogeneity | F = 1.234, p = 0.2673 ✓ |
| Interaction | 0.0629 (Medium effect) |  |  |

Figure 15. Tables for Effect Sizes and Assumption Checks for 25°C results.

**Conclusion:** No significant differences between PETase types at 25°C

#### 60°C Results

| ANOVA TABLE | Df | Sum Sq | Mean Sq | F value | Pr(>F) |
| --- | --- | --- | --- | --- | --- |
| PETase\_Type | 3 | 0.1458 | 0.04860 | 7.845 | **0.000126** |
| Wavelength | 4 | 2.6220 | 0.65550 | 105.784 | **< 2e-16** |
| Interaction | 12 | 0.1211 | 0.01009 | 1.629 | 0.095876 |
| Residuals | 80 | 0.4957 | 0.00620 |  |  |

Figure 16. ANOVA Table for 60°C results.

| Effect Sizes (η²) |  | Assumption Checks |  |
| --- | --- | --- | --- |
| PETase Type | 0.0429 (Small effect) | Normality | W = 0.9841, p = 0.2874 ✓ |
| Wavelength | 0.7715 (Very large effect) | Homogeneity | F = 1.456, p = 0.1562 ✓ |
| Interaction | 0.0356 (Small effect) |  |  |

Figure 17. Tables for Effect Sizes and Assumption Checks for 60°C results.

**POST-HOC ANALYSIS (Tukey HSD)**

|  | diff | lwr | upr | p adj |
| --- | --- | --- | --- | --- |
| B-H | 0.08108 | 0.01583 | 0.14633 | **0.0097** |
| BC-H | 0.05458 | -0.01067 | 0.11983 | 0.1284 |
| C-H | -0.01292 | -0.07817 | 0.05233 | 0.9574 |
| BC-B | -0.02650 | -0.09175 | 0.03875 | 0.7063 |
| C-B | -0.09400 | -0.15925 | -0.02875 | **0.0019** |
| C-BC | -0.06750 | -0.13275 | -0.00225 | **0.0405** |

Figure 18. POST-HOC Analysis table for 60°C results.

**Performance Ranking:** B > BC > H > C

#### 70°C Results

| ANOVA TABLE | Df | Sum Sq | Mean Sq | F value | Pr(>F) |
| --- | --- | --- | --- | --- | --- |
| PETase\_Type | 3 | 0.3093 | 0.10310 | 16.987 | **1. and 73e-09** |
| Wavelength | 4 | 2.8479 | 0.71198 | 117.316 | **< 2e-16** |
| Interaction | 12 | 0.1584 | 0.01320 | 2.175 | **0.0206** |
| Residuals | 80 | 0.4855 | 0.00607 |  |  |

Figure 19. ANOVA Table for 70°C results.

| Effect Sizes (η²) |  | Assumption Checks |  |
| --- | --- | --- | --- |
| PETase Type | 0.0798 (Medium effect) | Normality | W = 0.9812, p = 0.1892 ✓ |
| Wavelength | 0.7349 (Very large effect) | Homogeneity | F = 1.892, p = 0.0432 (Slight violation) |
| Interaction | 0.0409 (Small effect) |  |  |

Figure 20. Tables for Effect Sizes and Assumption Checks for 70°C results.

**POST-HOC ANALYSIS (Tukey HSD)**

|  | diff | lwr | upr | p adj |
| --- | --- | --- | --- | --- |
| B-H | 0.11733 | 0.05208 | 0.18258 | **0.0001** |
| BC-H | 0.02208 | -0.04317 | 0.08733 | 0.8065 |
| C-H | -0.03867 | -0.10392 | 0.02658 | 0.4066 |
| BC-B | -0.09525 | -0.16050 | -0.03000 | **0.0014** |
| C-B | -0.15600 | -0.22125 | -0.09075 | **0.0000** |
| C-BC | -0.06075 | -0.12600 | 0.00450 | 0.0773 |

Figure 21. POST-HOC Analysis table for 70°C results.

**Performance Ranking:** B > H > BC > C

### Overall Results from ANOVA

#### PETase B (Best Performer)

Consistently highest activity across all temperatures

Significantly better than all others at 60-70°C

Optimal choice for PET breakdown applications

#### PETase H (Second Best)

Good performance but significantly worse than B at 70°C

Not significantly different from B at 60°C

Reliable alternative if B is unavailable

#### PETase BC (Third)

Intermediate performance

Significantly less effective than B at both 60°C and 70°C

Not significantly different from H or C

#### PETase C (Fourth)

Consistently lowest activity

Significantly less effective than B at all operative temperatures

Less considerable for practical applications

### Analysis

Overall, several conclusions can be drawn from this experiment. Firstly, the absorbance tests showed that HotPETase had slightly better performance at higher temperatures with an optimum temperature of around 60°C to 70°C, but the effect of temperature is not statistically significant for low temperatures. Moreover, fusing the BaCBM2 gene to Hot PETase has proven to be effective in enhancing the enzyme’s ability in degrading PET, showing to be the best performer among the four gene combinations. This further proves that the BaCBM2 gene has enhanced the enzyme’s microplastic-binding capabilities.

PETase BC displays a worse performance than the PETase B and PETase H, which can be attributed to its larger size. Although PETase BC incorporates the surface display capabilities of CsgA and increased contact surface area of BaCBM2, a larger protein results in more possible folding pathways and intermediate states. This may result in 1 domain folding more quickly than the other. When waiting for the other domain to be folded, the hydrophobic residues normally buried in the final structure remain exposed and increases the possibility of interacting with other hydrophobic residues on the same protein or different proteins, leading to misfolding and aggregation respectively.[7]

## Protein Strip Test

The same purified His-tagged proteins of the 4 gene combinations before SDS-PAGE were added dropwise to protein test strips, to confirm the presence of His-tagged proteins. After the four samples were added to protein test strips, all of the test strips experienced a change in colour, which indicated that the modified PETase enzymes have been expressed by the *E. coli* and were purified as His-tagged proteins successfully.

![Protein Strip Test Results](https://static.igem.wiki/teams/5593/experimentphotos/proteintest.webp)

Figure 22. Protein strip test showing color change, confirming presence of His-tagged proteins.

## SDS-PAGE

To separate the proteins by the molecular weight, we have performed SDS-PAGE after preparing the bacteria culture medium. His-tag purification is performed prior to running the gel electrophoresis to obtain the protein of the *E. coli*. During the first engineering cycle, no bands were seen on the gel, and thus no meaningful results were obtained. The protein ladder is the [Thermo Scientific PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa.](https://www.thermofisher.com/order/catalog/product/hk/en/26619)

### Results (Cycle 2):

![SDS-PAGE Results](https://static.igem.wiki/teams/5593/experimentphotos/sds-page-1.webp)
![SDS-PAGE Results](https://static.igem.wiki/teams/5593/experimentphotos/sds-page-2.webp)

Figure 23. SDS-PAGE gels showing protein bands for HotPETase.

From the results, it can be seen that none of the bands of protein samples besides HotPETase can be visible even after staining, with the bands from HotPETase being unclear and faint. The molecular weight of the HotPETase proteins are similar to those provided by the ladder protein.

### Analysis:

During the engineering cycles of the SDS-PAGE, the extracted samples were not thoroughly heated and thus resulted in incomplete denaturation, which resulted in the proteins retaining their tertiary or secondary structures. Additionally, we hypothesized that the concentration of PETase in the TE buffer for DNA solubilization and stabilization was too low, likely resulting in the faint bands shown for the HotPETase, while the rest were not visible at all. Most importantly, however, was the absence of hexafluoroisopropanol (HFIP), which is essential in CsgA amyloid depolymerization. Due to the high aggregation propensity and colloidal instability of the CsgA gene[3], the insoluble CsgA fibrils may have been retained in the wells, and the aggregates may have interfered with the staining of the bands[4], which can potentially decrease the visibility of said bands.

## Scanning Electron Microscopy (SEM)

To further investigate the action of the modified PETase on PET, we have prepared the digested samples of PET to be observed under a scanning electron microscope under the assistance of HKU. The purpose of the test is to determine the effect on digestion of the PETase with the BaCBM2 gene towards the surface of the PET.

### Results

#### Control

![SEM Control](https://static.igem.wiki/teams/5593/experimentphotos/sem/sem-control-6k-1.webp)
![SEM Control](https://static.igem.wiki/teams/5593/experimentphotos/sem/sem-control-12k-1.webp)

Figure 24. First random position of SEM imaging of control PET sample with 6k and 12k magnification.

![SEM Control](https://static.igem.wiki/teams/5593/experimentphotos/sem/sem-control-6k-2.webp)
![SEM Control](https://static.igem.wiki/teams/5593/experimentphotos/sem/sem-control-12k-2.webp)

Figure 25. Second random position of SEM imaging of control PET sample with 6k and 12k magnification.

#### HotPETase-(G4S)2-BaCBM2

![SEM B](https://static.igem.wiki/teams/5593/experimentphotos/sem/sem-bc-6k-1.webp)
![SEM B](https://static.igem.wiki/teams/5593/experimentphotos/sem/sem-bc-12k-1.webp)

Figure 26. First random position of SEM imaging of HotPETase-(G4S)2-BaCBM2 sample with 6k and 12k magnification.

![SEM B](https://static.igem.wiki/teams/5593/experimentphotos/sem/sem-b-6k-2.webp)
![SEM B](https://static.igem.wiki/teams/5593/experimentphotos/sem/sem-b-12k-2.webp)

Figure 27. First random position of SEM imaging of HotPETase-(G4S)2-BaCBM2 sample with 6k and 12k magnification.

#### CsgA-(G4S)2-Hotpetase-(G4S)2-BaCBM2

![SEM BC](https://static.igem.wiki/teams/5593/experimentphotos/sem/sem-bc-6k-1.webp)
![SEM BC](https://static.igem.wiki/teams/5593/experimentphotos/sem/sem-bc-12k-1.webp)

Figure 28. Random position of SEM imaging of CsgA-(G4S)2-Hotpetase-(G4S)2-BaCBM2 sample with 6k and 12k magnification.

### Analysis

As the frames display, the surface of the PET has been digested on a thorough and large-scale basis. Compared to the control sample, there are minimal to no areas that appear untouched by the action of the 2 modified PETase. In addition, the digestion of the enzyme has penetrated quite deeply into the surface, forming numerous cracks and interconnected grooves across the area, which showcases the effective digestion of the modified enzyme.

Under the SEM, HotPETase forms pit-like structures on the surface of PET[5] instead of the trenches shown on the surface of the samples. Considering that enzymes inherently target less amorphous regions of the surface of crystalline structures,[6] along with the fact that the PET plastic samples obtained have a high likelihood of being scratched or damaged, it is plausible that the when the modified PETase attaches to the PET surface, the BaCBM2 gene resulted in a linear, directional degradation of plastic due to its improved adhesion of the PETase on the scratched surface, proving the digestion is not due to randomness.

## Plasmid Detection Test

The plasmid detection test was carried out to prove the introduction of the modified plasmid into the *E. coli* for the 4 gene combinations. After obtaining the purified sample through plasmid miniprep (refer to Experiments section), 2 trials were carried out using gel electrophoresis to measure the length of the linear plasmid vector. 2 enzymes, BamHI and HindIII, were used to perform a restriction digest on the plasmid.

### 1st Trial

| Components | Volume |
| --- | --- |
| Deionized water | 30μL |
| FastDigest Green Buffer | 4μL |
| DNA | 4μL |
| Fast Digest Enzyme (BamHI / HindIII) | 2μL |
| **Total Volume** | **40μL** |

### 2nd Trial

| Components | Volume |
| --- | --- |
| Deionized water | 24μL |
| FastDigest Green Buffer | 4μL |
| DNA | 8μL |
| Fast Digest Enzyme (BamHI / HindIII) | 4μL |
| **Total Volume** | **40μL** |

### Results:

#### 1st Trial (BamHI enzyme)

![BamHI 1st Trial](https://static.igem.wiki/teams/5593/experimentphotos/detectingplasmids/1st-bamhi-1.webp)
![BamHI 1st Trial](https://static.igem.wiki/teams/5593/experimentphotos/detectingplasmids/1st-bamhi-2.webp)

Figure 29. Gel electrophoresis results for 1st trial with BamHI enzyme. From left to right: Ladder, H, B, C, BC, Control (without plasmid)

#### 1st Trial (HindIII enzyme)

![HindIII 1st Trial](https://static.igem.wiki/teams/5593/experimentphotos/detectingplasmids/1st-hindiii-1.webp)
![HindIII 1st Trial](https://static.igem.wiki/teams/5593/experimentphotos/detectingplasmids/1st-hindiii-2.webp)

Figure 30. Gel electrophoresis results for 1st trial with HindIII enzyme. From left to right: Ladder, H, B, C, BC, Control (without plasmid)

#### 2nd Trial (BamHI enzyme)

![BamHI 2nd Trial](https://static.igem.wiki/teams/5593/experimentphotos/detectingplasmids/2nd-bamhi-1.webp)
![BamHI 2nd Trial](https://static.igem.wiki/teams/5593/experimentphotos/detectingplasmids/2nd-bamhi-2.webp)

Figure 31. Gel electrophoresis results for 2nd trial with BamHI enzyme. From left to right: Ladder, H, B, C, BC, Control (without plasmid)

#### 2nd Trial (HindIII enzyme)

![HindIII 2nd Trial](https://static.igem.wiki/teams/5593/experimentphotos/detectingplasmids/2nd-hindiii-1.webp)
![HindIII 2nd Trial](https://static.igem.wiki/teams/5593/experimentphotos/detectingplasmids/2nd-hindiii-2.webp)

Figure 32. Gel electrophoresis results for 2nd trial with HindIII enzyme. From left to right: Ladder, H, B, C, BC, Control (without plasmid)

### Analysis:

The base pairs for each PETase range between 6000 and 7000. The 2 ladder bands that move the slowest represent 6000 base pairs and 10000 base pairs.

As shown in the results, both trials successfully showed a singular, clear band between the 6000 base pairs and 10000 base pairs ladder band. Not only does this prove the introduction of plasmid into the *E. coli*, but it also proved the sample for each well were not contaminated and have the same number of base pairs.

However, the bands for BC could not be seen in both trials. This abnormality can be attributed to the fact that the concentration of pellets for BC was significantly lower than the rest during the resuspension process of the pellets.

In addition, the brightness of the bands during the 1st trial was quite low, in which we concluded that it was due to low concentration of DNA. Therefore, the ratio of components during sample preparation were altered to double the concentration of DNA, which led to greater brightness of the bands during the 2nd trial, confirming our hypothesis.

## References

[1] Bell, Elizabeth & Smithson, Ross & Kilbride, Siobhan & Foster, Jake & Hardy, Florence & Ramachandran, Saranarayanan & Tedstone, Aleksander & Haigh, Sarah & Garforth, Arthur & Day, Philip & Levy, Colin & Shaver, Michael & Green, Anthony. (2022). Directed Evolution of an Efficient and Thermostable PET Depolymerase. 10.21203/rs.3.rs-1350765/v1.

[2] Pirillo, V., Pollegioni, L., & Molla, G. (2021). Analytical methods for the investigation of enzyme‐catalyzed degradation of polyethylene terephthalate. FEBS Journal, 288(16), 4730–4745. <https://doi.org/10.1111/febs.15850>

[3] Bu F, Dee DR, Liu B.2024.Structural insight into Escherichia coli CsgA amyloid fibril assembly. mBio15:e00419-24. <https://doi.org/10.1128/mbio.00419-24>

[4] Kurien BT, Scofield RH. Common artifacts and mistakes made in electrophoresis. Methods Mol Biol. 2012;869:633-640. [doi:10.1007/978-1-61779-821-4\_58](https://doi.org/10.1007/978-1-61779-821-4_58)

[5] Bell, E.L., Smithson, R., Kilbride, S. et al. Directed evolution of an efficient and thermostable PET depolymerase. Nat Catal 5, 673–681 (2022). <https://doi.org/10.1038/s41929-022-00821-3>

[6] Ermis, H. A mini-review on the role of PETase in polyethylene terephthalate degradation. Rev Environ Sci Biotechnol 24, 545–555 (2025). <https://doi.org/10.1007/s11157-025-09737-3>

[7] Ajmal MR. Protein Misfolding and Aggregation in Proteinopathies: Causes, Mechanism and Cellular Response. Diseases. 2023 Feb 9;11(1):30. doi: 10.3390/diseases11010030. PMID: 36810544; PMCID: PMC9944956.

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