Contribution

We registered a new antimicrobial peptide called CTX (BBa_250NI4BC). This part is a 21 amino acid cationic antimicrobial peptide secreted by the skin of a South American frog, Hypsiboas albopunctatus. It has homology with the ceratotoxin peptide family, which permeabilizes membranes and forms pores via a 'barrel-stave' mechanism. It exhibited biological activity against bacteria and fungi.

CTX showed strong activity against two major post-harvest citrus pathogens: Penicillium digitatum (blue mold) at 25 µM and Geotrichum candidum (Sour rot) at 50 µM. We also tested CTX against three potential production hosts: Escherichia coli, Saccharomyces cerevisiae, and Aspergillus oryzae. The peptide was effective against bacteria and filamentous fungi, while yeast displayed good resistance. We believe this information will be valuable for future iGEM teams interested in using or producing CTX in their projects. Full experimental protocols, results, and detailed notes are available on our Engineering page.

We developed a new composite part that enables safe CTX production in microbial cell factories by preventing its antimicrobial effect during expression.

The CTX (BBa_25ONI4BC) was fused to sfGFP (BBa_I746916) as carrier protein, due to its strong expression, ease of detection, and well-documented use in synthetic biology. We insert two TEV protease cleavage sites (BBa_K20750037) incorporated for precise post-expression release, and a 8x His-tag (BBa_K4422007) was added to facilitate purification. A small flexible 3×GS linker (BBa_J18921) was used to prevent structural disruption of sfGFP.

After expression, the sfGFP–CTX fusion protein was purified via Immobilized Metal Affinity Chromatography (IMAC) using the incorporated His-tag. To obtain CTX, the fusion was cleaved with TEV protease, releasing a small tag-free peptide suitable for downstream applications. Because of CTX’s small size (~2.3 kDa), we implemented ultrafiltration with a 10 kDa MWCO membrane, allowing CTX to pass through while retaining the larger carrier fragments (~30 kDa). This provided an efficient and straightforward size-based purification strategy. We express the composite part in two different hosts, Escherichia coli and Aspergillus oryzae.

Results in E. coli

• The sfGFP–CTX fusion protein was successfully expressed, extracted, and purified by column chromatography.
• After TEV cleavage, we obtained an estimated yield of ~2 mg CTX/L of culture.

Results in A. oryzae

• Integration of the construct in the genome of fungi was confirmed, and sfGFP–CTX production was detected by SDS-PAGE and fluorescence assay.
• Although we did not reach purification steps within the project timeframe, protein quantification suggested ~10 mg/L of CTX — about 5× higher than E. coli.

This composite part demonstrates a generalizable strategy for safe AMP production, overcoming host toxicity while enabling downstream recovery of peptides

Greening, or Huanglongbing (HLB), is currently the most devastating citrus disease worldwide. We recognized the importance of attempting to provide a solution to this challenge. However, working with the causal bacterium, Candidatus Liberibacter asiaticus, is extremely difficult since it is non-cultivable in laboratory conditions. Direct plant assays are also complex, time-consuming, and require large amounts of peptide. To overcome these obstacles, we partnered with the Centro de Citricultura de Cordeirópolis (IAC) to design an indirect but rapid test using infected citrus leaves.

• Leaves from infected citrus trees were collected.
• A baseline DNA sample was obtained by excising ~15 mg of tissue from the petiole tip.
• The remaining leaf tissue was incubated for 7 days in Falcon tubes containing 2 mL of treatment solution in room temperature.
• After incubation, another 15 mg tissue sample was excised, and DNA was extracted.
• Both samples (before and after treatment) were sent for qPCR analysis, enabling the comparison of CLas bacterial loads and the evaluation of treatment effectiveness.

In our first DBTL cycle, infected leaves were incubated with either water or culture medium. qPCR analysis confirmed that Candidatus Liberibacter asiaticus (CLas) remained detectable for at least 7 days in excised tissue, with only slight reductions in bacterial concentration. These results validated the feasibility of the assay, demonstrating that excised leaf tissue provides a stable and reproducible system for testing potential treatments.

Building on this framework, we tested CTX (100 mM) alongside tetracycline (100 µg/mL, positive control) by incubating infected leaves for 3 days. Conventional PCR confirmed the presence of CLas after treatment, while qPCR quantification is still pending due to delayed results. Therefore, it was not yet possible to determine whether CTX reduced bacterial load relative to controls.

The results indicated that there was no significant reduction in the bacterial population in the samples after treatment (table). However, this lack of reduction was also observed in the positive control, which should have shown a decrease, suggesting that the exposure time used in the experiment may have been insufficient We believe in the potential of this assay. The urgent need to find effective treatments for citrus greening, combined with the fact that CLas is uncultivable, makes this challenge particularly demanding. By establishing this strategy, we provide a practical starting point for future teams to test new solutions, refine the rapid assay, and ultimately contribute to saving oranges—or even extend the approach to other plant diseases.

We developed a versatile hardware platform designed for detecting volatile organic compounds (VOCs) associated with citrus greening (HLB). At its core, the system integrates a nanofabricated sensor array with commercial gas sensors, environmental monitors, and a flexible electronic circuit capable of being adapted to many different detection needs.

The distinguishing feature of this hardware is its modularity. Each sensor is designed as a replaceable module: users can swap the current sensors for others that better suit their specific application, whether that is detecting different plant diseases, monitoring environmental pollutants, or even exploring biomedical diagnostics. With this flexibility, the same hardware can become a universal detection tool, powered by custom training of the machine learning model with user-collected data.

We also created a detailed Handbook that documents every step of the design and fabrication process. It includes the physical principles behind the hardware, construction methods, circuit diagrams, sensor integration strategies, and testing protocols. This guide ensures that future iGEM teams or independent researchers can replicate, adapt, and expand our platform to their own contexts.

From the project's inception, our ambition for the computational model extended beyond Greening. We aimed to create not just a single-purpose simulation, but a flexible and extensible framework that could serve as a foundational tool for studying a wide range of phloem-limited plant pathogens. We believe our work is a significant contribution to the iGEM community for the following reasons:

1. Modular Architecture: Our model is built with a highly modular architecture. The core logic for each biological process is encapsulated in separate, well defined functions. For example, the update rules for bacterial propagation, host callose response, and therapeutic effects are implemented independently. This separation of concerns means that a future user could, for instance, replace our logistic growth function with a different one, or modify the callose production mechanism, without altering the rest of the simulation's structure. This design makes the model accessible to adapt and extend.
2. Parameter-Driven Design: The model's behavior is not hardcoded. All biological and therapeutic properties, from bacterial growth rates to drug half-lives, are controlled by centralized and well-documented configuration files (config.h and constants.h). This means that adapting the model to a new pathogen or host system is primarily a matter of changing the parameters, rather than rewriting the underlying code.
3. Open, Documented, and Accessible: The entire source code is thoroughly documented and publicly available in our open-source repository. Accompanied by the detailed technical description and results on our wiki, our work provides a clear and accessible starting point for any team interested in computational plant pathology.

We offer this framework to the iGEM community and the broader scientific world as a contribution: a robust, open-source tool to accelerate research, test in silico hypotheses, and design new strategies to protect global agriculture. It can be readily adapted to model other devastating phloem-limited diseases, such as Flavescence dorée in grapevines (caused by Candidatus Phytoplasma vitis) or Lethal Yellowing in coconut palms (caused by Candidatus Phytoplasma palmae), serving as a template for future projects in this critical area.