iGEM 2025 — Team thailand-ris

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## What Should this Page Contain?

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* 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

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

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# Engineering

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## Keys Achievements

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* Completed 2.5 iterations of the DBTL (Design-Build-Test-Learn) cycle
* We successfully transformed pGWB2 and pCAMBIA13021302 into Agrobacterium tumefaciens.
* Successful proof of concept that PETase can be expressed and secreted in a multicellular organism—plant.

## Work in Progress

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* Completed 2.5 iterations of the DBTL (Design-Build-Test-Learn) cycle
  + Stable transformation of our pCAMBIA1302 and pGWB2 vectors
  + Real-world implementation of the kill switches
  + Protein function in plant

## Introduction

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For this year’s project, our project objective is step one of our vision—prove that PETase can be expressed and secreted in plants. Since the expression of PETase in plants has never been done before, our first goal is to prove that PETase can be expressed in a model plant. Specifically, N. benthamiana is a viable option, as it is a common GM host that we have worked on in previous years’ projects. Once we validate the potential of step one, we aim to advance this project by examining the protein function of plant-expressed PETase. Finally, our long-term goal is to create a platform that will lead to a plant-based water filtration system, which will allow for bioremediation, leading to the endless cycle of plastic pollution spread. Before proceeding, we must address the fundamental question: why use plants?

## Why Plants?

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As mentioned, PETase was originally discovered in the bacteria Ideonella sakaiensis (Yoshida et al, 2016). This discovery led to a significant shift towards enzyme-based processes in the global vision for solutions to plastic pollution. Existing mechanical and chemical processes frequently exhibit high energy consumption and inefficiency, leading to this change (Hampson, 2016).

Nonetheless, our team viewed that employing PETase via a bacterial platform may be suboptimal in the long run. Instead, we believe that a plant model would be more suitable. We credit this decision to three main factors: efficiency, safety, and sustainability.

## Efficiency—Deploy PETase where plastics accumulate

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Plants can secrete PETase into the extracellular space using a signal peptide (for example, an alpha‑amylase SP), placing the enzyme precisely in the apoplast and rhizosphere where microplastics are intercepted, rather than inside a reactor or cell interior where mass transfer limits activity.

Continuous, in situ expression turns the planted area into a catalytic surface area, avoiding repeated enzyme dosing and the aeration, agitation, and sterilization loads that bacterial bioprocesses typically require.

PETase can also be tuned for high yield and targeted expression—all while running at ambient temperature and using residence time, not electricity, to drive throughput.

In practice, this means a filtration bed or floating mat can scale with channel length and planting density, instead of with tank volume and power budget.

## Safety—Can be contained and shut down when needed

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Releasing engineered bacteria into open systems raises concerns about horizontal gene transfer, biofilm formation on infrastructure, endotoxins, and uncontrolled spread beyond the treatment footprint; even when kept in reactors, accidental escape and resistance marker dissemination remain audit burdens. Unlike microbes, plants offer several built-in safety layers. We can choose non-food plants grown only in controlled areas, separate them with buffer zones, and place genes in the chloroplast (which is inherited only from the mother), make the plants sterile, and, if needed, remove them easily by harvesting the biomass.

A red‑light‑inducible and …-inducible kill switch adds an active shutdown pathway that triggers lethality under defined light conditions if plants leave controlled installations, providing a clear, testable failsafe.

Because PETase is secreted extracellularly, the intervention targets polymer surfaces rather than microbial community structure, lowering the chance of perturbing local microbiomes compared with introducing engineered bacteria.

## Sustainability—Operates with lower external energy and material output

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Plants power enzyme production with sunlight and inorganic nutrients, eliminating the organic feedstocks and electrical demand of bacterial fermenters and reducing operating emissions over long deployments.

Seeds and cuttings enable low‑cost, decentralized scaling across canals, stormwater inlets, and polishing wetlands, cutting transport of bulky plastic‑laden waters to central facilities and the embodied energy of tanks and blowers.

Planted systems also deliver co‑benefits—habitat support, bank stabilization, and carbon sequestration—while allowing planned end‑of‑life handling: kill‑switch activation or harvest to stop expression, followed by controlled processing of biomass and capture or polishing of PET hydrolysis products.

Over a full lifecycle, this shifts plastic remediation from energy‑intensive industrial processing toward a steady, field‑deployable service that is easier to keep running cleanly and cheaply at scale.

## Engineering Practices

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The steps of achieving step one of our vision include:

1. Design and cloning
2. Expression and transformation, and
3. Testing for expression.

Beyond that, our time also explored three aspects from step two: protein function investigation, stable transformation of our vectors, and kill-switch design.

## Design and Cloning

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The design centered on expressing I. sakaiensis PETase (IsPETase) in N. benthamiana and directing the enzyme to the extracellular space, where microplastics are encountered, to maximize contact between the catalytic surface and polymer while minimizing diffusion barriers inside cells (Abbasi et al., 2020)(Azeem et al., 2021)(Wang et al., 2022). PETase was chosen based on foundational work identifying PET-degrading activity in I. sakaiensis and subsequent characterization and deployment in other systems (e.g., bacterial secretion and, most importantly, photosynthetic microalgae expression), which collectively de-risked its selection as the first enzyme to port into a plant chassis for secretion (Kim et al., 2020; Rocco et al., 2023; Seo et al., 2019; Yoshida et al., 2016; Yoshida et al., 2021).

Firstly, N. benthamiana was chosen as it is confirmed as an effective and easy plant for the functional characterization of various plant proteins and enzymes (Sainsbury & Lomonossoff, 2014). Furthermore, it is a safe option that aligns with the 2025 iGEM Safety Guideline for high school teams, which limits plant options. Lastly, it is a plant that the Thailand-RIS team had worked with in previous years’ projects.

The CaMV 35S promoter, a constitutive promoter from the Cauliflower Mosaic Virus (CaMV), was used in pGWB2 and pCAMBIA1302 backbones because it allows for a high-rate and consistent expression profile that increases the chance of detecting secretion and activity in a proof-of-concept phase (Amack & Antunes, 2020). This choice reduces confounding from promoter strength and tissue specificity, enabling a clear readout of whether plant secretion and enzyme production are feasible before moving to tunable or tissue-specific promoters.

It was also used in previous
[Thailand RIS-Projects](https://2024.igem.wiki/thailand-ris/engineering).

Secretion was engineered by fusing a plant signal peptide to the N-terminus of PETase so the protein enters the Sec pathway and is delivered to the apoplast, where rhizosphere contact and microplastic interception occur in situ. The α-amylase 3 signal peptide (Amy3SP) from rice was selected because plant signal peptides, including α-amylase variants, have repeatedly targeted recombinant cargo to extracellular compartments and modulated secretion efficiency in plant systems, giving a literature-backed starting point with a high likelihood of correct routing (Chen et al., 2004; Huang et al., 2015; Seo et al., 2019; and Wang et al., 2018). Prior to lab testing, molecular [docking simulations](https://2025.igem.wiki/thailand-ris/model) were also performed at this stage to provide insights. More information on the simulation is available on the [Docking Simulation Page](https://2025.igem.wiki/thailand-ris/model).

Please refer to the [Part Registry and the Contribution Page](https://2025.igem.wiki/thailand-ris/contribution) for additional information about parts.

Note that either the pCAMBIA1302 or the pGWB2 backbone already incorporates parts of the mentioned component. For example, the pCAMBIA1032 already contains hygromycin resistance and kanamycin resistance for selection and a Green Fluorescent Protein (GFP) gene as a reporter for plant transformation. For pGWB2, the vector comes with the cauliflower mosaic virus (CaMV) 35S promoter for plant gene expression, a hygromycin phosphotransferase (HPT) gene for fungal selection, and nopaline synthase (NOS) gene terminator sequences. After cloning, the following are our implemented vectors:

#### IsPETase-Amy3SP in pCAMBIA1302:

![](https://static.igem.wiki/teams/5619/engineering/ispetase-amy3sp-in-pcambia1302.avif)

#### IsPETase-Amy3SP in pGWB2:

![](https://static.igem.wiki/teams/5619/engineering/ispetase-amy3sp-in-pgwb2.avif)

Please refer to the [Part Registry and the Contribution Page](https://2025.igem.wiki/thailand-ris/contribution) for additional information about parts.

To reduce translational bottlenecks, the PETase coding sequence (minus its native bacterial signal peptide) was reverse-translated and codon-optimized for N. benthamiana using host-preferred codons documented in codon usage tables, which improves ribosomal throughput and can increase steady-state protein accumulation under the same promoter. Computational screening with SignalP 6.0 supported the secretory design, indicating Sec/SPI-class signal peptides with high confidence and predicting a cleavage site that maintains the intended PETase N-terminus after signal peptide removal in the plant secretory pathway. More information about this section is available in both the Part Registry and Contribution Page.

In the process of cloning vectors into Agrobacterium, our team initially struggled to clone the pGWB2 vector. This was discovered as we had extracted out transformed Agrobacteria before running PCR and gel electrophoresis but detected no band correlating with the sequenced gene in pGWB2. From this failure, we tried a few more rounds of transformation until discovering success when transforming using higher-intensity electroporation and a higher plasmid-to-competent-cell ratio.

### Before:

![](https://static.igem.wiki/teams/5619/engineering/before-igem.webp)

Original Gel Electrophoresis Result: no IsPETase-Amy3S detected

### After:

![](https://static.igem.wiki/teams/5619/engineering/after-igem.avif)

Gel electrophoresis following PCR amplification for PGWB2 vector, showing the detection of the IsPETase-Amy3S gene we inserted

With a successful implementation of the DBTL engineering cycle as well as Agrobacterium transformation, plant transformation is ready.

## Expression and Transformation

For transient expression, agroinfiltration was performed in leaves because N. benthamiana leaves are accessible and can rapidly yield expression, making them ideal for de‑risking secretion and activity questions. Once step one is completed and it is successfully proven that PETase can be expressed in plant tissue, tissue-specific expression can then be performed.

Various measures were implemented to enhance expression. That information is available on the [experiment page](https://2025.igem.wiki/thailand-ris/experiments).

Most information on in-plant transformation and transgene expression can be found on the [Plant Synthetic Biology Page](https://2025.igem.wiki/thailand-ris/plant).

## Ongoing Tests:

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## Testing for Expression

Cellular localization is used to verify expression and secretion routing. Through the use of a confocal microscope (Zeiss LSM980), high-resolution images of our GM plant are captured, showing the precise location and intensity of fluorescent signals (emitted from the GFP in our GM plant).

Based on this test, we were able to identify successful expression and secretion of our gene, of which, in comparison to our control, our gene is entirely found in the extracellular matrix. With these results, our tested parts, the IsPETase-Amy3S Coding Part Codon Optimized for *Nicotiana benthamiana* ([BBa\_252Q5ZWV](https://registry.igem.org/parts/bba-252q5zwv)) as well as the plasmid IsPETase-Amy3SP in pCAMBIA ([BBa\_250B1A28](https://registry.igem.org/parts/bba-250B1A28)), are confirmed to work. As stated, we hope that by establishing a proof-of-concept for plant-based PETase expression, we can pave the way for scalable, sustainable bioremediation strategies that integrate the prevention and active degradation of plastic pollution. Visit the [result and contribution page](https://2025.igem.wiki/thailand-ris/results) for more details about our findings.

To reiterate, upstream construct verification (colony PCR across junctions, diagnostic gels, and Sanger sequencing) was completed before plant work to ensure that any negative activity results would not be confounded by misassemblies or frame errors. Together, these tests close the design–build–test loop: secretion confirmed by localization and function confirmed by HPLC, with stable transformation underway to carry these results into persistent lines for scale-up studies aligned with field-deployable, low-energy remediation.

### Protein Function Test

Biochemical assays using the High Performance Liquid Chromatography (HPLC) machine confirm functional PET hydrolysis products in plant extract.

Leaf lysates were incubated with PET powder (20 mg in 500 μL lysate) or PET film (0.5 × 1.0 cm in 1 mL lysate) at 30 °C for 1–4 weeks, with weekly lysate refresh to maintain enzyme activity over long incubations needed for solid polymer substrates. Parallel no-PET controls were run to measure any background signals from plant metabolites and buffer components under identical conditions, ensuring that peaks attributed to PETase activity reflect polymer hydrolysis rather than matrix effects.

Products were quantified by HPLC using a Kintex C18 (5 μm, 250 × 4.6 mm), a 50 mM phosphoric acid–methanol gradient at 1 mL/min, 40 °C, and 240 nm UV detection, with BHET and TPA standards for retention time and calibration. However, results are not yet positive. More information about the protein function test can be found in the [Experiment and Result Pages](https://2025.igem.wiki/thailand-ris/results).

### Stable Transformation

Stable transformation started at the same time as transient transformation, as it is a process never committed by our team. Such was so that we are able to learn and understand the process for large-scale utilization in step two of our vision.

Stable transformation via leaf discs was initiated using a cytokinin-forward regeneration regime, which supports shoot induction from transformed cells while antibiotics select for integrants and eliminate Agrobacterium. This process was developed by Davarpanah, Seyed Javad, *et al.* 2009). However, we encounter two main roadblocks in this process: growing the plant and inducing callus.

Although growing a plant sounds simple and straightforward, we struggled to foster the plant to a viable size without mold. Even with various rounds of the engineering cycle, results have not been satisfactory.

Initially, we tried to grow a plant in a loosely covered jar with Aga medium. However, mold soon grew.

![Growing Plant in Aga Medium](https://static.igem.wiki/teams/5619/engineering/jar-agar-medium.webp)

From this point, we then tried growing via a sealed jar. However, some samples are still moldy despite the sterilization and containment. For the samples that are not moldy, the rate at which plants are growing is abnormally slow. Thus, we also tried regular soil growth, but to no advantage.

![Regular Soil Growth](https://static.igem.wiki/teams/5619/engineering/soil-growth.webp)

With that said, what we did try, which led to advancement, was decreasing the agar concentration. We ran a total of three tests with three AGA concentrations: 0.8%, 0.6%, and 0.2%. The result indicates a negative relationship between the rate of growth and AGA concentration. Therefore, we will implement a low-concentration AGA growing medium as a successful design choice in the near future.

Despite the struggle, we still worked with the little plant sample that we had obtained. However, inducing a callus has proven difficult with two other roadblocks. Firstly, more mold began to grow when waiting for a callus to grow. Secondly, the Agrobacterium remained alive in some cases, contaminating the plant.

![](https://static.igem.wiki/teams/5619/engineering/peti-dish-igem.webp)

The mold struggles are assumed to be caused by a combination of incomplete asepsis and suboptimal culture physiology—residual surface and endophytic microbes surviving an insufficient sterilization pipeline and exploiting high-sucrose media and sealed, non-vented vessels (condensation, high humidity) to sporulate. While we will try our absolute best to tighten the sterilization workflow, mold is still unavoidable. Still, we will continue to experiment with larger samples and trials.

To deal with *Agrobacterium* contamination, we will look into our antibiotic choice and dosing. Currently, we use 200 ug/ml cefotaxime and 50 ug/ml kanamycin. From this benchmark, we can increase the dose or experiment with additional antibiotics like Timentin (ticarcillin-clavulanate).

## Kill Switch Design

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From the [2024 Thailand-RIS team](https://2024.igem.wiki/thailand-ris/safety), we’ve decided to continue our research on a potential kill-switch mechanism for plants.

Synthetic biology has brought transformative possibilities to genetic engineering across various fields, from agriculture and environmental conservation to industrial biotechnology. In particular, genetically engineered (GE) plants hold the potential to address numerous challenges—such as increasing agricultural yields, enhancing crop resilience, and aiding in environmental clean-up efforts, i.e., our plastic pollution solution.

The release of GE plants into open environments without effective containment measures introduces various ecological risks. Gene flow between engineered and wild plant populations could impact biodiversity by introducing novel traits that alter ecosystem dynamics, such as resistance to pests or environmental stressors. These changes could reduce biodiversity, disrupt local food webs, or enable the engineered plants to outcompete native species.

Ethical concerns could also arise from the potential contamination of non-GE crops, which could lead to economic and social consequences for farmers and communities relying on non-GE agriculture.

The broader public may also harbor reservations about the environmental impacts and safety of GE plants, making biocontainment strategies like kill switches not only scientific but also an ethical and social necessity.

Currently traditional biocontainment strategies for GE plants include physical containment methods, such as greenhouse isolation and buffer zones, which aim to prevent the spread of GE plants through spatial barriers. While these methods are valuable, they can be costly, labor-intensive, and often impractical for large-scale agricultural applications.

In our research on expressing PETase in N. benthamiana, the traditional biocontainment methods do not pose as great a risk. However, our vision of a potential large-scale water filtration plant that utilizes plants as filters amplifies the previously mentioned disadvantages. In fact, a plant that continuously takes in and releases natural resources, such as water, can result in our GE plant being released into the open environment. As such, a genetic kill switch provides a suitable alternative.

Genetic kill switches leverage engineered systems that trigger organismal death or prevent reproduction under defined conditions. Kill switches have shown promise in microbial systems, where simple genetic circuits can induce cell death in response to environmental triggers. Adapting these systems to plants offers the potential for enhanced containment, though significant technical challenges remain due to the complexity of multicellular organisms.

In the 2024 Thailand-RIS (last year) team, a potential kill switch was designed for plants. That system was designed such that when the engineered plant experiences red light, a Pfr-responsive promoter drives barnase expression whose activity (as a ribonuclease) degrades RNA, halting protein synthesis, leading to cell death by apoptosis.

#### The vector diagram of last year’s project is as follows:

![](https://static.igem.wiki/teams/5619/engineering/kill-switch.avif)

Several significant parts of this vector include:

* **pEAQ-HT-DEST1 vector** was selected due to its proven effectiveness in *Agrobacterium*-mediated transformation. This is mainly because it contains the P19 silencer gene, which reduces the possibility of host plant cells silencing the transfer complex.
* **Pfr-promoter**: light inducible and responds to the red wavelength of light.
* **Barnase**: a ribonuclease that causes degradation of single-stranded RNA.

Red light serves as a powerful inducer due to its controllability and well-characterized phytochrome system. However, its natural abundance in sunlight and instability under fluctuating light conditions make it unsuitable in various cases. At the same time, other inducers are also unsuitable. For example, one idea discussed for this year’s project includes using a chemical inducer: ethanol. Unfortunately, ethanol is among the most abundant organic compounds in indoor air (Nazaroff & Weschler, 2024). This implies that we can easily activate the system, even in its natural habitat.

In short, a red-light kill switch is viable, but we will tighten control by hardening the light logic. The three modes are defined as follows:

* **Operate** (continuous far-red bias in bays to enforce OFF),
* **Induce** (brief 660 nm red exposure in a contained zone to trigger kill), and
* **Abort** (immediate 730–750 nm far-red to revert).

In future implementation where our GM plant functions within a regulated facility, a far-red-biased lighting can be deliberately provided to the plant such that it can function normally. If the plant escapes the enclosed facility, the absence of far-red light and the presence of the general red light from the sun will automatically trigger the kill switch, resulting in the plant’s death.

To make this change, we looked into swapping the Pfr‑responsive promoter for a UAS/minimal promoter controlled by a PhyB–PIF split TF, so red light assembles the activator to drive barnase while far‑red rapidly dissociates it for a clean ON/OFF gate.

This design is supported by the research of Mena et al. (2018) and Larsen et al. (2023), which provided information on the engineering and optimization of phytochrome–PIF based transcriptional control systems. Mena et al. demonstrated that split transcription factors coupled to PhyB–PIF interactions can achieve highly reversible, red/far‑red light‑dependent gene activation at single‑cell resolution in plants, with minimal leaky expression when tuned appropriately. Larsen et al. expanded this concept by enhancing promoter strength and refining degradation sequences for rapid response times, enabling sharper ON/OFF transition dynamics. These studies confirm that a UAS/minimal promoter under PhyB–PIF control can deliver the precision needed for a hard‑gated light logic kill switch, as far‑red light actively disassembles the complex and halts transcription.

However, this design has not been implemented yet due to practical and technical constraints in our current project cycle. Specifically, integrating this system with our PETase expression vectors poses a cloning complexity that exceeds our existing timeline. As such, the approach remains a proposed enhancement for future Thailand‑RIS iterations, with this year’s work focusing on proof‑of‑concept containment strategies rather than fully deploying the split TF–barnase circuit.

## References

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* 23. Sinaga, Desyanti Saulina, Ho, S., Lu, C., Yu, S., & Huang, L. (2021). Knockdown expression of a MYB-related transcription factor gene, OsMYBS2, enhances production of recombinant proteins in rice suspension cells. [Plant Methods, 17(1), 99.](https://doi.org/10.1186/s13007021007992)
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## Overview

As the initial step of the project, we first designed a primer to transform our synthesised gene from the pCAMBIA1302 vector into the PGWB2 vector (more about vector design can be found in the part collection page).

Following, we used infusion cloning to transform our synthesised gene into the pCAMBIA13021302pCA vector and the PGWB2 vector. To confirm our success in transformation, we grew our bacteria in plates and ran colony PCR and gel electrophoresis to confirm that our inserted gene was present in the plasmids. In doing this, we had to purify our plasmid by using the plasmid extraction kit from Flavorgen. After confirming that our PGWB2 plasmid contained our synthesised gene, we continued to transform it from both the pCAMBIA1302 and PGWB2 vectors into Agrobacterium. Following, we grew the two transformed types of Agrobacterium in plates in preparation for colony PCR and gel electrophoresis. We picked out bacteria colonies from the plates to run colony PCR and gel electrophoresis to confirm that our synthesised gene is present in the Agrobacterium. After that, we inoculated the Agrobacterium for 3 days and agro-infiltrated the Nicotiana benthamiana leaves with the inoculated Agrobacterium -- the plants were left to grow for a week. Next, we freeze-dried the plant samples by using liquid nitrogen and mixed them with PET particles under a solvent. Following, we ran localisation tests on the agroinfiltrated leaves and ran HPLC analysis on the sample mixed with PET particles to test for gene expression and protein functionality.

## Methods

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# Results

The enzyme PETase plays a crucial role in degrading PET micro particles. With microplastics being highly populated in marine environments, we decided to synthesize the PETase gene into plants. Plants naturally absorb water up into their systems to photosynthesize, and are therefore a suitable host for us to synthesize the enzyme PETase into. The bioengineering of significant genes to combat global issues has become a hot area of research, especially for mediating serious issues like microplastics. This research aims to engineer PETase into *Nicotiana benthamiana* as a proof of concept for synthesizing PETase into plants.

Beginning the process, *N Benthamiana* was transformed using the PGWB2 and pCAMBIA1302 vectors, which contained the key genes responsible for the expression of the PETase enzyme and the localization of the enzyme -- PETase gene and the αAmy3SP signal peptide. These genes are then introduced into *N. benthamiana* to show that PETase can successfully be transformed into a plant alongside with being localised in the extracellular space, as microplastic particles are too large to enter the cell.

#### Transformation into *Agrobacterium*

After transformation, PCR amplification and gel electrophoresis were performed to confirm the successful insertion of the PETase gene in the PGWB2 and pCAMBIA1302 vectors. The presence of expected bands verified the genetic transformation in our engineered construct, and is also a key step before moving forward to the next step.

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![fig.1](https://static.igem.wiki/teams/5619/results/fig-1.webp)

###### Fig.1 Gel electrophoresis following PCR amplification for PGWB2 vector, showing the detection of the *IsPETase-Amy3S* gene we inserted.

![fig.1](https://static.igem.wiki/teams/5619/results/fig-2.webp)

###### Fig.2 Gel electrophoresis following PCR amplification for pCAMBIA1302 vector, showing the detection of the IsPETase-Amy3S gene we inserted.

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As shown in Figures 1 and 2, we ran colony PCR and gel electrophoresis to confirm the presence of our inserted genes. The red boxes in both results highlight the successful transformation of our gene into our two vectors. The bands that are boxed in red match the band in the positive control, therefore confirming our success in transformation.

#### Localisation

Following, after confirming positive results that our synthesized vectors had our genes, we agroinfiltrated *N. Benthamiana.* We chose to agroinfiltrate both vectors, pCAMBIA1302 and PGWB2, as pCAMBIA1302 contains GFP which then will be used for localisation. After agroinfiltration, we ran localisation tests to determine if our signaling peptide worked, and whether our gene, PETase, was expressed.

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![fig.3](https://static.igem.wiki/teams/5619/results/fig-3.webp)

###### Fig.3 Localisation of PETase in *N. benthamiana*, showcasing the successful transport of PETase in *N benthamina* out of the nucleus.

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From the localization analysis, we were able to conclude that our target protein was successfully expressed and localised outside of the nucleus, which was what we aimed for. In Figure 3, we can see that in the control, a lot of the portions in the sample is glowing, meaning the protein wasn’t localised anywhere in specific -- just scattered all around the cell, including the nucleus. However, for the sample with our synthesised gene, IsPETase-Amy3S, the visuals shown from the localised results show us that none of our protein was localised near the nucleus of the cell, all were localised in the extracellular space; therefore meaning the expression and localisation of PETase in *N. Benthamiana* was successful and effective.

#### HPLC analysis

In addition to showing that our agroinfiltrated *N. Benthamiana* has successfully expressed and secreted our target protein, PETase, we mixed the leaves’ crude sample with PET particles to test out if the PETase actually works.

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![fig.4](https://static.igem.wiki/teams/5619/results/fig-4.webp)

###### Fig.4 Top graph is the standard BHET and bottom graph is the TPA standard

![fig.5](https://static.igem.wiki/teams/5619/results/fig-5.webp)

###### Fig.5 HPLC results for incubating our N.benthamiana crude sample with PET particles under a solvent for **1 week**

![fig-6](https://static.igem.wiki/teams/5619/results/fig-6-1.webp)

###### Fig.6 HPLC results for incubating our N.benthamiana crude sample with PET particles under a solvent for **2 weeks**

![fig.7](https://static.igem.wiki/teams/5619/results/fig-35.avif)

###### Fig.7 HPLC results for incubating our N.benthamiana crude sample with PET particles under a solvent for **3 weeks**

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Comparing figures 5, 6, and 7 with figure 4, the standard, the HPLC results from all weeks 1, 2, and 3 shows that there was no BHET or TPA detected. This was identified by comparing the peaks in the standard sample graphs with the peaks in the results for each week. As BHET and TPA are the intermediate metabolites for PET, therefore the absence of BHET and TPA means the enzyme PETase in our plant, *N. Benthamiana* is not working properly yet.

However, because in our localisation analysis we were able to identify that our target protein, PETase, has been localised outside of the cell, we know that our protein has been successfully expressed in *N. benthamiana* and localised in the extracellular space. Therefore showing that PETase can be successfully expressed in plants and localised in areas where PET particles can populate, which is the extracellular space.

#### Stable Transformation

In addition to transient transformation, we also attempted to perform transgenic transformation, or commonly also known as stable transformation. However we were not able to successfully perform stable transformation as all our shoot cultures went moldy. After multiple attempts in trying to successfully grow a shoot culture, we were confronted with negative results as all the cultures keep on going moldy.

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![](https://static.igem.wiki/teams/5619/results/fig-8-1.webp)
![](https://static.igem.wiki/teams/5619/results/fig-8-2.webp)

###### Fig.8 Shoot cultures gone moldy

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