1.1. PROJECT SUMMARY

Of the nearly 26.6 tons of fruits and vegetables produced each year in Peru, around 12.8 million never reach markets or homes. When analyzing the supply chain of food, we realized that an average of 47.6% of the country’s annual food production is lost, which places us among the highest rates of food waste in Latin American [1]. We identified that this degradation process occurs mainly due to fruit oxidation and the opportunistic fungi during the transportation process. This problem represents a challenge for environmental sustainability and also affects our population in different ways. Farmers and distribution companies receive less income by not taking advantage of all the production. Likewise, the precarious state of the supply chain in remote areas of the country exacerbates the already existing problem of malnutrition. To help mitigate this problem, various technologies are used to help keep food fresher for longer, such as chemical preservatives, which can have adverse health effects when consumed in high concentrations and many times can be washed into nearby water bodies harming local wildlife. An alternative is constant refrigeration, however, this tends to be expensive and difficult to access, especially in remote areas of the country. Having all this into account, MikuyTec seeks to aid the agricultural and agro-export sectors, two of the largest economic sectors in the country, since it could greatly benefit from biotechnology.

MikuyTec’s main objective is to reduce product loss in transport and distribution stages by extending the shelf life of fruits and vegetables. We propose a large-scale recombinant production of antifungal and antioxidant peptides for application in a coating applied to crops. This would slow down the deterioration of food while keeping their desired properties and in turn reduce food waste. At a nutritional level, it would improve food safety and improve access to quality food in remote communities. At an environmental level, it would contribute to environmental sustainability in matters of land and water usage, reducing the number of greenhouse gasses (GHGs) produced by rotten food, an overall reduction of carbon footprint. Finally, from an economic approach, it would be possible to increase income for producers and distribution companies. 

In order to speed up the research process, we focused on three types of fruits, based on their rates of deterioration and their cultural value in Peru: strawberries (Fragaria x ananassa), tomatoes (Solanum lycopersicum) and aguaymanto (Physalis peruviana). Two main deterioration factors were found. One of these is a fungal infection, with Aspergillus spp, Penicillium spp. and Botrytis cinerea as the most predominant in these crops. The mechanism of action of our target fungi is the release of mycotoxins for the degradation of the external and internal tissue of the fruit. On the other hand, oxidation is another of the main factors of decomposition. Oxidation occurs due to the action of the tyrosinase enzyme, responsible for the enzymatic browning of food.

To achieve the antioxidant and antifungal activity of the product, the team decided to design two cassettes. Both consist of the strong constitutive promoter J23104, to increase the production of peptides in the bioreactor and optimize resource consumption. Likewise, the T7 terminator will be used due to its extensive characterization in the iGEM database. The first cassette codes for antifungal peptides to which a protein domain is added to improve the specificity of action towards fungi. This extra sequence is a chitin-binding domain (CBD) and its main function is to facilitate the binding of our antifungal peptides to the fungi of interest. It does this by generating an affinity tag towards the fungal membranes’ chitins so that the peptides can attack them. The peptides chosen are Rs-AFP2, PAF26 and PAFP-S, which have been found to have antifungal activity against Aspergillus sp, Penicillium sp. and Botrytis cinerea respectively [2]. On the other hand, the second cassette synthesizes the VS14 and NOP-1 peptides [3], antioxidants whose function is to inhibit the tyrosinase enzyme, responsible for fruit oxidation. Since it does not require any particular binding to the fungi, this coding sequence will not have the CBD. Finally, the chosen chassis is E. coli BL21 due to its extensive characterization for recombinant protein production and because it is not affected by the peptides produced.

For production, a Batch-type bioreactor will be employed, providing the appropriate conditions for the growth of E.coli colonies in order to generate high concentrations of the peptides. These will be extracted from the bioreactor by lysing the cells through centrifugation, and then lyophilized into powder for delivery to fruit and vegetable distribution companies. This powder can later be dissolved in water so that the peptides regain their biochemical activity and can be safely sprayed onto the crops. In this way, the antifungal and antioxidant action will be achieved in the sprayed fruit, extending its shelf life and protecting it during the transportation process without the need for chemical preservatives.

References:

[1] Bedoya-Perales, N. S., & Dal’ Magro, G. P. (2021). Quantification of Food Losses and Waste in Peru: A Mass Flow Analysis along the Food Supply Chain. Sustainability, 13(5), 2807. https://doi.org/10.3390/su13052807

[2] Thery, T., Lynch, K. M., & Arendt, E. K. (2019). Natural Antifungal Peptides/Proteins as Model for Novel Food Preservatives. Comprehensive Reviews in Food Science and Food Safety, 18(5), 1327–1360. https://doi.org/10.1111/1541-4337.12480

[3] Thaha, A., Wang, B. S., Chang, Y. W., Hsia, S. M., Huang, T. C., Shiau, C. Y., Hwang, D. F., & Chen, T. Y. (2021). Food-Derived Bioactive Peptides with Antioxidative Capacity, Xanthine Oxidase and Tyrosinase Inhibitory Activity. Processes, 9(5), 747. https://doi.org/10.3390/pr90507

1.2. PROMOTIONAL VIDEO

1.3. PROJECT PRESENTATION VIDEO

- Include a link to your project presentation video on iGEM Video Universe

1.3. EDUCATION AND COMMUNICATION INFOGRAPHIC

1.5. TEAM AND ATTRIBUTIONS

Students:

Jeremy Rodrigo Guerrero Alejos

• Innovation area, Bioprocess and Synthetic Biology

Piero Beraún 

• Synthetic Biology, Bioinformatics and Computational Biology, and Collaborations

Nadia Odaliz Chamana Chura 

• Synthetic Biology, Human Practices and Innovation and Entrepreneurship

Alexander

• Synthetic Biology, Collaborations and Biosafety & Biosecurity

Alexandra Ximena Valdez Lopez

• Synthetic Biology, Bioprocess, and Marketing and Design

Darwin Diaz 

• Synthetic Biologý, General Biology and Human Practices

Maria Andrea Gonzales Castillo

• Synthetic biology, Collaborations, and Entrepreneurship & Innovation

Rodrigo Gallegos Dextre

• Synthetic Biology, Bioinformatics, and Entrepreneurship & innovation

Marjorie Esquivel Noriega

• Collaborations, and General Biology

Sonaly Samanta Tomas Valeriano

• Synthetic Biology, Human Practices, and Bioprocess 

Gabriel Luis Dario Loayza Pretel

• Synthetic Biology, Human Practices, and Collaborations

Jhon Anderson Pérez Silva

• Computational Biology, Biosafety & Security and Collaborations

Tamara Fátima Ortiz Ruiz

• Synthetic Biology, Web developing and Marketing and Design

Miguel Angel Saturno Villanueva 

• Web developing, and Entrepreneurship & innovation area

Gustavo Muro Marchani

• Synthetic Biology, General Biology and Marketing and Design

Mentors & Instructors:

Roles by committee:

• Human Practices 🙌

This committee connected with those involved in the problem through interviews and activities. We met with farmers, agro-export companies, merchants and buyers from the market, and children, all the stakeholders involved in the project. With the information collected, we will validate our assumptions and needs. We also evaluated if our project complied with the regulations of Peru.

• Synthetic Biology 🦠

This committee implemented synthetic biology in the project. Its function was to design the genetic circuit according to the needs found. The committee also chose the standardized elements to build the circuit. Based on the design, the committee chose the assembly method and protocols needed to build the system.

• Innovation and entrepreneurship 🚀

This committee was in charge of shaping the project into a business. To do this, we used innovation tools such as the Model Business canvas to improve details to establish ourselves as a business. We planned a Gannt diagram with future goals to continue as a venture. The committee helped analyze the market and thus better understand the consumer of the product.

• Computational Biology 💻

This committee used computational biology and bioinformatics tools, such as structural biology servers and Matlab in-house scripts to conduct an analysis of our synthetic antifungal and antioxidant proteins from a structural approach. We then used mathematical models to simulate the behavior of different parts of our project, obtaining results that were useful in the iteration and validation of our proposal.

• General biology 🍄

This committee identified the bacteria we used as chassis for our process and the fungi that we have to attack with our product. We studied the complete mechanism of the polyphenol oxidase enzyme responsible for enzymatic browning in order to propose a suitable antioxidant protein. Based on a bibliographic review we were also able to identify the antifungal proteins that would serve as the basis for our product as well as the selection of those peptides that could be safe for organisms with chitin wall which are not our target. 

• Marketing and design 🎨

The marketing and design committee was in charge of communicating and promoting our project’s advances and activities in a visually appealing way. This committee was in charge of managing and producing content for our social network accounts in Instagram, Facebook and Linkedin, as well as establishing our overall visual identity. Additionally, marketing and design produced all of the graphic content used for the activities we organized, such as flyers, infographics, powerpoints, illustrations and any other additional visual content needed.

• Bioprocess 👩‍🔬

This committee was in charge of investigating and developing the design of the bioprocess for the production of antifungal and antioxidant peptides with our recombinant E.coli. It is for this reason that we focused on the research for the bioreactor and the optimal conditions to obtain the maximum production possible, in this way, we were able to model the growth of our bacteria in Matlab. We also defined the protocols based on the already existing literature for the processes of bacterial growth, purification of peptides, and their lyophilization.

• Collaborations 🤝

In order to create partnerships and exchange knowledge with other synthetic biology students in Latin America, we collaborated with many different teams during our time in the competition. The collaborations committee was in charge of contacting and organizing constant meetings with other teams, as well as planning collaboration ideas and keeping track of everything discussed in each meeting. By the end of the Design League, we had formed meaningful partnerships with four other teams and attended different meetups and events.

• Biosecurity 🦺

This committee was in charge of the areas of Biosafety, Biosecurity and Bioethics. We looked for risks that our project may encompass as well as possible concerns that our proposal may raise, during the experimentation stage and later with the release of the product. We also looked for counseling from other professionals for their feedback about our proposal, which helped to improve our understanding of the topic.

• Mentorship 👨‍🏫

During the development of the competition, we received great support from our instructor and Mentors. Ursula Rodriguez, who helped us to contact stakeholders as well as offering mentorship in the bioprocess area. Msc. Gustavo Sandoval, whose contributions were greatly helpful for the bioinformatics committee. Miguel Camacho, who lends us his experience and knowledge on the synthetic biology committee, to overcome the problems that arose during our design stage. PhD. Luis DeStefano, whose suggestions and counsel helped on the refinement and development of our final product.

• Web developing 💻

The web developing committee was in charge of designing and producing Mikuytec’s landing page. The landing page was used to provide useful information for the general public and potential work partners.

Attributions:

Special thanks to:

• Kyle Alexander Post
• Santiago Bustamante Villa
• Noemí Bravo Aranibar
• UTEC Pride

2.2. DESIGN ROADMAP

As part of our project design, we have incorporated project management techniques that have helped us in the implementation and development process of our project. Here we present some of the techniques that we have taken into account: we implemented the design thinking methodology, created a roadmap, a storyboard, actively used brainstorming techniques and used theory of change methodology.

First, we used the Theory of Change methodology.

To see more details click here.

For the development of our project, we followed the design stages of the design thinking methodology. We searched for new solutions to our problem, taking our final user as the main focus of attention. In the first stage 'empathize', we evaluate the user's needs through interviews, small talks and continuous visits. Then, in the 'define' stage, taking into account the needs of the user, we made a statement of the problem to be addressed. In 'ideation', we brainstormed the possible solutions for use in addressing our problem. After obtaining varied solution proposals, we selected one which we have been developing and improving since this methodology is iterative, so it is always in constant improvement.

As a team, having a roadmap has allowed us to visualize the changes and developments that are needed to reach our final objective, in addition to showing the results that are expected to be achieved in a specific period of time. In this way, all team members can stay informed about the activities that we need to develop as a team, having a macro-level view of the project. We consider this to be an important project management tool, as it also helps in developing a more comprehensive project plan. In our roadmap, we divided the project into four major stages: Research, Design, Marketing and Risk Evaluation of results. In the research stage, we obtained relevant information by searching for current research papers and conducting a market study about the problem we are trying to solve in our community. In the design stage, we emphasized the construction of our genetic circuits and the processes to follow in order to bring this value proposition to a possible market. In the marketing stage, we emphasized communicating our idea to local communities through public exhibitions, social media activities, and workshops. Finally, for the risk evaluation stage, we emphasized the impact that our value proposition could have on the environment and therefore the considerations needed for its design and implementation. Under this roadmap strategy, we ensure that everyone is aligned and working towards the same goal.

Brainstorming and storyboarding are other techniques that we apply. As we mentioned earlier, in the 'ideation' stage of design thinking we collect quite a few proposals to address the identified problem, each of them being evaluated. Using this tool has allowed us to think freely and go beyond our imaginations, encouraging our members to generate a great number of innovative ideas that could be merged to create the solution. In addition, we also use this tool to propose possible activities to make our idea known to other people, starting with our community. This has allowed us to land on innovative proposals of activities in social media, events and workshops. We have carried out some of them which are detailed in the Educational and Communication section.

To see more details click here.

To see more details click here.

2.2. Excellence in Biological Engineering Design

In MikuyTec, we are engineering E. coli BL21 to produce recombinant antifungal and antioxidant peptides, aiming to reduce spoilage and extend the shelf life of fruits and vegetables. These will be freeze-dried and then activated with water to be used as a spray coating for the fresh produce.

To do so, we designed two cassettes to express both our antifungal and antioxidant peptides. The common elements in both cassettes are an ompA secretion signal (BBa_K208003) to allow increased production of the peptides, a His-Tag (BBa_K1223006) sequence to purify them, and strong RBS (BBa_B0034) for the production of different proteins in the same cassette. The first cassette is composed of the T7 promoter (BBa_I719005), two antioxidant peptides: VS14 and NOP-1, and the T1 terminator (BBa_B0010). The VS14 and NOP-1 peptides inhibit the tyrosinase enzyme, responsible for fruit oxidation (enzymatic browning) in the fruits selected (strawberries and tomatoes). The second cassette is composed of the J23104 promoter (BBa_J23104), three antifungal peptides: Rs-AFP2, PAF26 and PAFP-S, and a T7 terminator (BBa_K731721). In this cassette, the peptides selected have activity against Aspergillus sp, Penicillium sp. and Botrytis cinerea, correspondingly, which base their mechanism of action on the destruction of the wall of these fungi. Additionally, we bound each of the peptides from the second cassette to a chitin-binding domain (CBD) (BBa_T2028) through an SGS linker. This was done to facilitate the action of the peptides towards the fungi and improve the specificity of the product. Finally, we used pJK515 (GenBank ID ACT43674) as our plasmid vector backbone for high copies of our strain with the Gibson Assembly technique.

Both cassettes will be expressed in E.coli BL21 (DE3), a strain extensively characterized for recombinant large-scale protein production. Colonies will be bred in a Batch type bioreactor (BioFlo 3000) to reach high peptide concentration, then it will be extracted by centrifugation, purified through Immobilized metal affinity chromatography(IMAC), and then lyophilized into powder for delivery and distribution.

2.2.1 Standardization

• Parts

The following parts utilized in this project can be found in the iGEM Registry of Parts:

ompA secretion signal (BBa_K208003)

The ompA is a secretion signal which can attach to other proteins, to target them for exportation outside the cytoplasm. This characteristic aids in slowing down the saturation within E.coli by the peptides being constitutively produced inside. Consequently, this boosts the overall amount of peptides produced, before the lysis, extraction and purification of the final product.

His-Tag sequence (BBa_K1223006)

The His-Tag sequence is useful during the purification process, as it eases the task of peptide recognition during this step. Additionally, it must be noted that the default stop codon within the His-Tag was removed, for it to be suitable for the Gibson Assembly procedure.

Strong RBS (BBa_B0030)

The RBS is the RNA sequence upstream of the start codon that affects the rate at which a particular Open Reading Frame (ORF) is translated. We selected a strong RBS that contains more adenines in its sequence upstream to boost the expression of the peptides and allow the production of each one of them separately on the same cassette. This part is well characterized due to the extensive information available about it and its 1001 uses.

T7 promoter (BBa_I719005)

The T7 promoter is a constitutive promoter derived from the T7 bacteriophage and allows high expression of proteins only when the T7 polymerase is present. We selected it because it does not need an inductor, which could elevate the cost of the large-scale production of the peptides. Additionally, it has 262 uses in the iGEM competition so its broad characterization is demonstrated. Finally, it was convenient for the team to work with this promoter as it was already present on the selected backbone.

T1 terminator (BBa_B0010)

This is a transcriptional terminator from E. coli rrnB, consisting of a 64 bp stem-loop. It has been used 1064 times in past iGEM projects, making it a well-characterized terminator.

J23104 promoter (BBa_J23104)

This promoter is part of the J23100 through J23119 constitutive promoter family, out of which it showed the highest constitutive expression strength in K. rhaeticus according to the KEYSTONE_A 2020 iGEM Team. Additionally, it has had a total of 111 uses up to date.

T7 terminator (BBa_K731721)

T7 terminator is a sequence consisting of 48 bp from bacteriophage T7 which allows efficient transcription termination suitable for E. Coli. This terminator has been used 114 times in past iGEM projects.

Chitin binding domain (CBD) (BBa_T2028)

This is a CBD domain that was incorporated in the design next to each of the antifungal peptide sequences to have greater specificity for the peptide action towards fungi. Chitin-binding domains are a type of affinity tag that are usually used to purify proteins, convert chitin to chitosan, and degrade chitin. Nevertheless, in this project, we are using them as a tag of recognition towards fungal chitins. This part contains 156 bp and it has been used 5 times in past iGEM projects.

The next set of parts are the genetic sequences for the antioxidant and antifungal peptides which are not currently in the iGEM Registry of Parts:

PAF26 (iDLBB_001701)

PAF26 is a synthetic hexapeptide that has a relevant effect as an inhibitor of conidia germination and posterior growth of the mycelium, targeting certain filamentous fungi such as Botrytis Cinerea. It works by altering fungi cell walls' permeability.[1]

Rs-AFP2 (iDLBB_001702)

Rs-AFP2 is a defensin extracted from Raphanus sativus (radish). It works by binding to sphingolipid glucosylceramides in the fungal membrane, thus permeabilizing it and causing alkalinization of the growth media. It has high specificity towards filamentous fungi such as Aspergillus sp.[2]

PAFP-S (iDLBB_001703)

PAFP-S is a knotin-type antifungal peptide from Phytolacca Americana (American pokeweed). This thermostable peptide, which maintains its activity even after heat treatment, has activity restricted only to plant pathogens and does not affect other organisms (such as E. coli) growth. It bases its activity on its amphipathic and cationic surface and uses it to interact with the fungal membrane’s cationic sections. [3]

VS14 (iDLBB_001704)

VS14 is an antioxidant peptide derived from tuna-backbone protein that has a great effect at inhibiting tyrosinase, an enzyme responsible for enzymatic browning.[4]

NOP-1 (iDLBB_001705)

NOP-1 is a synthetic octapeptide that delays ripening in fruits, inhibiting the ethylene signaling system responsible for fruit senescence and aging, by binding to the ethylene receptors in the fruit. [5]

Plasmid pJK515 (GenBank ID ACT43674)

This part comes from the vector backbone pET23a for bacterial expression. We selected this plasmid because it contains the T7 promoter and the 6XHis-Tag, two of the genetic parts we wanted to use. We also selected it as it allows a high number of copies of the plasmid in the Escherichia coli BL21(DE3) strain.

Different uses:

Our antifungal and antioxidant parts can be used in other settings apart from fresh produce protection like we do in Mikuytec. 

Antifungals: The antifungals peptides can be used in any setting where Aspergillus, Penicillium or Botrytis cinerea strains would grow. This can be to protect crops and other plants in any stage of growth, like they are used in this project. They can be applied in an indoors setting as well, as a way to prevent mold growth on clothes, wallpaper and/or carpeting. Additionally, we know of other teams such as iGEM Panama that have used defensin peptides on paper materials to stop fungi from destroying the paper.

Antioxidants: Regarding the two remaining peptides, NOP-1’s and VS14’s antioxidant action can work in any other crop with a high concentration of the polyphenol oxidase enzyme (such as bananas, pears, avocados or mangoes) to delay ripening.

All of the genetic sequences for our parts and devices can be found here.

References:

• [1] Muñoz, A., Lopez-Garcia, B., & Marcos, J. F. (2006). Studies on the Mode of Action of the Antifungal Hexapeptide PAF26. Antimicrobial Agents and Chemotherapy, 50(11), 3847–3855. doi:10.1128/aac.00650-06 
• [2] Aerts, A. M., François, I. E. J. A., Meert, E. M. K., Li, Q.-T., Cammue, B. P. A., & Thevissen, K. (2007). The Antifungal Activity of RsAFP2, a Plant Defensin from Raphanus sativus, Involves the Induction of Reactive Oxygen Species in Candida albicans. Journal of Molecular Microbiology and Biotechnology, 13(4), 243–247. doi:10.1159/000104753 
• [3] Shao, F., Hu, Z., Xiong, Y.-M., Huang, Q.-Z., Chun-Guang Wang, Zhu, R.-H., & Wang, D.-C. (1999). A new antifungal peptide from the seeds of Phytolacca americana: characterization, amino acid sequence and cDNA cloning. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1430(2), 262–268. doi:10.1016/s0167-4838(99)00013-8 
• [4] Je, J.-Y., Qian, Z.-J., Byun, H.-G., & Kim, S.-K. (2007). Purification and characterization of an antioxidant peptide obtained from tuna backbone protein by enzymatic hydrolysis. Process Biochemistry, 42(5), 840–846. doi:10.1016/j.procbio.2007.02.006 
• [5] Klein, S., Fiebig, A., Neuwald, D., Dluhosch, D., Müller, L., Groth, G., … Hunsche, M. (2019). influence of the ethylene related signal-inhibiting octapeptide NOP-1 on postharvest ripening and quality of “Golden Delicious” apples. Journal of the Science of Food and Agriculture. doi:10.1002/jsfa.9613 

• Expression cassettes

Cassette 1 (iDLBB_001706):

Our first cassette (iDLBB_001706) presents the genes necessary to express the antioxidants VS14 and NOP1. These peptides are separated with strong Ribosome Binding Sites (BBa_B0030) to allow correct translation of each protein. This construct is also bound with an OmpA secretion signal peptide (BBa_K200803) for export out of the cell cytoplasm and a 6XHis-Tag sequence (BBa_K1223006) to aid in the purification process. In addition, we used the T7 promoter (BBa_I719005), a constitutive promoter to ensure constant and large-scale production, and the T1 terminator (BBa_B0010).

Cassette 2 (iDLBB_001707):

The second cassette (iDLBB_001707) contains the genes for three antifungal peptides: Rs-AFP2, PAF26 and PAFP-S, each bound with a chitin binding domain (BBa_T2028) through a linker for their adherence to fungal chitins. As in the first cassette, we used OmpA (BBa_K200803) and His-Tag secretion signal peptides (BBa_K1223006) for the same purposes described above. Genetic expression of the parts is driven by a constitutive J23104 promoter (BBa_J23104) and ends in a T7 terminator (BBa_K731721). In this second cassette, we used a different promoter than the first to avoid errors when assembling with Gibson Assembly, as the construct could be assembled incorrectly when primers bind to similar DNA segments. We omitted the use of random sequences in the construction, which could have allowed us to use the same promoters for both cassettes, but would have increased the length of our plasmid, which means a higher sequencing cost.

Our proposal presents modularity, as the parts used (excluding the peptides) are standardized. This allows us to be able to vary our genetic circuit by exchanging specific parts to improve the expression profile eventually obtained experimentally.

• Assembly technique

The sequences of the biological parts chosen were uploaded to and annotated in Benchling. Additionally, we built the genetic circuits in BioRender according to the schematics.

When first working on our system, we initially designed a single cassette. However, we then realized that using only one promoter in the production of several peptides could potentially lead to weakened gene expression further downstream and thus compromise the efficacy of the final product. To address this, we decided to express the peptides in 2 different cassettes, one for the antioxidant and another for the antifungal peptides. Regarding assembly, our biological devices are composed of several different parts. This means that techniques that use restriction enzymes would raise the assembly cost considerably. Moreover, it would be necessary to increase both the number of reagents and DNA segments needed for the RE(restriction enzymes). This is why we selected the Gibson assembly method, as it allows us to assemble our cassettes in a short amount of steps and does not leave any scars.

2.2.2 Optimization

Computational Biology

Following our first project concept, we combined some of the parts (Table 1) described before creating chimeric protein sequences (Table 2) to analyse which combination would lead to better antifungal and antioxidant effects.

Table 1. Parts that will be translated into amino acid.

• Computational modelling of proteins

We produced predicted 3D models for all our proteins (Table 2) with a diverse set of computational biology tools, to analyze their biophysical properties and viability after being secreted by bacteria.

Firstly, by using Python in-house scripts with the MODELLER tool we got homology models of the 7 chimeric antifungal proteins, using the experimental models of some parts from the Protein Data Bank as templates: (PDB: 1ED7), RsAFP2 (PDB: 2N2R) and PAFP-S (PDB: 1DKC). 

Then, using the Phyre2 and Robetta software we created models of all our chimeric proteins by threading and ab initio respectively. We gave special care to modelling our antioxidant proteins as we could not previously do it with the MODELLER tool due to the absence of its experimental structure (NMR solution or X-Ray crystallography).

Finally, we decided to try modelling our proteins using the latest AI algorithms of protein modelling, AlphaFold and AlphaFold 2, to compare the quality of models given by each approach (Homology, Threading, Ab initio and AI).

Table 2. Sequences for potential chimeric proteins.

• Quality assessment of 3D models with ModFold

ModFold is a useful computational tool that allows users to assess the quality of models predicted. It does so through the use of functions that allow us to generate rankings of multiple 3D models for the same protein target, according to predicted model quality and predictions of the local quality (per-residue error) within multiple models.

Therefore, as we got more than 10 models per sequence, we submitted all the models as a .tar.gz file to the ModFOLD8 server to get a trustworthy ranking of the 3D models.

We selected the first result of each ranking of models (9 in total) to conduct the molecular docking analysis (Table 3).

Table 3. Best models for each chimeric sequence.

• Molecular Docking of Antifungal proteins with Chitin

We conducted a molecular docking analysis of our chimeric antifungal proteins bound to a tetramer of chitin using the software DockThor, to analyze which combination of antifungal domains would allow the CBD to optimally bind to chitin. 

Finally, proteins with the highest affinity energy score (AFP4, AFP5 and AFP7) were selected to continue with the design of our plasmids.

Table 4. Best models for each chimeric sequence.

2.2.3 Mathematical modeling

• Gene expression model

To simulate the recombinant production of our proteins, we will use two algorithms: the first at a single cell scale and the second at a population scale. To simulate the behavior of protein production in a single cell, as some of the molecules involved in the process are in low quantities, and as there is only one plasmid with one promoter controlling the transcription of our protein, the model that best fits this situation is a stochastic one. The algorithm used is based on Gillespie’s work [1] and the implementation was previously done by Charlebois [2]. We are defining our processes as the two steps of the central dogma (transcription and translation), considering that other chemical species involved in the reactions are constants inside the cell (such as amino acids, RNA pol, nucleotides and ribosomes), the equations are as follows:

Where:

The values of the rate constants that we used are arbitrary and don’t represent the real biochemistry of our bacteria. Since our proteins are chimeric and designed by us, their constants are not reported in previous literature and we will need to calculate them in future lab experiments in order to show a behavior closer to reality. However, the arbitrary values still help us figure out the general behaviour of our system. The result of the stochastic simulation is shown in Figure 1: 

Figure 1. Stochastic simulation of protein production

To see what happens at a population level, we can use a deterministic simulation of our system, because now we will have all of our parts in large enough quantities to consider them as continuous variables. The equations are similar to the stochastic model, but now we have a system of ordinary differential equations based on [3]:

The values of the rate constants are the same, but now instead of the number of molecules, we have their concentrations. The deterministic curves are compared to the stochastic ones in Figure 2:

Figure 2. Deterministic and stochastic simulations of protein production

We can see that both models match well even though the stochastic one has a lot of noise. We can reduce the noise if we increase the number of cells we are using, so the values of the molecules normalize and tend to behave as the deterministic model. This effect can be seen in Figure 3:

Figure 3. Stochastic simulation of protein production with 20 cells

Now we can see that both models tend to the equilibrium values for Protein concentration and mRNA, which can be calculated as follows:

"Ktx" depends on the nature of the promoter, which remains the same for all constructs.  depends on the nature of the mRNA, which differs from one protein to another. "Ktl" depends on the affinity of the ribosome for the RBS sequence, which can also be considered constant as it is the same in all constructs. Finally  is different from one protein to another, and it is one of the most important values in our design for a couple of reasons. The first reason is that it is related to the lifespan of our peptide. The second reason is that it tells us the expiration time of our product and how long it will be active in case it comes in contact with nature, so the values of this constant will be among the first to be calculated in future lab work.

The conclusion of this virtual experiment is that the more cells we have in a volume, the more predictable the system will be. This not only applies to our cell cultures during experiments and the bioreactor but also to the liquid flows and samples containing cells.

• New genetic designs

In order to produce large quantities of different types of proteins, we planned to change our designs from single protein production to polycistronic genes that can produce more than one protein for each expression cassette. Our current mathematical model only predicts the behaviour of one gene with one protein sequence, so we need to add the new parts to our model. We can see a scheme explaining the new design in Figure 4:

Figure 4: New expression cassettes designed

Since all the constructs share the same promoter and RBS, Ktl and Ktx remain the same for all antifungal proteins and antioxidant peptides. The three proteins of the first cassette are transcribed together, so they share the same value of RNA degradation rate , the same situation occurs in the second cassette for the two peptides. The equations that control the behaviour of this new system are:

1st cassette transcription:

Antifungal protein 1:

Antifungal protein 2:

Antifungal protein 3:

2nd cassette transcription:

Antioxidant peptide 1:

Antioxidant peptide 2:

Where:

The resultant expression curves of both cassettes can be seen in Figure 5 and Figure 6:

Figure 5: Curves of expression of the antifungal proteins in the first cassette

Figure 6: Curves of expression of the antioxidant peptides in the second cassette

In the first graphic we can conclude that despite being in the same expression cassette, the curves and subsequently the equilibrium concentration of the three antifungals proteins are different. This means we can predict that during large-scale production, our protein powder mix will contain different amounts of the antifungal proteins. However, if the amount of proteins exceeds that necessary to have an effective antifungal effect, the product will meet its objective, and this needs to be tested in future lab experiments. We can also reach the same conclusion if we analyze the graph of the peptides.

• Cell growth and bioreactor models

To model the growth of the modified cells in our bioreactor, we will use a logistic growth model because all nutrients are constantly supplied and are therefore in constant concentrations. The cell population growth follows the equation:

Where:

We can obtain the values of K and of E. coli strain BL21 in a batch reactor from [4], assuming an initial OD600 of 0.5 (this value is entirely defined by us by adding a controlled amount of bacterial sample to the bioreactor), the simulation gives us the curve in Figure 7:

Figure 7. Logistic growth of E. coli BL21

We can calculate the numerical value of the time in which the culture reaches the exponential phase, by taking the second derivative of the cell growth function and finding its maximum value. The equation of this second derivative is:

When plotting the time behavior of this second order differential equation, we obtain the curve in Figure 8:

Figure 8. Derivative of the logistic growth of E. coli BL21

Where the time to reach the exponential phase is 9.66 hours. We can repeat this process for other values of the initial concentration, so we can plot the changes in one single picture, as shown in Figure 9. We can see that the higher the initial concentration, the faster it reaches the limit value K.

Figure 9. Growth curve at different initial concentrations of cells

Then we can calculate the time to reach the exponential phase for every curve, the values are shown in Figure 10:

Figure 10. Time to reach exponential phase for each initial concentration of cells

This last graphic shows us that after an initial concentration of approximately 0.5 OD600, the time to reach the exponential phase doesn’t vary much, so we can keep this as our value to continue with the rest of the modeling. 

To keep the best performance of protein production in our bioreactor, we need to maintain the cell culture in a permanent exponential phase, so we need to filter some of the cells from the medium continuously in order to maintain a constant concentration at this phase. To ensure that we are not losing part of the protein produced, we need a filter with the pores small enough to capture cells but no macromolecules. The final system without including feed streams and other features is shown in Figure 11:

Figure 11. Biorreactor scheme for the cell-containing flows. Made in BioRender

Considering that the volume of the reactor does not change through time, and that the biomass filtered doesn’t represent a considerable loss of volume; we can calculate the volumetric flow rate to be filtered with the following equation:

Where:

At the equilibrium, the rate of change in the cells concentration equals 0, so the equation reduces to:

Then we can replace the values of the cell growth constants and the volume of the bioreactor in our design at laboratory scale V = 12L. The value of the volumetric flow rate to be filtered is:

This value will be used in the future design of the bioprocesses and is useful to calculate the amount of production of our proteins during the economic analysis of our proposal.

• Fungal growth inhibition by antifungal proteins

In order to model the inhibition of fungal growth in a liquid medium, we are going to take the following considerations:

• The growth of filamentous fungi in liquid medium follows the kinetics of the Contois equation according to [5]:

  Where:

  X = fungal biomass

  S = amount of limiting substrate

  Kd = death rate of X

  umax = maximum growth rate

  Kc = Contois saturation constant

  mx = specific maintenance demand for substrate

  Y = growth yield (gr biomass/gr substrate)

• The term in the equation corresponding to the consumption of substrate for the production of a product "P" is discarded.
• The antifungal activity of our proteins occurs by contact between the proteins and the fungi, so it is directly proportional to the amount of both.
• The decay of protein activity is of equal probability for all of them, so it is proportional to its amount in the medium.

With all the considerations mentioned above we can define the following 3 differential equations of the system:

Where:

P = amount of antifungal protein

Kp = degradation constant of P

 = Killing constant of X by P

Values for most of the constants for Penicillium are available in [5], excepting Kp, Kd and . For the last 3 constants we will be using arbitrary low values.

The objective of this experiment in the lab is to find the minimum inhibitory concentration (MIC) of the proteins, which means the minimal amount at which fungi can not grow, in other words, that during their population growth curve they never exceed their initial biomass value. In fact, we can perform this experiment in silico thanks to the equations raised above, and observe how the curves will behave so that we can expect the same during experimentation. Assuming an initial fungal biomass concentration of 0.5 OD492, we can run the experiment in matlab at different initial concentrations of antifungal protein, as we can see in Figure 11:

Figure 12: Fungal growth over time in presence of an antifungal protein

We can now easily spot that the MIC in this virtual experiment is 1 uM of antifungal protein. It is important to notice that in the curves of higher concentrations of P, we can see a little boost in the growth of penicillium at 40 hours; this occurs due to the degradation of the antifungal proteins over time. This is where we realize the importance of calculating the half-life of our product, since it allows us to approximate an expiration date and, therefore, the time that our product is capable of protecting fruits and vegetables.

References:

[1] Gillespie, D.T.: ‘Exact stochastic simulation of coupled chemical reactions’, J. Phys. Chem., 1977, 81, (25), pp. 2340–2361 

[2] Daniel Charlebois (2021). Gillespie's Direct Method Stochastic Simulation Algorithm (https://www.mathworks.com/matlabcentral/fileexchange/34151-gillespie-s-direct-method-stochastic-simulation-algorithm), MATLAB Central File Exchange. Retrieved October 17, 2021.

[3] U. Alon, Introduction to systems biology. Boca Raton, FL: Chapman & Hall, 2006, pp. 9-15.

[4] Phue, Je-Nie & Noronha, Santosh & Hattacharyya, Ritabrata & Wolfe, Alan & Shiloach, Joseph. (2005). Glucose metabolism at high density growth of E. coli B and E. coli K: Differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and Northern blot analyses. Biotechnology and bioengineering. 90. 805-20.

[5] Chetan T. Goudar; Keith A. Strevett (1998). Estimating growth kinetics of Penicillium chrysogenum by nonlinear regression. Biochemical Engineering Journal, 1(3), 191–199.

2.2.3 Build and Test

This page details the protocols followed in the biological laboratory to develop our final recombinant antifungal and antioxidant peptides. 

1. Bacterial transformation
2. Peptides PCR
3. Protocol to amplify the peptides ordered to be synthesized. It is based on the PCR Protocol for Phusion® High-Fidelity DNA Polymerase (M0530) by New England BioLabs Inc and the Experiments wiki page by iGEM UPCH (CrioProt) with minor modifications. You can find more information about our protocol here.
4. PCR products purification
5. Protocol to purify the DNA fragments of PCR. It is based on the GeneJet PCR Purification Kit by Thermo ScientificTM and the Experiments wiki page by iGEM UPCH (CrioProt) with minor modifications. You can find more information about our protocol here.
6. Gibson Assembly Protocol
7. This is the protocol for Gibson Assembly using the Gibson Assembly® Cloning Kit (E5510) with minor modifications. More information from NEB can be found here. You can find more information about our protocol here.
8. Bacterial transformation
9. Get started by giving your protocol a name and editing this introduction. It is based on BL21(DE3) Competent Cells from Thermo ScientificTM. More information about our protocol here.
10. Colony PCR Introduction
11. Protocol for performing colony PCR to evaluate the presence of the DNA insert in the bacteria carrying the expression vector. It is based on the Protocol for Phusion® High-Fidelity DNA Polymerase (M0530) and the Experiments wiki page by iGEM UPCH (CrioProt) with minor modifications. More information about our protocol here.
12. Peptides production
13. Protocol for Protein Expression Using BL21(DE3). It is based on the Protein Expression Using BL21(DE3) (C2527) by New England BioLabs Inc and the Experiments wiki page by iGEM UPCH (CrioProt) with minor modifications. More information about our protocol here.
14. Antioxidant activity assay
15. 𝜷-Carotene bleaching assay for antioxidant activity
16. Heat-induced oxidation of an aqueous emulsion system of 𝛽-carotene and linoleic acid was used as the antioxidant activity test model. It is based on the β-Carotene assay revisited. application to characterize and quantify antioxidant and prooxidant activities in a microplate article from PubMed with minor modifications. More information about our protocol here.
17. Antifungicide assay
18. Microorganisms culture
19. Protocol to culture the target fungi (Aspergillus sp., Penicillium sp. and Botrytis cinerea) and the strain of bacteria to be used as the chassis (Escherichia coli BL21 (DE3)). It is based on the Antimicrobial properties of derivatives of the cationic tryptophan-rich hexapeptide PAF26 article from ScienceDirect with minor modifications. More information about our protocol here.
20. Growth inhibition assay
21. Protocol to quantify fungal growth inhibition. It is based on the Antimicrobial properties of derivatives of the cationic tryptophan-rich hexapeptide PAF26 article from ScienceDirect with minor modifications. The antimicrobial activities of the peptides were determined using a microtiter plate assay. More information about our protocol here.
22. Fungicidal and bactericidal activity assay
23. A protocol to assess the peptide's microbicidal activity. It is based on the Antimicrobial properties of derivatives of the cationic tryptophan-rich hexapeptide PAF26 article from ScienceDirect with minor modifications. More information about our protocol here.
24. Chitin binding assay
25. Protocol to test chitin binding domain affinity to fungal chitin. It is based on the In vitro Chitin binding Assay article from Bio-Protocol with minor modifications. More information about our protocol here.
26. Cytotoxicity 
27. MTT Assay Protocol for Cell Viability and Proliferation for cytotoxicity activity
28. MTT assay protocol is used to determine cell viability, proliferation and cytotoxicity by MERCK. This protocol is a colorimetric assay based on the reduction of a yellow tetrazolium salt to purple formazan crystals by the metabolic active cells with minor modifications. More information about our protocol here
29. Medium culture for E.coli recombinant
30. Medium Culture
31. This protocol is to prepare the culture medium for the growth of the modified E.coli inside the bioreactor. It is based on the Recombinant protein expression in Escherichia coli: advances and challenges from Frontiers with minor modifications.  More information about our protocol here
32. Peptides purification
33. Reversed-Phase High-Performance Liquid Chromatography 
34. This protocol is based on Reversed-Phase High-Performance Liquid Chromatography from HPL of peptides and proteins. It involves the separation of molecules on the basis of hydrophobicity. More information about our protocol here
35. Peptides lyophilization
36. Freeze-Drying 
37. This protocol is for the lyophilization of peptides after being purified. It is based on Cryopreservation and Freeze-Drying Protocols. Methods in Molecular Biology. More information about our protocol here

Experiments :

Validating our project design is one of the most important steps in our project. In order to achieve this, we have selected and elaborated the following experiment protocols:

1. Peptide Insertion :

1.1 Peptides PCR (Link to benchling protocol here) [1][2]

1.2 PCR products purification (Link to benchling protocol here) [2][3]

1.3 Gibson Assembly Protocol (Link to benchling protocol here)[4]

1.4 Bacterial transformation (Link to benchling protocol here)[5]

1.5 Colony PCR Introduction(Link to benchling protocol here)[1][2]

1.6 Peptides production(Link to benchling protocol here)[2][5]

1.7 Testing genetic circuits (Link to benchling protocol here)

 2. 𝜷-Carotene bleaching assay for antioxidant activity (Link to benchling protocol here)

  3. Antifungal properties evaluation:

3.1 Microorganisms culture (Link to benchling protocol here)[6]

3.2 Growth inhibition assay (Link to benchling protocol here)[6]

3.3 Fungicidal and bactericidal activity assay (Link to benchling protocol here)[6]

3.4 Chitin binding assay (Link to benchling protocol here)

 4. MTT Assay Protocol for Cell Viability and Proliferation for cytotoxicity activity(Link here)[7]

 5. Medium culture for E.coli recombinant (Link here)

 6.  Peptides purification (Link here)

 7. Peptides lyophilization (Link here)

2.3. Human Practices

Theory of change - User Profile (before interviews)

As part of one of the applied design methodologies, Theory of Change, we evaluated the potential consumer profile. This would allow us to identify the situation, as well as the potential problems that we could solve.

First, we select our main stakeholder: Peruvian fruit and vegetable distribution companies (For more information about the process we follow to choose the stakeholder, review section 2.1). From this, we create a supposed profile of this type of company to empathize with the needs and experiences that this user has: Fruit distribution company "Frutiverso" with 10 years of experience in the market and headquartered in Cuzco, Peru. To begin with the idea proposals, it was necessary to review what the current goals of this company are: to be the leading company in the distribution of fruits and vegetables in Peru and to increase its economic income. We also had to delve into the current problems of this client, among which we highlight: economic losses for different reasons, distribution of products in poor condition and look bad with the company and/or buyers. From this we obtained information on how these types of users spend their time: looking for new ways to reduce economic losses, improving the company to provide better service and better products, and the logistics and organization of distribution for their products. Finally, we selected two main needs that are more likely to be solved and generate a greater impact on the user: offering a quality service (ensuring that the product arrives in optimal condition), and reducing economic losses due to products in poor condition. Here we highlight the importance of the famous phrase "falling in love with the problem and not with the solution" because the generation of assumptions was made before brainstorming ideas.

Validation of assumptions about needs and problems

To begin with the validation of the problem that represents a local issue, different questions were elaborated to initiate the interviews. Two models were proposed: one for farmers and the other for agro-export companies.

Farmers interview:

The objective was to find out if they had knowledge about the applications of biotechnology in agriculture. Likewise, we were interested in knowing the transport conditions of the fruits after harvest, if they had losses and what strategies or products they used to avoid damage to the fruit. In addition, we prepared a pitch about our solution explaining how it can extend the life of fruits and vegetables, within which we emphasized that our product is derived from a microorganism modified with synthetic biology. From this. we devised the following list of questions:

• What's the first thing that comes to mind when you hear biotech?
• What are the conditions of transfer of the product?
• What are the conditions for the transfer of your product?
• Do they leak their products before they are distributed?
• Do you know of any product to prevent fruits and vegetables from spoiling?
• What do you think about making a bacterium produce what you want?
• What do you think about a product that can extend the shelf life of fruits and vegetables?
• What questions do you have about a product of this type?

Agro-export companies interview:

The objective was to find out if they had knowledge about the applications of biotechnology in food security. Likewise, we were interested in knowing the transport conditions of the fruits and the methods used to preserve fruits for long periods of time. In addition, we prepared a pitch about our solution explaining how it can extend the life of fruits and vegetables. From this we devised the following list of questions:

• What are the regulations by country? 
• Are they related to the details of the patents?
• What legislation does SENASA propose?
• How much extension of shelflife is there? Can it be differential?
• Is there a synergy with similar methods or do they apply a cold chain?

Interpretation of the interviews:

Farmers:

In July of this year, we travelled to the province of Cañete, where the main economic activity of the inhabitants is agriculture. One of our team members is originally from this province and helped us to meet with a group of villagers who were dedicated to the cultivation of fruits such as apples, grapes, cucumbers, strawberries and blueberries.

We were able to speak with 12 villagers, 100% of them had more than 30 years of experience as farmers, and their families had been engaged in this activity for generations. After the interview, the following suggestions and queries were obtained:

• 100% of them had losses at the time of transferring the product, this involves the harvest, the selection of the most suitable fruits and the transport of the crates to the fruit market.
• Losses represented 15-20% of the total harvest, the fruits that were larger were those that suffered the most damage at the time of transfer.
• This fact affected the total kg of fruits suitable for the market and therefore, total profit from the sale since the payment is made per kg; In addition, the fruits with a larger size are those that have a higher price compared to the smallest ones.
• Some have chosen to outsource the harvesting process and receive a single payment for the total kg, which means that all fruits regardless of their size will have the same value. In this way, the buying company is responsible for the loss that may occur at the time of the transfer; however, your final profit turns out to be less.
• Although most had used insecticides or products that improve colour or sweetness, they did not know of any other alternative to avoid the rotting of the fruits; the best strategy was prevention.
• Most of them were not sure about the impact that the product would have on health, since they had the idea that transgenic products were harmful to the human immune system.
• Also, when mentioning that we were going to use a bacterium, most did not understand what the mechanism of action was going to be like; in addition, they thought that this bacterium could contaminate the fruits.
• The term biotechnology gave them the understanding that the production process involves very advanced technology, so the price of the final product was going to be too expensive.
• The idea of ​​being able to extend the lifespan of fruits and avoid their oxidation was something that would help reduce their losses; they were interested in the product but believed that its value was expensive

Based on these suggestions, we implemented an empathy map and did some iterations on the product and the other areas of the project:

• We decided to implement a divulgation strategy about our product, where we explain more about the field of biotechnology, giving synthetic biology as the main focus.
• Explain the existing myths around biotechnology and its applications in different areas, carry out scientific divulgation activities to the public.
• Likewise, explain the mechanism of the product's operation and detail with simple concepts for the different parts in the production process. This includes defining what a bioreactor is, the technique for modifying a microorganism, specifying what our product is made of and how it should be used.

Agro-export companies interview:

Based on a search for potential stakeholders, we were able to contact and interview César Zúñiga Delgado, Director of Manufacturing Integration of the CBC company. Director Delgado has had extensive experience in the field of agro-export of fruits and vegetables in different Latin American countries. Based on the problem presented about the economic losses of this type of company and their probable causes, César explained a little more about how the fruit and vegetable distribution flow worked and confirmed the problem we were addressing: "Vegetables such as onions or chilli peppers are usually transported with refrigeration so as not to spoil". Likewise, he told us that the current solution is used on a recurring basis to transport food between different countries and is usually expensive.

As the interview was conducted after having a better approximation of our solution, the proposal was presented and the following suggestions and queries were obtained:

• An investigation of laws that control its use by countries should be carried out. And in the case of Peru, regulations by SENASA could be further considered. Likewise, in relation to these laws, it was suggested to search for patents for similar products, since one could be found that would impede our distribution, causing the reduction of our market in Latin America and the world.
• It is necessary to determine the exact time our product can extend the food’s shelf life, which would be the main characteristic of the value proposition and the form of presentation to other companies. Likewise, the evaluation of current methods should be considered, such as cold chains, and comparing the different extension times.
• The application to products that have names in different stages must be evaluated: for example green and red chilli, and that represents a great loss with the change of colour in their maturation.
• Evaluation of other competitors such as dehydrated fruits. At this point, it should be noted that the distribution companies have adapted to the problem of having food waste even to the point of changing the version of their product. This would mean that this problem has a huge impact.
• Some considerations about the product itself were: effects of pH on the stability of the peptides because the properties of the water used to dissolve the lyophilized can vary between different places, as well as the pH on the surface of the fruits.
• It is necessary to define whether shelled fruits and vegetables will be used and if so, evaluate the implications for human health.
• As for the selected fruits and vegetables, he suggested adding a control fruit that is hard to spoil to check the effectiveness of the product even in those exceptional cases.
• After explaining a little more about how synthetic biology works, he stated that our solution is innovative and sustainable since there is no chemical synthesis, but we are taking advantage of natural resources. Also, he added that it could be a cheaper alternative to current solutions due to this new way of production, so he would be willing to work with us more closely.

Based on these suggestions, some iterations were made on the product and the other areas of the project:

• The biosafety and bioethics committee was created in MikuyTec and all the probable laws of Peru that would regulate our product were evaluated, and the protocols that SENASA has to combat the same problems with current methods were analyzed.
• As part of the experimental protocols, a way to evaluate the antifungal and antioxidant activity of peptides was added to have an approximate value of the shelf life of fruits and vegetables that it can extend. On the other hand, a bibliographic review was carried out on the other options to keep the products fresh.
• The final target fruits/vegetables are strawberry, tomato and aguaymanto. The latter was chosen as the control indicated by Cesar, a product that hardly spoils when exposed to fungi or due to oxidation.
• It was defined that the project would be designed for products with and without “shells” because the selected peptides are extracted from natural sources, so their effect on humans is almost nonexistent. Likewise, to support this characterization, a specific protocol was added to measure the cytotoxicity of the product in animal cells (in vitro).

We sought out companies that have to deal with the problem we want to solve and got feedback on the ideas planned so far. We scheduled a meeting with AGJ CORPORATION SAC to present this idea. The company emphasized they viewed berry fruits as problematic, since they have a short shelf life after harvest. With this, we could validate the idea about what kind of fruits presented this problem frequently, which indicated that we were on the right track in the design part. The interview also allowed us to learn more about the fruit supply chain in Peru. The most important point is that fruits (especially berries) go through cold processing in which around 10% of fruits are chosen not to be exported after being judged by appearance. Those fruits have to leave the cold chain, after which accelerated ripening occurs. This procedure is found as a competitor for our project, and we present ourselves as an alternative to this method. The representative of the company remained attentive to the proposal, considering it very innovative. A noteworthy point is that the interviewee mentioned that other students had previously approached them with a similar idea to ours, with the use of proteins for the same purpose. Our project differed from theirs in the use of synthetic biology. The interviewee highlighted that the project in question failed due to the large amount of capital they needed in the process. He also gave us a series of recommendations based on the current infrastructure managed by the existing companies that participate in the supply chain. Most companies present gas chambers for the quick spreading of fruit and vegetable preservatives, since they use ethylene in their procedures. That is why we believed that our product could generate a greater impact on the market if it was adapted into gas form. This is the next step we are going to evaluate. He also recommended that we should further investigate the specific needs of fruits that have a cold or hot destination and how our bioactive would respond to these stimuli.

Theory of change - User Profile (after interviews)

After having validated the needs and problems of the user, and received feedback on the solution, we carry out a new analysis on the client's profile. In order to eliminate all erroneous assumptions and add new information from interviews and bibliographic research.

To see more details click here.

In order to better empathize with the profile, the personal information of a supposed CEO of a fruit and vegetable distribution company was again noted. Subsequently, the challenges it faces within the company were evaluated to verify that our product is helping to cover at least one of them. Those that we can highlight are: routine losses during transportation processes, fast ripening of berries after freezing period, large scale freezing of berries is too expensive, preserving the berry's bloom integrity, grape stem fast degradation and fermentation, transportation losses towards the north part of the country and the large working volume to not have losses. It is important to note that the evaluation is being done to a specific user and having a greater amount of details helps us to internalize the problems, to later extrapolate these behaviors to our entire market segment.

On the other hand, in this profile we decided to identify the personal professional goals of this user, which are: Improve their lucrative activity in the local fresh produce market, try to expand towards the international export market and achieve enough economic resources for their retirement. Once we were clear about the problems and objectives of the potential clients, we established the ways of how to help them from our proposal. MikuyTec can potentially slow down the ripening of fruits and vegetables in a more economic way than the freezing option. It is also a more effective way than the coating alternative and is a healthier option than preservants. Additionally, it facilitates the entrance of agroexport companies to the international market by preserving the quality of the products.

Based on this evaluation, we can affirm that our solution will be effective for the main stakeholders: fruit and vegetable distributors and farmers, since it covers different needs in the most efficient way possible. Likewise, it proposes a cheaper and healthier alternative than what is currently used. It should be noted that synthetic biology is an extremely new topic for the companies related to our product and the farmers we interviewed, so we try to share the information in the simplest way possible and compare our proposal with others in terms of positive and negative effects that relate to their behavior. And based on that we can affirm that MikuyTec is the right solution to reduce food waste.

2.4. Integrated Human Practices

2.4.1. Public engagement

Markets

We were looking for a way to reach the people who were most affected by post harvest fruit losses. To do so we aimed to explain what our project is in an illustrative and appealing manner, for which we produced the following infographic. To access the infographic click the following link.

We looked for representative markets where the problem was exemplified clearly. As our first choice we went to the main fruit market located in the Victoria district, in Lima, Peru. This is a wholesaler market that provides supplies for multiple fruit businesses dedicated here in Lima. 

We positioned ourselves inside the market with our printed infographic and observed people’s reaction towards it. It was a rather large poster hence it was hard to miss, we observed who stayed reading it and who showed interest in it. We then further explained our project and advances. From these interactions we could listen to the main issues both customers and sellers faced. We listened and took note of their opinions and recommendations regarding our product. Finally, we were able to collect several surveys reviewing the way in which we communicated the information and if the infographic had been helpful to do so.

FINDINGS:

There were certain questions raised given some people confused our proposal with a new chemical alternative, which is why we realized we must take some points into account to improve the public’s perception of our product.

• The product must be explained simply and clearly. What is it for? Why is it different from damaging chemical alternatives?
• Fruit transport in Peru is a big problem given it increases the overall price of goods, there is a distrust towards new chemical products, and this can be a barrier. In order to surpass this obstacle we must ensure effective communication and well-rounded media coverage.
• Thinking on how to generate trust within the population for our product must be a key point before we start the commercialization stage. 
• Right now we are offering a solution to the problem but we must evaluate what is left to do in order to be the best solution there is. 

Taking people’s recommendations into account, we are trying to improve the way in which we communicate our product, such as portraying the bacteria as a fantasy character to stray away from the damaging chemical product lane. We think selling our product directly to post harvest transport and distribution businesses is the best option to start off until we can resolve our own transport costs.

We identified a generalized fear in the population towards new products, which is why we are aiming to promote science communication and educational initiatives focused on synthetic biology. The first step to create trust is assertive communication. It is essential for the people to know we are producing a safe product.

Workshop for children 

In section 2.3, we were able to evaluate our product with the main stakeholders. However, we consider the proposal review by more people of different age ranges is more important. One of these groups was the 10-12 year old children from the school "3097 Institución educativa la Nación de San Judas Tadeo". As part of the strategies in section 3.7, Arts and Creativity, we found the easiest way to explain the project: through a cartoon coloring story.

In the first part, we try to describe the problem of the waste of fruits and vegetables and empathize with the children that it is a problem that affects them and their families. That is why we chose a scene from everyday life: a chinchilla with rotten food, a situation that we validated with the interventions they gave.

Later, we delve more into the causes of the problem. So they can have a bigger vision of what we are looking to solve. The idea was not to give too many scientific details, though, the same children began to ask more about the microorganisms that attack fruits: fungi, and we had no problem explaining it to them.

In the final part, we decided to add a short concept that represented how synthetic biology would help our project through a working bacterium producing our peptides. From which a large number of doubts arose, which we were able to absolve simply:

• How long do the bacteria that work for you live?
• Do bacteria die?
• Do bacteria or other animals suffer in the process of your project?
• Is the magic powder from MikuyTec those things that E. coli produces?
• Is the name of the bacteria E. coli?
• How many bacteria are needed to produce this dust?
• How do they produce the dust?
• How do they modify E. coli?
• Could they produce other things we need?

With the doubts resolved, the children understood the project better. In addition, they became very interested in synthetic biology and mentioned their own ideas. They enthusiastically requested a continuation of the workshop with a series of future classes. We were in agreement with the idea, also that we are more clear about the topics of interest to the children and with this, we can plan more interesting classes for them.

2.4.2. Interactions and optimization of the design 

Storyboard

After both activities, we made some iterations in the original design of our storyboard, since although it identified that the product would reduce the rotting of the fruits, we did not focus on the factor that differentiates it from the chemical products that are already in the market. Likewise, we must put more emphasis on detailing the mechanism of operation, since people still relate the products produced by synthetic biology as unhealthy.

To see more details click here.

Storybook

In section 2.3, we were able to evaluate our product with the main stakeholders. However, we consider the proposal review by more people of different age ranges is more important. One of these groups was the 10-12 year old children from the school "3097 Institución educativa la Nación de San Judas Tadeo". As part of the strategies in section 3.7, Arts and Creativity, we found the easiest way to explain the project: through a cartoon coloring story.

In the first part, we try to describe the problem of the waste of fruits and vegetables and empathize with the children that it is a problem that affects them and their families. That is why we chose a scene from everyday life: a chinchilla with rotten food, a situation that we validated with the interventions they gave.

Later, we delve more into the causes of the problem. So they can have a bigger vision of what we are looking to solve. The idea was not to give too many scientific details, though, the same children began to ask more about the microorganisms that attack fruits: fungi, and we had no problem explaining it to them.

In the final part, we decided to add a short concept that represented how synthetic biology would help our project through a working bacterium producing our peptides. From which a large number of doubts arose, which we were able to absolve simply:

• How long do the bacteria that work for you live?
• Do bacteria die?
• Do bacteria or other animals suffer in the process of your project?
• Is the magic powder from MikuyTec those things that E. coli produces?
• Is the name of the bacteria E. coli?
• How many bacteria are needed to produce this dust?
• How do they produce the dust?
• How do they modify E. coli?
• Could they produce other things we need?

With the doubts resolved, the children understood the project better. In addition, they became very interested in synthetic biology and mentioned their own ideas. They enthusiastically requested a continuation of the workshop with a series of future classes. We were in agreement with the idea, also that we are more clear about the topics of interest to the children and with this, we can plan more interesting classes for them.

2.5. Impact on the Sustainable Development Goals (SDGs)

#2 End hunger, achieve food security and improve nutrition and promote sustainable agriculture

Our project finds its origins in the global food-waste problem. The main objective is to extend the shelf life of plant products, which constitutes an important amount of the food being wasted.1 Food waste has many contributing factors including poor food management and spoilage at different stages of the food processing chain.2 By tackling one of these problems directly, we can increase the food available for more people on one side as well as increase the income of the producers and thus give them more resources to achieve food security, better nutrition and even a better life.

The way in which we are trying to solve this problem involves using alternative antifungal control and antioxidant molecules which are biodegradable. This creates another alternative to mainstream unsustainable options constituted of non-biodegradable agents that have an important impact on the surrounding ecosystem.3 This is why Mikuytec contributes directly to SDG #2. This impact can be measured by determining the amount of food being saved from spoilage after applying our method comparing it to a control group with several plant products.

#12 Responsible consumption and production

By helping with the reduction of food waste we are indirectly contributing to other SDGs. The spoilage of food reduces the amount of food available and thus requires the use of more landfill area (deforestation), water (used for irrigation), energy (used to keep adequate conditions), and fertilizer (energy use, especially during the Haber-Bosch process). These extra unnecessary activities can be skipped by reducing the amount of food lost that is already accounted for when the production process begins. Our project will also help at the end of the food production chain when the product is already purchased. This is because people tend to buy more than what they need and food will get spoiled. By extending the shelf life of the product we can help buffer this problem providing more time before the product is no longer apt for consumption. We will have to test the degree to which our project helps this area as this is more of a social problem than a biotechnological one.

#13 Take urgent action to combat climate change and its impacts

Finally, food waste has a hidden side effect that most people don’t know about. When food spoils and is not adequately composted (anoxygenic conditions, e.g. inside bags) produces methane which is a strong greenhouse gas(GHG), 25 times worse than CO2.3 Food waste accounts for around a quarter of the global GHG emissions,4 and thus reducing the amount of food being wasted has an important contribution towards fighting climate change. We consider that measuring the amount of GHGs produced by a determined amount of spoiled food can be compared to the amount of food being saved in other posterior studies and thus giving us an important measure of our impact in this area. Our project also contributes to decreasing energy use as some of the methods of keeping products fresh that we try to replace (such as refrigeration) require huge amounts of energy and produce a lot of heat. This is why Mikuytec has a strong contribution not only to food security and responsible production but also to helping stop climate change through one of its biggest contributors, food waste.

3.2. Collaboration

Throughout our time in the Design League, we have collaborated with four iGEM Design League and iGEM Global teams in different manners.

3.2.1 iGEM Panama

We held 5 meetings with the iGEM Panama team. Our collaboration was focused on the design stage and modelling sections of our respective projects. Our objective for the first meeting was to discuss our project ideas and share knowledge, as we had previously noticed that our projects were similar in that both teams aimed to produce antifungal peptides.  

The first couple of meetings consisted of the exchange of antifungal biomolecules databases and papers to help us decide what proteins of interest should be included in our expression cassette. In these meetings, we discussed possible antifungal peptides, enzymes and other options and collectively decided that using a defensin peptide as an antifungal agent would be favorable. Because of our collaboration, we had consolidated what parts to use in our expression system.

In those first meetings, we also shared ideas about the next steps we would have to take after our peptides get expressed, which gave us some insight about the approach we were taking to present our product. This resulted in us deciding we would present our peptides in a freeze-dried, powder form when we transition into commercial use in the future (See 3.3 Entrepreneurship and Innovation).

In our third meeting, as we discussed our respective teams’ progress and gave each other updates, we realized we could help them with their computational modelling. That is why from the fourth meeting and onwards, we shifted the focus to their team and helped them develop a bioinformatic modelling system using the MATLAB tool.

From here on, we were able to mentor them on theoretical systems biology and modelling. For this, we showed and explained a presentation developed by one of our team members about the topic. He also created a MATLAB file to be used as a step-by-step annotated guide for their own team’s model. Both our teams were able to get an understanding of each other’s model, especially after the fifth meeting, in which we went over and gave feedback on the progress they had made on their model by following the guide.

Overall, the collaboration with iGEM Panama benefitted both teams in a critical aspect of our projects: design and computational modelling. We consider that thanks to our communication during the competition we could identify the specific points in which we were lacking knowledge and managed to help each other overcome those obstacles towards the interest of both teams.

3.2.2 Ollin Synbio IPN

We joined with teams Ollin Synbio and UAM in order to create a Biosafety and Biosecurity Evaluation Assessment Group. This initiative was led by the Ollin Synbio team, (Details about the announcement can be seen here) that would provide recommendations in order to ensure the safety and integrity of our respective projects. We held several meetings throughout September to coordinate and structure the points that would be covered and to contact experts in the field that would assess our proposals and provide recommendations in order to ensure the safety and integrity of our respective projects.

We submitted a draft on these topics. and after review, a feedback on our proposals, which contributed to our understanding of the topic and the development of these sections in our safety form and later in our JOGL page. 

3.2.3 FCB-UANL

We joined an initiative led by FCB-UANL and Calgary teams to create a storybook for kids that explained the problems each team was trying to solve in an educational manner (the final storybook can be viewed here.). As part of this effort, we created an illustrated short story explaining our project by exposing the role of fungi on fruits and vegetables spoilage. This collaboration was a big resource to develop every participating team’s Human Practices, Arts and Education areas.

For our story, we tried to give our own personal approach to our project through the design of the characters (Tamilla the Chinchilla, among others) and the illustrations (hand-drawn by our art team). Our objective was to share our vision about the food waste problem and help children understand our project from a simpler perspective. We used this short story as part of one of our activities in the school IE 3097 la Nación de San Judas Tadeo.(More details about these activities can be consulted in 3.4 Education and Science Communication).

While Spanish is the most widely spoken language in Peru, there are several other native languages that are extended throughout the country. We consider that language shouldn’t be a limitation in order to share knowledge about synthetic biology and biotech, and that it would be helpful if this short-story could be shared to as many people as possible. So we made efforts to make our short story available in Spanish and Quechua (Each version can be accessed here) and we are, at the time being, looking for someone to do the translation to Aymara.

3.2.4 iGEM IXORA

Similar to our collaboration with iGEM Panama, we had the opportunity to mentor team iGEM IXORA with the help of P(D)PANA. We had three meetings, the first in which we shared our project ideas and went over the details of their project proposal. This meeting allowed us to pinpoint specific topics that could be covered in a future mentoring session, which we agreed to discuss in the next meeting. In the second meeting, we went more in-depth into giving feedback about their plans for a genetic construct and experimental design. However, we still had more to go over in terms of their circuit implementation. We met one more time to solve these doubts, and in this third session we finally were able to figure out a method to accomplish what they were looking for and chose the gene to be knocked out as well as the one to be enhanced in their cyanobacteria. Throughout the competition, we kept up communication with them through chatting applications to assist them and review the progress on each project iteration.

3.2.5 Synthetic Biology Course

As a joint effort between many iGEM Design League teams (Synthetic Biobots, iGEM Bolivia, Ollin Synbio IPN, iGEM UAM, iGEM UNAM), and some iGEM Global current and past teams, we organized and promoted a month-long synthetic biology course “Reaching a New World with Synthetic Biology”. The course featured 12 different experts in synthetic biology fields, such as Drew Endy and Pamela Silver, and all of the funds obtained from the entrance tickets went towards every iGEM Design League organizing team’s competition inscription fee. The event details can be found here.

As organizing teams, we were in charge of promoting the inscription to the course so more people could attend. We published posts and Instagram stories as well as carried activities such as bingos and lotteries to incentivize as many people to get a ticket as we could. The funds obtained would be divided equally between every team regardless of the amount of people each team could reach, thus forming a true collaboration between everybody towards a common goal: affording iGEM inscription. 

3.2.6 Other collaborations & meetups

In addition to the collaborations described above, we also participated in meetups and initiatives organized by other iGEM teams:

• MycoExpo: Organized by iGEM IISER Thiruvananthapuram, MycoExpo is a photo exhibition to showcase fungi diversity. We documented and photographed fungi grown in a wet chamber and sent the pictures to the team for their use in the showcase. Details can be seen on the IISER team page.

• Meetups: We participated in the meetups organized by teams Ollin Synbio IPN and Biotech-EC, in which many teams from Design League shared our projects, personal experiences and got to know each other for future collaboration opportunities.
• We participated in Fire Detective game day, an activity organized by iGEM FCB-UANL to test a board game they designed centered around their problem.
• Some of our members attended Women in STEM -diversity and inclusion workshop- a workshop about women's implication in STEM through history, organized by teams iGEM Concordia, ULaval, Patras and Thessaloniki

3.3. Entrepreneurship and Innovation

In MikuyTec, we plan on extending our project beyond iGEM. This is why we decided to start looking into the development of a start-up to take our idea into the real-world market. To achieve this, we have consolidated our business model through the following activities:

Participation in iGEM EPIC

We participated in both the Venture Creation Lab and Mentorship Programme organized by the iGEM Entrepreneurship Program Innovation Community (iGEM EPIC) earlier this year. We were selected from a range of candidates to attend the workshops and networking opportunities organized by EPIC for the VCL program and spent a couple of months iterating our business model and getting feedback and advice from experts in the Mentorship programme. More information on each program can be found below.

Venture Creation Labs (VCL) : 

The VCL programme consisted of 3 weeks of workshops and lectures, in which at the end of each week a deliverable was due.

Week 1 deliverables required us to define our value proposition, market analysis and lean business model canvas, as well as a script for a possible future interview to a customer in order to refine our product. This helped us consolidate our business idea as well as settle in a specific market. 

Week 2 deliverables included development of a Gantt chart, defining objective Key Results and elaborating a Skills and Gaps Analysis. These allowed us to pay attention to our product development roadmap and the steps we needed to take in order to appropriately start the company. Even though the Gantt chart was not as detailed back then, it served as a base for making a better, updated version of it later (see Gantt section below).

Finally, for Week 3 we needed to have a pitch deck ready for the Demo Day. We pitched with 30 other teams from around the world in front of judges like Jun Axup of IndieBio, Darren Cooke of Berkeley Skydeck and Francia Navarrete of Protera. Unfortunately, we did not win any medals, as this was the first version of our pitch, even before we had a good idea of the biology behind our project. However, we still had the opportunity to go into the Mentorship Programme in which we got assigned mentors to further develop our idea.

You can find all of our work and deliverables for the VCL programme here.

iGEM EPIC Mentorship Programme:

After the 3 weeks of preparation in the VCL program, the iGEM EPIC assigned us 2 mentors, during 2 months approximately, to guide the next steps of the project: Mona Oliveira and Minerva Castellanos. 

With our first mentor, we iterated the pitch deck. We elaborated the first draft of the Business Model Canvas and the financial plan, the local and global market analysis, competitors analysis, MikuyTec distribution, and the manufacturing process of the product. With Minerva we organized the activities to participate in iGEM Design League and received feedback to iterate our pitch presentation. Finally, we did a thorough review of different funds that we could apply to.

 First pitch version

Second pitch version

Market analysis

According to the October 2020 Exterior Commerce Monthly Report by MINCETUR, there are over 1900 agro-export companies within Peru and 91.3% of them revolve around fruits and vegetable exports. This translates into a US $3.5 Billion dollar market (MIDAGRI: Agroexportaciones crecieron 26% en junio y en primer semestre del año ventas llegaron a US $3,480 millones, 2021). As MikuyTec is already working on the refinement process of the product with local distributor companies, we plan to capture 15% of the local market in the following year after its official release date.

Our main competitors are traditional servants, the use of industrial freezing and protective coatings. However, these preservation methodologies have numerous disadvantages when compared to that of MikuyTec. Firstly, MikuyTec does not have long-term repercussions on the consumer’s health(Traditional chemical preservatives); secondly, it is an economically viable solution, as the raw materials needed for the bacterial cultures to grow are much less expensive than the freezing of several thousand tonnes of fruits. Finally, as minutes interact mainly with metabolism mechanisms, it does not wear off as a protective coating would. Furthermore, according to the CEO of AGJ corporation (Peruvian distribution company), some companies have already tried the protective coatings alternative and they found mainly problems and inefficiency in them due to the extremely large volumes of food they work with. Regarding this, a new opportunity opens in the local market, as traditional solutions are expensive and new ones have failed until now.

Business Model Canvas

Once the problematic and the market was identified, we started developing our Business Canvas Model(BCV). Through this process, we passed through 3 iterations of a BCM:

Model 1: Traditional Business Model Canvas

The first model helped us define for the first time the key activities required for our project to generate revenue, as well as our key partners and cost structure. This iteration was particularly helpful to realize a lot of the costs that are going to be involved in bringing this idea to reality, as well as pushed us to start working on developing our distribution channels, such as becoming more prevalent in social media and the creation of a website (it’s a work in progress).

To see more details click here.

Model 2: Sustainable Business Model Canvas

Subsequently, we decided to fill out a sustainable business model canvas chart to help us take into consideration some of the eco-social concerns present if we were to establish our project as a startup company. We realized we would need to take special measures to make sure our environmental impact and carbon footprint is kept in check as well as think of a strategy to reduce waste in our production process.

To see more details click here.

Model 3: Lean Business Model Canvas

The design of the Lean Business Model Canvas (LBMC) was done with the focus of solving the problems at hand in a way that meets the demands of the stakeholders. We also designed it in a way that can be easily applied by them so the product can be brought to market.

To see more details click here.

Market

Customer profile 1:

To see more details click here.

At the beginning of our project, we had this image of our principal customer. We defined our customer as the whole company, which we called “Frutiverso”. This is a popular distributor company in Peru. Here, we made a simple description of what the company needed and what they wanted. These are the points that would lead us to present our product firstly as a powder that could be dissolved for easy integration into their supply chain. As we could see later, this option was not the best solution and was changed. Also, we realized that their most important objective was having a good company image in front of their clients and their consumers. This “challenge” for them was strongly related to two more challenges: distributing products in poor conditions and economic losses. These are the points where the use of our product shines.

Customer profile 2:

To see more details click here.

For the second iteration of the customer profile, we defined an actual person, which is the CEO of the company. By doing this, we are adding more layers to this profile, based on their real needs and concerns. We focused on the CEO because in Peru the normal approach to companies is through them. Furthermore, the meetings with the CEOs of the companies interviewed allowed us to determine the information needed for a more precise customer profile.

Pitch 

It is said that the pitch is everything for a startup, as it defines whether or not they are going to get the investors’ attention in this competitive environment. Creating and delivering a good, inspiring pitch is key to achieve a startup’s goal of raising capital and scaling up. After many iterations, we have decided that this pitch and this presentation is our chance to succeed.

Start-up company

Gantt Diagram

To help our stakeholders and investors see how our team is doing in the project, we have designed this Gantt. We have worked hard to make this a realistic timeline so we can complete every step of our project as efficiently as possible. Here you will be able to find how long each step of our project will take, and an approximation of the operating costs and raised funds that we will find along the way. 

As you will see, we have envisioned the key results and expertise needed for each one of the tasks at hand until the end of 2025. We have planned to revise and update this Gantt each 2 to 3 months in an executive meeting where all the team, our advisors and stakeholders are present. You can find the full document here.

Budget

Based on the calculations made through mathematical modelling in the bioprocess part, it was possible to roughly determine the number of resources that will be used when peptides are produced on a large scale. We were also able to do the first draft of the budget. It contains the expenses due to the purchase of equipment, laboratory materials, research work, mentoring, marketing, salaries and other operating expenses. Likewise, the initial investment and future sales that were predicted based on the previous market analysis were considered. In this way, we were able to evaluate the viability and sustainability of the company in a 5-year cash flow, in which we had positive income from the second year in October. To see the complete budget click here.

3.4. Education and science communication

Infographics and exposure to the general public

We believed that public exposure would be the best way to communicate our product at the places where it will be more useful. Following this thinking, we went to several crowded markets like the Victoria Fruit Market to explain our project and its importance to the general public. We obtained a lot of feedback for the product through surveys, and we were able to identify what the fears of the population and their concerns are regarding the product. This helped us to identify better ways to communicate our product concept for future public exhibitions.

As we carried out more exhibitions, we improved our communication techniques allowing people to understand more about our product. In that way, we could explain something relatively complex such as synthetic biology to citizens without academic training, at least in its basic concepts, which for us was a form of democratization of knowledge.

The material is aimed at farmers, marketers and distributors of fruits and vegetables in markets of Lima Peru. Through this material they will be able to obtain general information about our product and then give their opinion and dialogue with us exhibitors. From here we can clarify their questions regarding the product, or respond to their concerns about the application of this new product which uses synthetic biology.

We sought to hold exhibitions in various places so that more people can be exposed to our project. We think this helped people realize that there are a lot of young people in Peru who want to solve real problems through first-rate technology, which through their help, opinions and contributions will help us get closer to a new era of agribusiness.

Frutifridays

As the goal of our project is to reduce the food waste of our country and promote the local production of fruits and vegetables, we wanted to produce an educational initiative that allowed people to know more about our fruit and vegetable diversity. We did this by posting weekly summaries of facts about some of the most important agricultural products in Peru, many having a South American origin, on our Instagram page. These posts were designed to be visually appealing while providing relevant information in an easy-to-digest format.

The material is educational and easy to share in networks so that it reaches all audiences, it is designed for all people interested to learn more about our Peruvian flora. Through the comments made on social networks, we seek to improve the posts, shorten them for accessability and make communication more efficient. We have accessed the public of all ages, which is recorded in the statistics of our networks such as Facebook and also residents of most of the regions of our country.

Bioscience symposium

This activity brought together researchers of different areas in biosciences, each speaker made short presentations on their area of expertise. The activity was open to the general public for two days so that everyone could participate and listen to the talks on various scientific subjects. In addition to this, we also offered certificates of participation along with the AIChE organization. The money collected allowed us to fund the inscription costs to iGEM for the MikuyTEC team. Around 35 people entered the symposium, and 15 chose to obtain a certificate. They tried to make it simple and clear so that anyone with basic knowledge could understand most of the content without problems. After the event, the team gave a certificate to the speakers as gratitude for their participation in the symposium.

Children’s workshop and storybook

Long before iGEM we believed that synthetic biology should be taught freely from our first years of school, nevertheless it was really difficult for us to think of a way to teach this age group given the topics can be complicated to understand. Inspired by the experience some of our team members had had when volunteering with children in different educational activities, we designed the workshop: “El ABC de la biología sintética” for children 10 - 12 years old from the school “Institución Educativa 3097: la Nación de San Judas Tadeo”. The objective was to explain basic synthetic biology concepts and the simpler aspects of our project. 

In the first part of the activity, through a Google slides presentation, we focused on how synthetic biology can allow us to change the colors of plants. The main strategy we used to interact was making questions to capture the attention of the kids. For example: Which colour would you like the tree to be? Which other things do you think we can change? Other aspects we analyzed were the ethical implications of modifying other living beings such as insects. Lastly, we asked some questions to arouse the curiosity of the children: What is the difference between us and a rock? Why are we living beings? What is a living being? Most were easily answered by them because they already knew the majority of these basic concepts. We started focusing more on synthetic biology itself, asking questions such as: What is DNA? How are genetic modifications made? What is a GMO? Do these exist in real life? Is this science fiction?

In the second part, we explained the main applications of synthetic biology. For instance, the use of GMOs to generate the color change of a fruit. This brings up several other questions, going from the security of eating this kind of food to the contributions other modifications could have. For example, making new apples with higher nutritional content. Furthermore, a discussion that brought up our attention was their interest in knowing where this technology is being made. We explained the restrictions there are in Peru that prohibit GMOs and we were happily surprised to see that they firmly believe there should be no restrictions to this type of science because we can improve things for the benefit of society. Another example was the use of modified bacterias for ecosystem remediation. In this part, the children added new proposals like the purification of the Rímac river, a Peruvian river, or other places like the Amazon rainforest. They said it could also help with recycling and plastic waste.

In the last part, we explained the way synthetic biology can make science fiction real through genetic modifications. This generated some new questions about modifying the human genome to avoid the aging of grandparents, curing illnesses such as cancer. Finally, we showed the storybook with our illustrations to tell them about the problem we are trying to solve and our project. Thanks to the clear drawings used, children could understand what we are doing in MikuyTec and ask for more information and details about the production of our peptides.

The main results of this workshop were shown by the last questions and opinions: “When are we going to have another workshop like this? I want to be a scientist! How can I study your career? I like these classes! You teach science so well!” Based on this opinion, we believe we can create a big impact on society through synthetic biology, but it is time to implement more activities like this with more people, even if they are not the main stakeholders of the project.

Agrotalks

To improve our project and help other initiatives of scientific diffusion, we decided to talk with agro experts to share their experiences and opinions about our project.

Clemente Huachan - Agricultural engineering of Universidad Agraria La Molina

Principal project: Radar is a podcast to interview different scientific experts with the easiest language to achieve a wide audience. It is mostly related to rural agriculture.

Research: The bioremediation of soil with Cadmium based on sunflower plants.

Inspiration: He recognized that there is a lot of misinformation about science, and there are a lot of things to do in the rural areas of Peru.

Problems to research in Peru: More economic help for research in university and the public and private management.

Biotechnology: It would be the solution for climate change, for example: creating drought-resistant plants.

Opinion on MikuyTec public acceptance: It seems difficult because of the GMOs moratorium, but it is not impossible.

Karim Salazar - Environmental engineering of Universidad Agraria La Molina

Principal project: Tachyon Peru is a company that was born to provide an innovative solution to problems in crops, food contamination and low productivity on farms due to pests. For her, it was a bit difficult to have a start-up in Peru due to the lack of resources.

Biotechnology: Students need great economic support to develop this kind of project.

Opinion about MikuyTec: There are a lot of impediments when we are talking about transgenics. You first have to convince experts related to agriculture about biotechnology. The implementation of new members from different areas to have a multidisciplinary project is important.

After the interview, the video was edited and posted on MikuyTec Instagram since it was the easiest way to reach a large number of people. The impact of this initiative was measured through the number of views and the content viewers.

Vocational talks for high school students

The MikuyTec team and Kunay organized a vocational talk about biological engineering. The workshop had two parts, with Miguel Camacho directing the first part. He is a Peruvian researcher working in biotechnology in France. Miguel taught about the profiles necessary for the career of biotechnology, along with his experience and some tips. The attendees, who were around 12 people, asked the speaker different questions. The second section of the talk complemented the ideas developed in the first part, which consisted of a practical workshop where the participates would experience an introduction to genetic circuits and observe their operation. For this, we created interactive pieces to cut out and teach them how to use them with a simulator. The pieces have the modular design of the components of synthetic biology and thus help you better understand the term. We shared the material so that they could do it for themselves and experiment.

Past activities

Mikuytec’s activities started long before the beginning of the iGEM Design League competition. Our iGEM team was founded in 2018 with the aim of democratizing knowledge in synthetic biology and spreading the value of biotechnology all over the country. With this in mind, it was that we decided to launch the first completely free theoretical/experimental course on synthetic biology in Peru, with the help of Joan Campaña (FabLab) and Fabricio Espinoza (BioUTEC) we officially started our journey to join the iGEM world. This course was taught by us every year until 2020, with many improvements and growing in public more and more.

At the end of 2020 we had the opportunity to make a huge change in the educational field in Peru when one of our members was contacted by MAB learning, an education company that works with students in urban and rural areas. He was able to update the national school curriculum of the biology course and we supported him in developing a new chapter that introduces high school seniors to the world of genetic engineering and synthetic biology. Now, more than 20,000 students can learn about biotechnology and see the incredible applications it has to solve the problems of today's world.

3.5. Policy, biosafety and/or biosecurity.

To cover details in this section, we looked at the regulatory framework related to our product. Similarly, an assessment of biosafety procedures and possible risks in design and experimental stages, as well as the ones that would come related to the public and environmental safety once the product is released were performed.

3.5.1 Law and regulation

In Peru, the use and development of Genetically Modified Organisms (GMO) is covered under law number 29811, which follows the guidelines established by the Cartagena Protocol on Biosafety. This law forbids the release of any GMO in the environment. Exceptions to this law are the use of GMOs for research, veterinarian or pharmaceutical products, and food processing. In order to comply with these regulations, we made several modifications throughout our design process to constrain the limit the use of GMOs and to avoid their release. Therefore, for our final design we considered limiting the use of them (our modified E. Coli) to only grow and express our peptides inside a bioreactor, which will be then extracted and purified. This way, we would ensure to release only the isolated peptides as a product.

3.5.2 Safety Assessment

3.5.2.1 Design Stage

Regarding our peptides: PAF26, Rs-AFP2, and PAFP-S, which we chose for their antifungal activity, are all plant-derived. Rs-AFP2 coming from Raphanus sativus, PAFP-S from Phytolacca americana and PAF26, a synthetic peptide. As for our intended antioxidant peptides, VS14 is a protein obtained from tuna backbone, and NOP-1 is a synthetic peptide based on Arabidopsis thaliana. All these are innocuous to humans, and in the case of the antifungal ones, are highly selective toward certain filamentous fungi such as Botrytis Cinerea and others. As these components are on its majority well-characterized, and none of our peptides have known pathogenic activity, nor can cause disease to humans or animals, we have classified them into Risk Group 1, for which we consider working in a BSL-1 lab.

3.5.2.2 Experimental Stage

We considered risks related to the protocols we would be using during our experimental process. We also identified certain risks related to the use of CaCl2 and Triton X-100. According to the CaCl2 datasheet, misuse or mishandling can cause chemical burns and severe ocular irritation. For this we considered the use of safety gloves and goggles in our protocols. Additionally, it is further recommended to use a protective mask as well, in case of inhalation of the substance. On the other hand, Triton 100-X, which has heavy oral toxicology implications and its inadequate disposal represents a threat to marine life. As with CaCl2, its safety measures include the use of protective gloves and goggles.

Another part of our experimental stage comprises the use of a bioreactor. Possible health risks that are involved in the use of bioreactors are divided into two parts. Firstly, we have the risks of using a mechanical machine that requires an electricity source; such as the risk of electric shock, ruptures due to excess pressure, wear of parts, or exposed hot areas. We can reduce the risks with the correct training in risk prevention and the proper use of Personal Protective Equipment (PPE). Secondly, there are biological risks when working with E. coli, there is the probability of contact with the personnel and possible infection with a genetically modified strain. To reduce the risks, the parts and the interior of the bioreactor must be autoclaved during the periods when it is not in operation, in addition to having qualified personnel work with adequate biosafety and biocontainment measures. It is therefore extremely important to tightly regulate bioreactor cleaning procedures to prevent uncontrolled batch contamination and maintain product purity.

3.5.2.3 Public and Environmental safety

Regarding public safety, while we know that individually neither of these peptides is harmful or capable of colonizing the gut of human beings, we are not certain of side effects that may result from their direct consumption. Considering these risks to team members and the public, we would conduct in vitro and in vivo tests to identify potential side effects of consuming our peptides in order to avoid risks from malicious misuse of our product.

While we will try our best in order to avoid the release of our modified E. Coli, in the event of an unintended release, gene transference may benefit certain strains with antagonistic activity with the fungi we analyzed. This could potentially affect ecosystem equilibrium in unexpected ways. We try to reduce this risk to the minimum by observing handling and manipulation protocols during the transformation, as well as carefully disposing of any residues of extraction and purification processes. In this way, we expect to deliver only the peptide products, limiting usage of modified E. Coli to controlled conditions.

There is also a small concern regarding effects on arthropods (specially marine ones and other not targeted organisms with similar physiology) due to their exoskeleton composition made up of chitin. There is low suspicion that our solution designed to attack microorganism cell walls might interfere with other animals with chitin in their structures but this is just an extension of the possible risks. We are planning to conduct experiments in the future regarding concentration of our molecules in controlled environments with arthropods, specially aquatic ones due to their vulnerability in the scenario of water drainage.

3.5.3 Future activities

As part of our collaboration in the Biosafety and Biosecurity Evaluation Assessment Group (more details of this collaboration can be seen in 3.2 Collaboration) we submitted a draft of our thoughts on the topic for consideration of the teams. The feedback from the experts that formed this committee gave us insights on the shortcomings that our current proposal may have and gave us ideas for improvement on the topic.

It was noted that while we detail possible concerns about the possible release of our modified E. Coli., there is still a lack of detailed physical, chemical or biological procedures for biocontainment of the organisms used in our project. Measures such as the use of an inducible promoter to limit peptide production outside controlled conditions or the introduction of a kill-switch are considered as measures to further reduce the possible effects of unintended release of any modified strain.

Similarly, it was recommended that a detailed proposal for a protocol or methodology that would allow us to characterize the probabilities as well the exact amounts of risks would be of great help to further strengthen our biosafety and biosecurity component.

Finally, we noticed that our project fell into a regulatory void, and there were no specific details on how to handle projects such as ours. Consequently, we thought of producing a detailed proposal for the regulation and risk assessment of recombinant peptides produced using GMOs. This could serve as a guideline that similar projects may follow to fall under national regulations, as well as to inform them of the methods of procedures that could assure a correct handling of biosafety and biosecurity matters.

We consider such a regulation could set a precedent for the development of similar projects in the context of our country, which has a strict moratorium on the production and release of GMO, and help to regulate the application and possible effects of such products on the ecosystem and biodiversity. While we do not intend to establish a definitive method, we expect to deliver an outline that can outline or ease the development of new policies related to this topic and to pave the way to the introduction of biotechnological solutions in the country.

In order to achieve this kind of regulation, we plan to seek further assessment on legislation for recombinant peptide production and distribution, as well as the mechanisms to take our proposal up for evaluation by regulatory entities such as the Environment Ministry (MINAM). We want to do this to refine and structure a proposal according to the standards of national policies.

References:

• [1] Ministerio del Ambiente (2017) Ley que establece la moratoria al ingreso y producción de organismos vivos modificados al territorio nacional por un período de 10 años. https://www.minam.gob.pe/wp-content/uploads/2017/04/Ley-N°-29811.pdf
• [2] Sigma Aldrich (n.d) Calcium Chloride Dihydrate Safety Data sheet. https://www.sigmaaldrich.com/PE/en/sds/mm/1.02382
• [3] Millipore Sigma (n.d) Safety Data Sheet for Triton® X-100 108603. https://www.emdmillipore.com/US/en/product/msds/MDA_CHEM-108603?bd=1

3.6. Diversity and inclusion

3.6.1. Diverse and inclusive team

We know that inequality and discrimination in science are social prejudices that have limited a large percentage of people, that is why Mikuytec as a team has implemented strategies to close down these social gaps. For example, our team member selection process only considered motivation and professional competencies as evaluation criteria. Likewise, to preserve the privacy of each applicant, we avoid requesting personal information, we only focus on requesting information related to the specific requirements of the position. At the end of the process, an anonymous survey was carried out, in order to know the members of Mikuytec. As a result, it was obtained that 58% of the selected applicants reside or come from a different region than Lima, 93% identify as people with hispanic ethnicity, 100% speak Spanish and English, 46.6% speak French, 20% know German and 27 % speak native languages ​​such as Quechua at a basic level, 37% are women and 40% belong to the LGBT+ community. In addition, we discovered that Mikuytec members have different academic backgrounds, including studies in biology, bioengineering, electronic engineering, agro-industrial engineering, genetics and biotechnology. These varied backgrounds allow us to address our problems with a multidisciplinary approach.

3.6.2. Gender Equality

Following the same principle, the election of the leaders of each working committee focused on the percentage of participation and performance during the development of the project. Although 37% of our team, including students and instructors, is made up of women; they lead 60% of the working committees, such as synthetic biology, human practice, marketing, bioprocesses, collaborations and entrepreneurship.

3.6.3. Safe environments

Mikuytec knows that the populations most vulnerable to acts of discrimination are women, the LGBT+ community and people with an ethnic group other than Caucasian. For this reason a safe space was created for each member of our team; especially for our female members and those who belong to the LGBT+ community. In our first meeting, a statute was elaborated in a collaborative way, where it was highlighted as infractions: any lack of respect, physical or psychological aggression, as well as endangering the physical integrity of any member will be sanctioned with direct expulsion from the group. By signing 100% of our team on the document, Mikuytec committed to fostering a culture of respect among its members. This document is available at the following link.

3.6.4. Inclusions in the synthetic biology field

As part of our scientific dissemination initiative "Democratizing Biotechnology", which believes that anyone interested in learning synthetic biology can access it, since gender, sexual orientation, ethnicity, professional trajectory and years of study is not a limitation to learn about this area of ​​science. That is why we carry out workshops on basic concepts of synthetic biology for 5-year-old children, vocational talks for high school students on the Biological Engineering career emphasizing on the area of ​​synthetic biology and a symposium on biosciences and research in Peru and agrotalks, which are a series of interviews that seek to publicize the work of entrepreneurs and researchers in agronomic development.

3.6.5. Activities implemented to foster diversity and inclusion

A general meeting was scheduled where the different points of our research were discussed and various strategies were proposed to improve our research proposal. Each of these meetings was led by a democratically elected moderator. Likewise, all the opinions and contributions of each team member were taken into account in each part of the project development process. In addition to researching and appointing functions in the weekly meetings, Mikuytec focused on creating a recreational space, where all members can live together and create a bond. That is why at the end of each meeting, we designated a space to play or talk about how our week had been.

3.7. Arts and creativity activities and results.

We decided to use arts and creativity to promote the understanding of synthetic biology by making an approachable short printable children’s book. The main goal of the book was to explain our project, the problem it aims to solve, and the scientific concepts involved. The plot is centred around a chinchilla that is struggling with spoiled fruit. The story explains the mechanisms through which fruit gets rotten and explains the basics concepts of synthetic biology pertinent to our proposed solution for the problem. This book is accompanied by coloured digital artwork illustrating the narration, as well as line art versions for the children to colour. This has the objective of incentivizing the children to get involved with the story and further comprehend it.

When creating the story we decided to use animals native to our country, particularly from the Peruvian Andes, as they are not usually represented in this kind of media. We chose the chinchilla as our main character given it consumes fruits and plants, and it is also a cute and appealing animal, further approachable for children. The story was written as a group and had several drafts in order to find the best way to explain the concepts we were trying to portray. We struggled on finding a way to explain fruit degradation and protein synthesis processes in a simple way but were able to do it while maintaining the fantastical and kid-friendly aspect of the story. 

Also, with the aim of making our story accessible to more children, we decided to make different versions of it. The storybook idea started as a collaboration project and was originally written in English, but we decided to develop it further and extend the story, as well as produce translated versions in Spanish and Quechua, two of the most spoken official languages in our country. We also used the storybook in one of our children’s educational workshops and it allowed a much better understanding of the topic. The children had many questions about the characters in the story and the scientific concepts mentioned, it was much easier to explain things with a visual aid and story at hand. You can access the full storybook here.

Examples of the illustrations you can find in the book

Storybook example

Link to our PDF JOGL template: 

https://docs.google.com/document/d/1PlRAL12Nqci-iZso8AhswBr0Ne0Woqc8/edit?usp=sharing&ouid=111097239090418999922&rtpof=true&sd=true