Integrated

Why We Reached Out:

• Expertise in plant symbiotic relationships, including interactions with arbuscular mycorrhizal fungi and rhizobia, as well as multiple and concurrent interactions on the same host plant.

• Professor Tsikou specializes in molecular and developmental plant biology, making her the ideal expert to consult before advancing our agricultural research at the molecular level.

What We Discovered:

• Professor Tsikou advised that while many fungicides are available to combat plant pathogenic fungi, it might be more beneficial to target a pathogenic bacterium instead of a fungus, since bacterial infections in plants are harder to treat and antibiotics are often too expensive for agricultural use.

• She suggested that we focus on crops important to Greece, like olive trees, which can be affected by Pseudomonas savastanoi, a bacterium that causes galls.

• If the team continues targeting fungi, she recommended focusing on Verticillium dahliae, a major fungal disease affecting olive trees for which there are no effective chemical treatments available.

Our Reflection:

• Our team has decided to focus on crops that are central to the agricultural economy and the livelihood of farmers.

• Our main concern is the overuse of fungicides, which can have detrimental effects on entire ecosystems.

• Excessive application of fungicides can disrupt soil microbial communities, negatively impacting biodiversity and long-term soil health.

• This underscores the importance of exploring more sustainable and eco-friendly alternatives for disease management in agriculture.

Why We Reached Out:

• We chose Dr. Papadopoulou for the agronomy topic due to her strong academic background in Plant Biotechnology and Agricultural Biology.

• Her research expertise focuses on the molecular interactions between plants and microorganisms, with a special emphasis on symbiotic organisms.

• Her research into the role of rhizosphere microorganisms in plant communities and their relationship with soil-borne phytopathogenic fungi makes her an ideal candidate for providing valuable insights into sustainable disease management strategies in agriculture.

What We Discovered:

• Dr. Papadopoulou emphasized the relevance of our focus on fungi, as environmental changes are expected to increase fungal diseases.

• She noted that climate change makes it crucial to advance research to develop effective and sustainable antifungal strategies for future agricultural practices.

Our reflection:

•Dr. Papadopoulou, along with Dr. Tsikou's input, highlighted that our focus on fungi affecting olive trees is driven by the impact of climate change.

• As fungal pathogens are expected to spread, the use of fungicides will likely increase, leading to greater environmental and health consequences.

• Our work aims to address these challenges by mitigating the need for excessive fungicide applications and reducing their negative effects on the environment and agricultural practices.

Why We Reached Out:

• We selected Dr. Tzamos for his expertise in plant pathology and biological control of plant diseases, particularly his work on managing Verticillium dahliae biologically.

• His knowledge and experience are highly relevant to our integrated approach.

• Dr. Tzamos's background makes him well-suited to offer helpful ideas and practical solutions for managing plant diseases in an eco-friendly manner.

What We Discovered:

• Dr. Tzamos helped us understand the fungus, including its biological cycle, significance, and impact on farmers' lives.

• His insights were crucial in understanding how the fungus affects crops and how to address these issues through sustainable practices.

• His expertise also provided valuable knowledge on effective biological control methods and their application to improve agricultural outcomes.

Our reflection:

•We gained insights into the biological cycle of the fungus, including its effects on plant health and its broader impact on agricultural practices.

• This understanding helps us see its implications for crop management and the livelihoods of farmers.

Why We Reached Out:

• Professor Tsikou specializes in molecular and developmental plant biology, making her the ideal expert to consult before advancing our design.

• We were seeking her insights on the practicality and effectiveness of using modified bacterial BCAs to combat V. dahliae, as our original idea, with the pathogen-activated pathway

• Her expertise could help us understand how our proposed BCA might interact with plants, ensuring our solution would be both feasible and impactful.

What We Discovered:

• She informed us, that a growing trend in managing phytopathogens is the use of double-stranded RNA (dsRNA)

• She encouraged us to explore dsRNA as a promising alternative to chemical treatments, suggesting it could be more effective and safer than our initial idea of using antifungal peptides.

Our Reflection: (Re-design)

• Professor Tsikou found our idea of using bacteria to fight the fungus quite intriguing!

• We began exploring the effectiveness of RNAi technology against phytopathogens in the literature.

• We started searching and thinking of ways to incorporate RNAi into our project, aiming to enhance its impact and sustainability.

• We communicated with the professors Professor Tsikou suggested

Why We Reached Out:

• Specializes in RNA interference mechanisms and the role of small RNA molecules in plant growth and defense against pathogens like fungi.

• We needed more insights and guidance on RNAi technology and its applications.

• We considered designing RNA to target multiple genes for increased efficacy and sought confirmation from an expert, making Professor Dalakouras perfect to validate, refine, or dismiss our idea.

What We Discovered:

• He introduced us to various RNA-Based Gene Silencing Modalities, helping us choose the most suitable one.

• He focused particularly on two RNA delivery strategies:

1) Virus-Induced Gene Silencing (VIGS): Advised against it, as it targets the olive tree's phloem, and V. dahliae enters through the xylem

2) Spray-Induced Gene Silencing (SIGS): Also not recommended due to its low efficiency.

• He suggested we pursue bacterial-mediated RNAi (bmRNAi) because:

1) Other silencing technologies would be ineffective for our purpose.

2) Bacteria can protect RNA molecules from degradation more effectively than even the best nanocoating

• Fully supported our idea to target multiple genes, emphasizing that targeting more than one gene reduces the chance of resistance.

Our Reflection: (Integration)

• We decided to pursue bmRNAi technology due to its promising efficacy and RNA protection, and immediately began gathering more information.

• With our multi-gene targeting strategy approved, we expanded our project to include the search for specific target genes (which is mentioned further on).

Why We Reached Out:

• A previous iGEMER with postgraduate experience in V. dahliae and of course this year’s instructor, she was the perfect person to provide us with valuable insights on this specific fungus.

• We sought her advice and attention to important details.

• We wanted to confirm the effectiveness of the target genes we identified through literature review.

What We Discovered:

• She confirmed that the AAC and RGS1 genes are excellent targets due to their lethality, a fact she knew from her studies.

• While she hadn't studied the THI20 gene, she agreed with us that it is a good gene- target, seeing its potential after our explanation.

• Advised us to search for virulence genes that reduce hyphal growth, germination, or are lethal to ensure the effectiveness of our targets.

Our Reflection: (Integration)

• We retained the AAC and RGS1 genes, which she confirmed.

• We found that THI20 plays a role in colonization, hyphae morphology, and conidia germination, making it a suitable target gene, validating our initial idea.

Why We Reached Out:

• A plant pathology expert with experience in gene silencing for V. dahliae, making her a valuable resource for our project.

• We sought her confirmation on the target genes we had selected.

What We Discovered:

• Ideal RNAi targets are genes crucial for fungal pathogenicity that do not have homologs in the host plant or non-target microorganisms.

• Pathogenicity genes they’ve studied are involved in pathogenicity, but the pathogen can still cause mild to moderate symptoms even after silencing.

• She suggested that genes essential for fungal survival would be the best RNAi targets and confirmed our gene choices, emphasizing that lethal phenotypes in knockouts are ideal for RNAi.

• She agreed that targeting multiple genes with different mechanisms likely enhances effectiveness and reduces resistance.

• She highlighted the importance of over 90% homology for effective silencing, but noted that even 70% can have an impact, depending on the target region.

• It is important to ensure RNA doesn’t cause toxicity to plants or humans, advising bioinformatic analysis to select suitable gene sequences and avoid toxicity.

Our Reflection: (Better understanding)

• She found our project and ideas for tackling V. dahliae to be original and innovative!

• Following her advice, we conducted bioinformatic analyses (BLAST) to ensure none of the selected genes were homologous to plant genes or other microorganisms in the plant’s rhizosphere. This was crucial to safeguard plant biodiversity, the plant itself, and human health.

• We finalized our gene and sequence choices based on positive results from our bioinformatic analyses (BLAST), as avoiding toxicity was a top priority for us.

• We began searching for specific gene sequences to target, ensuring no homology with other gene regions in the plant or microorganisms in the rhizosphere.

Why We Reached Out:

• Recommended by our PI due to his iGEM competition experience and he is also an honorary guest professor of Synthetic Biology at the department of Biochemistry and Biotechnology

• We sought his advice on whether dsRNA or hpRNA would be more effective, as we were leaning toward the latter

What We Discovered:

• He advised producing dsRNA instead of hpRNA because:

• The polymerase would need to transcribe much less, given the shorter length of dsRNA.

• Unlike hpRNA, dsRNA doesn’t require a loop, allowing more space for our target sequence.

• Fitting all three genes in a single hpRNA cassette would be challenging due to its size, but they would fit in a dsRNA cassette, if needed.

Our Reflection: (Re- Design)

• We decided to follow his advice and selected dsRNA production over hpRNA, shifting our approach accordingly.

Why We Reached Out:

• His interest in plant molecular biology, particularly his research on the phi6 system , which we discovered through the scientific paper by Niehl, A., et al. [1], caught our attention. The phi6 system is centered on the replication of double-stranded RNA (dsRNA) by an RNA-dependent RNA polymerase, enabling the production of high-quality, long dsRNA molecules. We were searching a way of producing dsRNA in our bacteria, so we reached out to gain further insights.

• Our idea included using PGPR (Pseudomonas spp) for bmRNAi and applying the phi6 system in plant roots, so we searched for his opinion on its feasibility.

What We Discovered:

• He recommended starting with in vitro dsRNA production, which included using the T7 RNA polymerase to transcribe a PCR fragment with T7 promoter overhangs, creating the double-stranded RNA molecules, rather than using the phi6 system.

• The phi6 system requires extensive DNA cloning, specific P. syringae strains (our chassi is P. putida strains), specialized equipment, and a multi-team effort over several years.

• He encouraged us to consider using the T7 Pol System instead of the phi6 system.

• He emphasized that the phi6 system is designed for upscaling and should be applied only after demonstrating the functionality of the dsRNA sequences with in vitro produced dsRNA.

Our Reflection: (Better understanding)

• We decided to pivot from the phi6 system and focus on alternative dsRNA production methods.

• We began exploring the Pol T7 system for further development.

Why We Reached Out:

• We discovered him through his scientific paper Elston, K. M., et al [2], we believed his expertise in symbiont-induced RNAi could be invaluable.

• We also wanted his opinion on whether the T7 Pol system was indeed the most effective for our needs.

What We Discovered:

• He confirmed the T7 Pol system is highly effective.

• However, he warned that it might be unstable within a plasmid and suggested integrating it into the bacterial chromosome instead, because many copies of the T7 pol system would cause unnecessary stress in the cells

• He validated our idea of using two T7 promoters—one sense and one antisense to our target gene—and emphasized the need for a terminator since transcription might not conclude effectively without it.

• bmRNAi is superior to genetically modified plants due to its longer-lasting silencing effects and modularity.

Our Reflection: (Re - design)

• His validation of the T7 Pol system motivated us to proceed with its implementation in our design!

• His insights led us to explore the integration of T7 Pol system into the bacterial chromosome and consider terminators based on the scientific literature he recommended.

Why We Reached Out:

• As we continued exploring the T7 system and the production of dsRNA, we needed clarification regarding the placement and necessity of spacers in dsRNA production. To gain insight, we turned to scientific literature, which led us to the work of Chen, Z., et al. [3], prompting us to reach out to Dr. Chen for further guidance.

What We Discovered:

• He agreed with our choice of T7 promoter, emphasizing its strong promoter activity, its wide use and its appropriateness for our system.

• For terminators, he noted that the terminators sequence is mainly determined by the host cell.

• He suggested that spacers might not be necessary and encouraged connecting the terminator directly to the end of the target sequence.

Our Reflection: (Integration)

• We decided against adding spacers between the terminators and the promoters

Why We Reached Out:

• We discovered PhD student Tianxin Liang through his scientific article Wu, M., et al. [4], and decided to pursue communication with him.

• We looked for his expertise on the T7 Pol system and the potential effectiveness of its chromosomal integration.

• We also wanted his opinion on the most suitable type of promoter for T7 polymerase production.

What We Discovered:

• T7 RNA polymerase is serving as a powerful tool, plays a crucial role in enhancing gene expression levels and results in higher protein expression levels for improved whole-cell catalysis efficiency.

• He suggested that T7 polymerase and LacI could be expressed from a single genome copy, simplifying the process.

• He told us that constitutive promoters are preferred for constructing expression systems like ours, and he specifically suggested using a constitutive promoter during the exponential phase for T7 polymerase expression.

• This approach allows bacteria to grow unimpeded initially, and T7 RNA polymerase expression is gradually initiated once stable cell density is reached, mitigating the potential negative impact on cells

Our Reflection: (Integration)

• We decided to use a constitutive promoter of exponential phase and began an in-depth literature review to identify the best options.

• This reaffirmed our decision to proceed with confidence in implementing the T7 Pol system in our design.

Why We Reached Out:

• We discovered him through a paper on dsRNA production in bacterial cells [13] and wanted his expert opinion on gene silencing in fungi.

• We had lingering questions about regulating dsRNA production in relation to bacterial growth.

What We Discovered:

• He emphasized the importance of ensuring sufficient dsRNA concentrations.

• V. dahliae exists in different structures throughout its life cycle, and we needed to know which structures would be effectively targeted by dsRNA.

• He confirmed our idea of using different regions of each gene, which was already part of our plan!

• dsRNA production and bacterial numbers must be well-aligned to achieve the desired effect.

Our Reflection: (Inspiration)

• We concluded that using a growth-phase-specific promoter would be ideal

• Our decision to adopt this approach was reinforced by his advice on harmonizing dsRNA production with bacterial growth, as it would ensure gene silencing effectiveness.

• We also got some inspiration from his work with dsRNA production in bacterial cells

Why We Reached Out:

• DDuring our literature review, we came across the scientific paper Hung, M. E., et al. [6] which was relevant to our idea of loading dsRNA into vesicles via a chimeric protein. Without hesitation, we reached out to the corresponding author, Joshua N. Leonard, as the paper seemed very interesting and relevant to our idea!

• We wanted to ask if the dsRNA encapsulation in vesicles described in his scientific article was mediated by a signal pattern and whether we could adapt something similar for our project.

• Given his expertise in using chimeric proteins for RNAs encapsulation in vesicles, we also sought his opinion on our initial idea.

What We Discovered:

• Βacterial vesicles differ significantly from mammalian ones

• In gram negative bacteria like Pseudomonas putida, the RNA is typically restricted to the cytoplasm.

• He informed us that outer membrane vesicles (OMVs) might not incorporate cytosolic contents, since they bud off the surface of the periplasm

• He highlighted the importance of getting RNA into the periplasm, in order to ensure it's encapsulation into the OMVs

• He suggested looking into viral mechanisms that might have evolved to address this challenge and recommended another article of his for further reading.

Our Reflection: (Inspiration + Integration Better understanding)

• We decided to delve deeper into the system Dr. Joshua N. Leonard used in his research.

• Inspired by his work, we designed a chimeric protein with a dsRBD and a domain tailored to integrate with the OMV membrane.

• We began exploring methods to move cytoplasmic material—dsRNA—into the periplasmic space to ensure effective encapsulation in bacterial vesicles, as advised by Dr. Joshua N. Leonard.

Why We Reached Out:

• We found the paper Ahmadi Badi, S., et al. [7], the corresponding author of which is Professor Andrea Masotti, and believed he could significantly enhance our project, so we reached out to him.

• We sought his opinion on whether our idea of a chimeric protein with two domains would be effective.

What We Discovered:

• He was enthusiastic about our idea of creating a chimeric protein!

• He suggested we could also try to create fusion proteins that link OMV-associated proteins with RNA-binding domains to increase dsRNA binding and encapsulation.

Our Reflection: (Better understanding)

• His feedback helped us refine our design, and we began considering ways to create fusion proteins linking OMV-associated proteins with RNA-binding domains.

Why We Reached Out:

• In our search for more information on chimeric proteins, we found the scientific paper Mascali, F. C., et al. [8] by Professor Rodolfo Rasia and decided to consult him.

• We needed more details about the dsRBD of our chimeric protein and the potential constraints posed by the outer membrane localization domain, and we thought it would be appropriate to address him as he is working with double dsRBD proteins

• We also wanted his advice on:

• Whether a specific linker was necessary if we used more than one dsRBD to maximize RNA binding efficiency

• What should we consider when designing the linker for the chimeric protein so as not to disturb the folding of the two different domains all while ensuring the efficiency of the dsRBD and outer membrane domain.

What We Discovered:

• He confirmed that most dsRBDs do bind dsRNA and would be in principle suitable, but advised us against using dsRBDs evolved to perform different functions as HYL1 dsRBD2 and TRBP dsRBD3.

• A linker is going to be needed if more than one RBDs is going to be used!

• He assured us that fusing to the outer membrane localization domain would pose no limitations, as long as the linker is long and flexible enough.

• A linker is going to be needed if more than one RBDs is going to be used! He recommended a linker at least 15 residues in length (40-50bp long) with a sequence predicted to be disordered to maintain domain independence and an attempt to achieve a neutral overall charge.

• He advised avoiding serines and lysines in the linker and suggested modeling the construct and the dsRNA using AlphaFold to check for potential conformational issues.

• If the linker is too short (he proposed using the RNase III linker), we can repeat the linker, however, not with the exact same sequence in fear of this creating a loop with itself.

• We can also use a linker from another protein and run it in silico in order to check if it is the right one, but he encouraged us to try an already existing linker in the cell

Our Reflection: (Inspiration + Integration)

• We opted to include a single dsRBD, using a linker - RNAse III as he suggested - between the two domains of our chimeric protein

• His guidance was a crucial source of inspiration for our project.

Why We Reached Out:

• We discovered her through the scientific literature Niño-Sánchez, J., et al. [9] and wanted her opinion on whether OMVs or autolysis was the better option for dsRNA delivery.

What We Discovered:

• She supported OMVs as a better option than autolysis.

• As we explained our project design, she supported our decision to use a phase specific promoter, specifically stating it was the best choice!

Our Reflection: (Integration)

• We decided to further investigate OMVs for dsRNA delivery but planned to gather additional opinions to confirm this choice.

Why We Reached Out:

• We reached out to her as the corresponding author of the paper Cai, Q., et al. [10] which is related to extracellular vesicles (EVs), directly relevant to our project since OMVs are included in EVs..

• We wanted to ask whether EVs were indeed the most efficient RNA delivery method and gather as much information as possible.

What We Discovered:

• She confirmed that EVs are one of the most efficient RNA delivery methods, as they are used by mammalian cells, plants, and fungi, to deliver such molecules, in nature.

• She supported our use of EVs in our project, as they are suitable for dsRNA delivery.

Our Reflection: (Better understanding and Integrated)

• Her insights deepened our understanding of how EVs function and provided the information we needed to start designing our OMV-based system

Why We Reached Out:

• We were exploring ways to induce OMV production in our bacteria and were inspired by the iGEM UZurich 2021 system. We considered using the TolB-TEV system to control OMV production and encapsulate dsRNA, but sought expert confirmation on its effectiveness.

What We Discovered:

• She loved our idea!

• The TolB-TEV system sounded efficient to her

• She explained that in this system, TEV cuts the periplasmic TolB protein, triggering OMV formation.

Our Reflection: (Better understanding)

• Her approval reinforced our confidence in the TolB-TEV system, but we didn’t stop there. We sought additional expert input to fully validate the approach before integrating it into our design.

Why We Reached Out:

• She was our go-to mentor for insights into the TolB-TEV system, offering expert guidance that was invaluable to our project’s progress.

What We Discovered:

• She advised us to find TolB’s 3D model in AlphaFold to visualize the active site and introduce the TEV sites.

• She recommended integrating TolB with TEV sites directly into the chromosome.

• OMVs should be generated and released only after dsRNA production is complete.

Our Reflection: (Integration)

• We followed her advice to incorporate TEV sites into TolB and integrate it into the chromosome.

• To ensure OMVs are produced after dsRNA synthesis, we considered using a stationary-phase promoter for TEV protease production.

Why We Reached Out:

• Having decided to regulate OMV production post-dsRNA synthesis, we needed to clarify another key point: Can V. dahliae accept OMVs, and can dsRNA be released into its cytoplasm? Given his extensive experience with OMVs, we turned to Dr. Andrea Masotti for answers.

What We Discovered:

• OMVs can indeed be taken up by V. dahliae!

• Receptor-mediated uptake might be challenging because it requires identifying the specific protein and receptor.

• He suggested that the release mechanism could be similar to that in humans with liposomes or exosomes: when a vesicle enters a cell, an influx of protons (H+) causes swelling and releases the content within the cell.

• There are 2 possible mechanisms for releasing dsRNA in Verticillium (based on extracellular communication)

1) Vesicles behave like exosomes: The membranes fuse together to release dsRNA into the cytoplasm.

2) Vesicles enter the cytoplasm via endocytosis.

Our Reflection: (Better understanding)

• With confirmation that V. dahliae can indeed accept OMVs, we could move forward with the final part of our system!

• His insights helped us better understand how OMVs enter V. dahliae and release dsRNA into the fungus’s cytoplasm.

Why We Reached Out:

• One of our main concerns was about choosing to use fungal or bacterial chassis. We were leaning towards the second, but wanted to consult an expert before making a final decision.

• Professor Tzima, with her plant pathology background, was ideal to provide insight on V. dahliae control.

What We Discovered:

• She really liked and supported our decision to use bacteria for RNA production and delivery to V. dahliae!

• She warned us that using fungi as a chassis could cause gene silencing both V. dahliae and to our modified fungus!

Our Reflection: (Integration)

• With her confirmation, we focused solely on bacterial chassis options and discarded the fungal chassis.

Why We Reached Out:

• As an expert in plant biotechnology, we wanted her input on which microorganisms have been most effectively used as chassis.

What We Discovered:

• She recommended using an endophytic organism for our project.

• She suggested considering Pseudomonas spp., because:

1) It is a strong chassis option

2) of its PGPR properties

Our Reflection: (Inspiration + Integration)

• We kept Pseudomonas spp. in mind as a potential chassis and continued gathering input to ensure we would choose the best option for addressing V. dahliae.

Why We Reached Out:

• We consulted Professor Dalakouras again to get more insights into the chassis selection, particularly regarding Pseudomonas spp., as recommended by Professor Papadopoulou.

What We Discovered:

• The bacterial chassis must be RNAse III deficient to prevent the RNA from being degraded before reaching V. dahliae.

• The bacteria must deliver the RNA in its original form, so V. dahliae can cut it with its DICER into 20-21nt segments for gene silencing.

• Bacterial chassis is the best choice because:

1) Even the best nanocarriers can’t protect RNA as effectively as a bacterial chassis.

2) Bacteria offer auto-replication, which may present an economic advantage.

• Pseudomonas spp. offer significant advantages, as these species:

1) Can be placed in the soil, unlike E. coli.

2) Introduce novelty into synthetic biology, moving beyond the common use of E. coli.

3) Some Pseudomonas strains are endophytic, an important trait as V. dahliae infects the olive tree via its roots.

• Pseudomonas spp. constitute a good chassis as they are well-studied bacterial species

Our Reflection:

• With the requirement for RNAse III deficiency, we ruled out Bacillus spp. and Pseudomonas fluorescens as options.

Why We Reached Out:

• After leaning towards Pseudomonas putida as our chassis, we wanted expert opinions on its interactions with soil and olive trees. Dr. Blanco, an expert in agroforestry microbiology, was the perfect candidate to consult.

What We Discovered:

• He confirmed that Pseudomonas could likely be used for bmRNAi in the olive tree rhizosphere.

• He emphasized that gene targeting must be precise, as Verticillium dahliae’s pathogenicity and virulence rely on various traits (a point highlighted by Professor Tzima and PhD candidate Katsaouni).

• The impact on the rhizosphere microbiome is more related to changes in microbiome interactions than drastic shifts in microbiome structure and composition.

• He referred us to literature for further study on Pseudomonas species and their role in the rhizosphere.

Our Reflection: (Integration Better understanding)

• With his positive feedback on Pseudomonas putida, we began fully developing our system.

Why We Reached Out:

• Our instructor, Maria-Tsampika Manoli, recommended we study the paper Zobel S., et al. [11] on T7 pol insertion into the chromosome, then consult with PhD researcher Esteban Martinez for guidance on integrating it into Pseudomonas putida

• He also serves as a provider of RNAse III deficient P. putida strains, which we needed for T7 integration.

What We Discovered:

• He confirmed that transcriptional units for dsRNA production must be integrated into the chromosome, as Dr. Jeffrey E. Barrick had also advised.

• He detailed the need for four bacterial strains for chromosome integration and provided us with relevant literature and step-by-step guidance for lab work!

• Emphasized the importance of using a vector with an ori compatible with P. putida.

Our Reflection:

• We identified the pBBR1 ori of pSEVA vector as a suitable option and began working with the literature he shared.

Why We Reached Out:

• Dr. Karas is conducting postdoctoral research at the Department of Biochemistry and Biotechnology at the University of Thessaly.

• His research interests focus on the isolation and identification of microorganisms from soil samples.

• He has designed and constructed five incubators for the Department of Biochemistry and Biotechnology, contributing to the enhancement of the laboratory’s equipment.

What We Discovered:

1) Humidity Control:

• Humidity is controlled with a humidifier that sprays mist from purified water. It can be hard to keep the humidity levels steady.

2)LED Lighting:

• The LED lights are categorized into "growing" and "blooming" types, each emitting specific types of light or colors that the lamps give off to support different stages of plant development. The lights do not have a light level; instead, they turn on one by one to create a light pattern that resembles the middle of the day. Typically, the photoperiod is 16 hours of light followed by 8 hours of darkness.

• Modern LEDs can handle both growth and blooming phases and consume 36W per 50cm strip.

3) Ventilation:

• Ventilation uses fans to pull in fresh air and expel used air.

4) Materials:

• The outside can be made from Teflon for insulation. Inside, a reflective material covers the walls, the shelves are made of stainless steel, the cables are waterproof, and all connections are covered with insulation.

Our Reflection:

As a team, we carefully considered all these factors and made efforts to incorporate them into our device. We adjusted the design to ensure effective humidity control, appropriate LED lighting for different plant growth stages, efficient ventilation with fans for air exchange, and suitable materials for insulation and durability.

Why We Reached Out:

We chose Dr. Doulgeraki due to her expertise in microorganism characterization, studying factors affecting microorganism survival, growth, and death, as well as the behavior and interactions of microorganisms.

What We Discovered:

1) Dividers Between Plants: It is suggested to add dividers between the pots using the same material as the outer structure (e.g., plexiglass). Dividers will:

• Protect against the transfer of microorganisms between plants.

• Allow experiments to be parallelized under uniform conditions.

• Ensure conditions are sufficiently similar, especially between the control and experimental plants, to obtain reliable data.

2) Sterile Environment:

• A sterile environment is only needed for experiments requiring specific conditions.

• The device aims to simulate field conditions where continuous plant-microorganism interactions occur, so sterile conditions are unnecessary for most experiments.

3) Use of Inert Substrate:

• It is recommended to use soil rather than an inert substrate for more realistic and "processable" data.

• Soil similar to that in areas affected by Verticillium should be used.

• Soil is needed to support plant growth.

4) Airborne Contamination:

• A filter or sterilization point is required where air enters and exits the device to prevent airborne contamination.

5) Theoretical Extension of the Project:

• The project could explore how two or more plants interact with each other and with bacteria over time.

Our Reflection:

• Dr. Doulgeraki helped us recognize the device's usefulness, especially for studying plant-pathogen interactions.

• We decided to keep the dividers and filters, finding that sterile conditions are not required for our setup.

• After an extensive literature review and advice from Professor Tsikou, we have decided to use an inert substrate instead of soil. Although soil may provide more realistic data, the inert substrate offers more controlled conditions, which are essential for the accuracy and repeatability of our experiments [12].

• We rejected the theoretical approach for co-culturing due to its complexity, but we believe it has the potential for further development and scaling up, in the future.

• This aligns with the iGEM competition’s focus on developing practical and scalable solutions for real-world challenges.

Why We Reached Out:

• We selected Dr. Koudounas for his role in our device's hardware development due to his academic background and research interests.

• Due to his interests in the molecular and biochemical characterization of enzymes, molecular biology, and plant biotechnology, his expertise would greatly assist us in the hardware development of our project.

• Dr. Koudounas is recognized for his work in areas related to the chemistry of Volatile Organic Compounds VOCs and their use in various applications, such as environmental monitoring and analysis.

What We Discovered:

1) Chamber Size / Number of Pots:

• 8-10 plants are required per condition.

• Therefore, the chamber should be 60x30x50 cm.

2) Dividers:

• There is no significant risk of contamination from volatile microorganisms.

• Low dividers should be placed between plants to prevent cross-contamination through irrigation waters.

3) Sterilization:

• A hospital-grade filter should be sufficient to prevent any substances from entering or leaving the chamber.

4) Sensors:

• A camera should be installed to capture plant phenotypes, with the data automatically processed, by a machine-learning model. This can be presented as a theoretical extension, or a camera mount can be included in the design.

• Avoid using a UV camera, which could damage the plants despite providing useful information.

5) Controllers:

Lighting:

• Use high-quality LEDs that cover the full light spectrum.

6) Cooling:

• Design a smart cooling system to prevent the chamber from overheating due to the cooling mechanism.

• Overheating is a common issue with incubators in Greece.

Our Reflection:

• According to Mr. Koudounas, we carefully considered everything we discussed, including the filter, LEDs, and have now incorporated them into our design.

• However, the question was raised about whether the dividers are necessary, as there was a disagreement with Professor Doulgeraki. As a result, we started researching this further based on the literature and found that dividers are not essential [13], [14].

• The capacity may be limited to accommodate 8-10 plants, with restricted space for access or modifications, particularly for tasks such as watering or inspecting the plants.

• Overheating from the cooling mechanism is not relevant for a small greenhouse. A small greenhouse doesn't generate enough heat to require such a complex system.

• The camera has the potential to be integrated into the system as we scale up the project in the future, allowing for more advanced monitoring and analysis capabilities.

Why We Reached Out:

• Interested in microbial communities, which involves studying the interactions and dynamics of microorganisms within their environments, we considered Dr. Vasileiadis suitable for our device.

• His professional experience in plant biotechnology and environmental science enhances his expertise, making him highly qualified to contribute to the development of our device.

What We Discovered:

1) pH Control + Organic Matter:

• The soil does not change its pH naturally, and organic matter changes over 5-10 months.

• Experimentally, pH and organic matter management is done by selecting specific soil.

• In other words, instead of regulating pH and organic matter within the setup, we choose soil that meets the experimental needs.

2) Inert Substrate:

• It could be used to control pH and other specific conditions required by each experiment.

• Additionally, inert substrates are commonly used in hydroponic agriculture.

3) Data Loggers:

• Multiple repetitions are required to study plant-microbe interactions properly.

• Although there are existing lab processes for studying these interactions, they require numerous experiments and repetitions.

• Hence, there is a need for automated data processing.

Colonies are visible on petri dishes, but in soil, mixed microbial populations make it challenging to observe the growth and spread of specific microorganisms in real-time. The goal is to measure the microbial population in environmental samples, which is difficult both experimentally and practically. To determine the level of participation of our organism in a microbial community, a identification method is needed, such as:

1) Fluorescence:

• The most common method involves fluorescent proteins. Recognition of the population is achieved through enzyme or protein detection via biochemical reactions or chromatographic methods.

2) VOCs:

• A specific VOC marker could be useful, but the challenge is that soil contains around 50,000 bacterial species per gram, meaning many strains of Pseudomonas putida will produce VOCs. Therefore, the signal may not be distinct in practice.

3) Specific VOC/Nullomer:

• Another approach is to engineer the organism to produce a unique VOC or Nullomer for measurement. Although this could be theoretical rather than actual genetic modification, it might simplify the process.

4) Quorum Sensing (QS):

• QS can act as a qualitative indicator, where bacteria produce signals when they sense a quorum, when their population exceeds a certain limit.

5) Activation of Metabolism:

• Bacteria could be engineered to activate their metabolism only in the presence of Verticillium dahliae.

6) Use of a Reporter Bacterium:

• Using a reporter bacterium is another idea, but it’s tricky because of the soil’s complexity. Problems include detecting the bacterium if the soil is full of bacteria or if the bacterium is present in very low amounts, which might be too close to the background noise.

Monitoring microorganisms in situ is quite challenging due to the fact that many microbes produce a variety of different metabolites. As a result, it is difficult to correlate the population of a specific bacterium with the concentration of a particular metabolite. Additionally, detecting fluorescence within soil is also very difficult due to the inherent complexities.

Our Reflection:

• We have concluded specific soil selection, choosing the appropriate soil is crucial for meeting the specific needs of our experiments.

• Implementing automated data processing is necessary to efficiently manage and analyze the data collected during experiments.

• Both the use of specific soil and automated data processing will be integral components of our device, enhancing its effectiveness and accuracy.

• VOCs and monitoring present significant challenges; the other options were dismissed because we preferred not to make further modifications to the system.

Why We Reached Out:

• Expertise in Electrical Energy Systems and Power Distribution Automation.

• Insights on optimizing temperature control and ventilation systems.

• Knowledge relevant to improving system efficiency and output.

What We Discovered:

1) Temperature Control:

• Consider a small heat pump, as it can provide both heating and cooling with effective performance.

2) Ventilation:

• Determine the airflow needed for the chamber and use the ventilation system only when required to minimize energy use.

Our Reflection:

• Rejected the use of a small heat pump because it is too expensive. Instead, we chose Peltier modules, which are a more affordable and efficient option for maintaining temperature control in a small chamber [15], [16]

• Integrated a system to activate ventilation only when needed to reduce energy consumption.

Why We Reached Out:

• Dr. Trent Northen is an expert in environmental genomics and systems biology, with extensive experience in studying microbial community interactions and metabolism.

• As the Environmental Simulations Science Co-Lead at Lawrence Berkeley National Lab, he manages the implementation of the most advanced and innovative research making him highly knowledgeable in advanced technologies.

• His research focuses on understanding community structure, gene functions, and regulation, which aligns perfectly with our project's goals to study microbial interactions in a controlled environment.

What We Discovered:

• They recommend using a simpler model plant, like plants of the genus Setaria, which can be studied more easily in a lab, instead of challenging plants like olive trees.

• Working with Verticillium might require special permits, so they suggest choosing a different model pathogen that's easier to work with.

• They propose using transparent soil to observe interactions between plants and microbes by detecting fluorescence with a gel imager.

Our Reflection:

• Setaria was chosen over olive trees for its simplicity and ease of study in a lab environment.

• Working with Verticillium might require special permits, so it is considered to be potentially more complex and less realistic than using a different model pathogen.

• The significant difference between soil and gel made it impractical to use transparent soil with a gel imager for watching plant-microbe interactions [17].

Why We Reached Out:

• Expertise in plant symbiotic relationships, including interactions with arbuscular mycorrhizal fungi and rhizobia, highlights her deep understanding of complex plant-microbe relationships.

• Professor Tsikou specializes in molecular and developmental plant biology, making her an ideal expert for advancing our research at the molecular level.

What We Discovered:

• Experiments requiring high precision can be carried out in Magenta boxes, which prevent infection from the environment.

• Magenta Boxes are sterilized boxes often filled with inert substrates, such as sand or perlite. Nutrients are added to these substrates to ensure that the nutrients provided to the plant are strictly controlled.

• Professor Tsikou recommended using an inert substrate if we examine plant-bacteria interactions.

Our Reflection:

• Following Professor Tsikou's advice, we decided to begin our device as a Magenta box. It provides a controlled and sterile environment, which is crucial for high-precision experiments.

Why We Reached Out:

• Knowledge in molecular mechanisms that control plant resistance to abiotic stress can be applied to enhance microbial control strategies. Understanding these mechanisms can help develop safer strategies for managing and containing microorganisms or genetically modified organisms. This knowledge could support biocontainment.

• Skills in genotyping can be crucial for identifying and characterizing microbial strains and their interactions with plants. As the accurate identification and understanding of microbes can help control and limit their spread, which is related to biocontainment

What We Discovered:

1) Monitoring Device Considerations:

• No similar device known for monitoring as required.

• Uncontrolled growth is not expected for these bacteria, as Plant Growth-Promoting Rhizobacteria (PGPR) typically do not cause issues with excessive spread. However, extensive research is still required regarding engineered bacteria growth.

2) Fluorescence:

• Soil is not see-through, which makes it challenging to measure subsurface conditions.

3) Volatile Organic Compounds (VOCs):

• All bacteria, including endophytic strains of Pseudomonas putida, produce VOCs, which we can detect.

4) Optical Density (OD) and Monitoring:

• Optical Density (OD) measures bacteria in a laboratory culture.

• Results from our monitoring device can be compared with OD measurements to provide an objective assessment of the device’s effectiveness and bacterial population.

Our Reflection:

• We chose to measure VOCs, as these measurements prove to be a simple, yet effective method to monitor bacterial growth.

• Our results from the VOC measurements can then be compared to those from an OD experiment.

• Fluorescence was not used because the opacity of the soil blocks the light needed for effective fluorescence detection, which makes it hard to see the fluorescent signals [18]

Why We Reached Out

• approached Konstantinos Daniilidis due to his background as a Postgraduate Student in Computational Biomedicine and Informatics.

• His academic focus on computational methods made him an ideal candidate to assist with data-driven and machine learning approaches.

• We believed his expertise would be valuable in guiding the development of our model and enhance the biological relevance and accuracy of our model's predictions.

What We Discovered:

• Recommended designing only the Receptor Binding Domains (RBDs) instead of the entire chimeric protein to avoid using unnecessary memory on non-essential data during the protein data collection phase.

• Proposed the integration of existing APIs, such as Alpha Fold, for predicting the secondary structure of the protein, rather than building our own model from scratch.

• Encouraged us to test the significance of other variables, such as hydrophobicity, for future prospects of our neural network.

• Strongly advised us to choose machine learning over neural networks, specifically using the ensemble training technique.

Our Reflection:

• Contributed significantly to the development of the model both during the design phase and in the process of building and structuring it.

• Provided crucial guidance in creating the appropriate dataset for our model and actively assisted in its development.

• Suggested the right tools, which became key pillars for setting up our model.

Why We Reached Out:

• Professor Braliou Georgia specializes in Molecular Biology and Genetic Epidemiology, fields that are closely related to our project’s focus on biological interactions and genetic mechanisms.

• Her expertise in genetic data analysis and epidemiological modeling provided valuable insights for the biological modeling aspect of our work.

• Her research in statistical genomics and modeling genetic risk factors aligned with our need for creating predictive models in the context of plant pathology.

What We Discovered:

• Dr Braliou emphasized the importance of proper hybridization between siRNAs and their target mRNAs. She introduced a bioinformatics tool, siRNA Design Tool by Eurofins Genomics to identify the most suitable siRNAs for effective gene silencing.

• She thoroughly explained the appropriate inputs for our model regarding domain testing of the chimera, with the model's output being: "Most effective binding domain due to binding strength."

• Highlighting the challenges of achieving successful gene silencing and suggesting that we showcase the mechanisms behind it in our project.

• Also proposed using ROC curves to assess parameters beyond binding strength for optimal domain selection. Additionally, she recommended using siRNA prediction tools to design the most effective sequences.

Our Reflection:

• We followed Professor Braliou’s advice closely, as her expertise in genetic modeling and molecular interactions provided essential guidance for refining our approach.

• Her contributions were not only constructive but also pushed us to think more critically about our experimental setup and the implications of our findings.

Why We Reached Out:

• We consulted Professor Stergiopoulos after discovering his paper Niño-Sánchez, J., et al. [18] on dsRNA production in bacterial cells. We were thinking of developing a biocontainment system for Pseudomonas putida to ensure safety when applied. His experience with V. dahliae and dsRNA production made him the perfect expert for our concerns.

What We Discovered:

• He recommended elaborating a mechanism to induce stress in bacteria, as this would reduce their population.

Our Reflection: (Integration)

• According to him, the production of OMVs already induces significant stress, which in itself serves as a containment mechanism, although it may not offer much more in terms of safety.

Why We Reached Out:

• With his expertise in soil microorganism interactions, we wanted to ensure that our biocontainment system would preserve biosafety from an environmental perspective.

What We Discovered:

• He emphasized the importance of ensuring that other soil microorganisms are not affected

• Those phylogenetically closest to our target are at the greatest risk of being affected.

• One way to ensure safety is to induce highly specific gene loci silencing in V. dahliae

• To achieve this, we would need to identify homologous genes in the fungus and soil microorganisms and induce targeted RNAi for V. dahliae.

• He suggested that this could be predicted through in silico analysis.

Our Reflection: (Integration)

• We ensured all the necessary criteria were met to protect plant rhizosphere biodiversity and the plant itself through conducting BLAST analysis to detect possible target sequences, avoiding environmental toxic effects arousal

Why We Reached Out:

• As we explored biocontainment strategies for Pseudomonas putida, we considered leveraging the LuxR-AHL quorum sensing system in combination with a toxin-antitoxin kill switch. This mechanism would ensure that if an individual bacterium were to escape from the colony, it would activate an endogenous autolytic pathway, leading to its self-destruction and preventing uncontrolled spread.

• We reached out to our instructor, PhD candidate Katsaouni, for guidance on this idea.

What We Discovered:

• She advised against the LuxR/AHL system, because it would only work if a single cell escaped.

• If an entire population moved, the complex (LuxR-AHL) would continue to be produced, allowing the cells to survive.

Our Reflection: (Re - design)

• Following her guidance, we incorporated auxotrophy into our design, eliminating the need for kill switches to increase system efficiency.

Why We Reached Out:

• Unable to find studies on the olive tree microbiome, we decided to take matters into our own hands; we consulted Professor Vasileiadis, an expert in plant-microbe interactions, to guide us.

What We Discovered:

• He explained that the lack of literature on olive tree microbiome diversity stems from limited knowledge in the field.

• Research has mostly focused on pathogens or industrially significant microorganisms, leaving a gap in understanding the broader soil microbiome.

• To ensure safety at the soil and microbiome level, we must consider the soil's biodiversity

• Since soil can be unpredictable, he suggested we conduct metabarcoding to gather essential insights.

• He recommended making use of auxotrophy for vitamin B12 in our bacteria, a strategy he had worked with before and considered highly effective.

Our Reflection: (Inspiration and Integration)

• We valued his advice on soil metabarcoding and proposed that it could contribute to the evolution of our hardware by integrating the soil samples from these analyses into our hardware device. This would allow us to study the soil and uncover its microbiome.

• We decided to follow his advice and use auxotrophic strains of our bacteria for vitamin B12, restricting their growth under specific conditions. This ensures they won’t proliferate uncontrollably, adding a layer of safety to our system.

Why We Reached Out:

• We sought a containment system that would prevent our engineered bacteria from spreading, ensuring biosafety if released into the environment.

• Ms Kalliampakou Aikaterini as a synthetic biology professor, she was the ideal person to advise us on biocontainment strategies for synthetic systems.

What We Discovered:

• She rejected the use of a toxin-based system, as it would put too much stress on the bacterial cells.

• She advised against using chemical triggers, as they would not be effective.

• After reviewing our system, she recommended we explore chemotaxis-dependent mechanisms as a containment approach.

Our Reflection: (Integration)

• We rejected the chemical death triggers, as we wanted our solution to be the best possible—sustainable and free of chemicals.

• Since this was the second expert suggesting auxotrophic strains, we decided to incorporate this into our system

• Her suggestions were valuable, sparking further exploration into sustainable biocontainment strategies.

• We decided to align bacterial function with a chemotactic agent specific to Verticillium dahliae, leading us to design a highly specialized logic gate, as suggested by prof. Papadopoulou further on.

1) Copper Application: Copper-based solutions were applied but yielded no results.

2) Lime Application: Lime was applied, again without success.

3) Solarization Method: Covering the soil with black plastic to raise the soil temperature, aiming to disinfect microorganisms. This also proved ineffective.

4) Mycorrhiza Application: Mycorrhizal fungi were introduced to the soil, but this did not lead to any improvement.

5) Bordeaux Paste (Copper): Excavation down to the central roots of infected trees was followed by the application of Bordeaux paste (a copper-based solution) and lime. Although no further harm was observed, this treatment also failed to yield positive results.

1) Tree Uprooting and Burning: The most effective management approach considered was the complete uprooting and burning of the tree’s branches and trunks. However, this results in the loss of the tree’s potential future yield.

2) Cessation of Soil Tillage: Farmers halted all soil tillage as it was found to worsen the problem by spreading infected soil to healthy trees.

3) Pruning and Burning of Dry Branches: The management practice eventually adopted was cutting and burning the dried-out branches. However, this was more a containment measure than a solution.

• Farmers have observed a notable increase in symptoms during the early spring, extending through the summer and autumn months. Generally, in years with warmer winter temperatures, the problem appears more strongly. Given the significance of these critical periods, we decided to take advantage of this timing and apply our solution when the issue is most pronounced, ensuring maximum effectiveness.

• In lowland areas, geographical features complicate the implementation, and the lack of irrigation points can further hinder efforts. However,they really need a solution no matter what as they are willing to attempt root irrigation, demonstrating their commitment to eliminating the issue. In coastal regions, symptoms are more pronounced, leading to a greater urgency for effective solutions.

• For irrigated crops, root irrigation is more feasible, as these areas already have established systems in place. Conversely, in dryland farming, the lack of an existing irrigation system poses significant difficulties, requiring additional equipment and incurring a greater financial burden for installation.

• A common challenge faced by producers with large tracts of land is that root irrigation can be quite difficult to manage. Nevertheless, they have indicated a willingness to explore its use even in these cases.

Why We Reached Out:

• One of our biggest challenges was determining how our solution would be applied in the real world. We had several ideas but knew the application would need to focus on the tree’s roots. We consulted Professor Dalakouras to resolve this question and determine the most effective application method.

What We Discovered:

• He suggested that root irrigation would be the most practical and effective method. Since Verticillium dahliae enters the plant through the roots, applying our solution directly to the roots via irrigation ensures it reaches the future infection site efficiently. Additionally, Pseudomonas putida is endophytic to olive trees, making it a perfect match for this method.

Our Reflection: (Integration)

• Before proceeding, we decided to contact additional experts to ensure we were choosing the optimal application method.

Why We Reached Out:

• To explore more application methods for our solution, we consulted Professor Tzima again, given her expertise in plant pathology.

What We Discovered:

• For an endophytic pathogen, spraying is not sufficient since it doesn’t reach the roots.

• Root irrigation is necessary, and she considered it a viable solution.

Our Reflection: (Integration)

With her recommendation aligning with root irrigation, we began seriously considering this method.

Why We Reached Out:

• As we moved forward with implementing Thaelia, we sought additional guidance from Professor Papadopoulou to refine our system even further. Our goal was to ensure that the production of dsRNAs, would only occur in the presence of the V. dahliae fungus. In essence, we wanted guidance into how to precisely induce the activation of our system when needed.

What We Discovered:

• She suggested adding a Logic Gate related to the extension of the lag phase, ensuring the rest of our system operates in the presence of V. dahliae, triggered by the presence of a specific molecule.

• This strategy ensures that our bacteria remain inactive and in the lag growth- phase until the fungus is detected, preventing early dsRNA production and OMV release.

The advantages?

• Modularity maintenance: The logic gate acts as an independent module, adding an extra layer of regulation to our system without interfering with dsRNA or OMV production.

• Measurability maintenance: It allows precise control over system behavior and activation in the presence of V. dahliae, guaranteeing accuracy and efficiency.

• Orthogonality maintenance: Extending the lag phase is autonomously without interfering with the other parts of our system

• It helps reducing the frequency of application as activation of our system occurs only when it’s needed; when V. dahliae is present. Such a tier of regulation makes our product more competitive!

• The trigger for this system doesn't need to be species-specific for V. dahliae, providing flexibility in the signals used for activation, as bacterias can identify the fungus due to their chemotacticity. Besides, the “molecular dialogue” between V. dahliae and the plant is yet to be discovered, making it impossible to find a species - specific molecule to induce our system.

Our Reflection: (Integration)

• Recognizing the value of this suggestion, we immediately began integrating it into our solution. This was an extremely useful addition, as it allows better regulation of our system and ensures there’s no activation in the absence of V. dahliae, which could otherwise deplete energy resources and harm our bacterial chassis.