1.1 Project Summary 

Guatemala’s biggest and strongest economic sector is agriculture, positioning itself as one of the world class producers of cardamom, banana, sugar, and coffee. At the same time, 40% of the Guatemalan population lives in poverty and 15% in extreme poverty. Many of these people choose to grow their own food or use crops as a means of income. At igroBio we understand this situation and are convinced that synthetic biology can help a large part of the population. One of the main problems with Guatemalan soil is poor bioavailability of phosphorus. Our project is focused on this issue.

Monocultures such as sugar cane represent a serious problem for biodiversity and soil health; However, sugarcane cultivation is also one of the activities that contributes the most to the gross domestic product in Guatemala and it is the direct source of income for dozens of thousands of families. For this reason, we believe that it is possible to find balance between economic profiting and social responsibility. Solving this problem would reduce the use of fertilizers, protect the health of the soils and make sugar cane cultivation more efficient.

Furthermore, sugar cane production is highly dependent on the phosphorus available in the soil. It drives producers to apply high amounts of fertilizers during the cultivation process, even when the soil is naturally rich in phosphorus. However, the formation of phosphorus complexes that cannot be absorbed or metabolized by plants decreases the efficiency of fertilizers. The continuous advancement of microbiology and genetic engineering techniques creates opportunities to design more efficient strategies for optimal crop development. Under this approach, we propose using phosphorus solubilizing organisms or some of the components involved in the solubilization processes as a substitute for conventional fertilizer. For this reason, we partnered with Ingenio Magdalena, the fourth biggest producer of sugar in the world. We seek to produce a biofertilizer that can help Ingenio Magdalena improve their sugarcane production and also benefit several poor communities they work with by teaching them novel techniques of cultivation.

We propose using a rather wide set of phosphorus solubilizing genes in a genetic circuit to be expressed in an E. coli strain. We will be working with a phytase and a glucose dehydrogenase enzyme, which are involved in different metabolic pathways involved in phosphorus solubility, like acid microenvironment formations, chelation and mineralization. Also, we will incorporate a regulation system for the production of gluconic acid to reduce the risk of over acidification of soil. The system consists of the use of a pH dependent promoter and regulates glucose dehydrogenase transcription using Cas13 and ADAR protein. 

1.2 Promotial Video

1.3 Project Presentation Video

1.4 Education and Communication Infographic

1.5 Team and Attributions

2.1 Design Roadmap.

2.2 Excellence in Biological Engineering Design. 

2.2.1 Standardization.

• asr pH-responsive promoter: Part:BBa_K1231000

The asr promoter is a native promoter from E.coli K-12 which induce gene transcription within an optimal pH range between 5.0-6.0 and has a much lower activity below or above those values (Li et.al, 2020). This promoter works using a two-component regulatory system phoB-phoR in the pho operon. This system is activated under phosphate starvation (Sužiede et.al, 1999

). This part was used previously by the team Northwestern/detectpH - iGEM 2013 to build a biological system to sense pH fluctuations in mouth microbiome.

• P2A peptides

2A peptides are viral sequences first identified in picornaviruses and allows the expression of multiple proteins from a single promoter and ORF. P2A can induce ribosomal skipping during the translation process. This peptide family has been studied for their applications in the production of recombinant proteins (Provost et.al, 2007). Although P2A -like sequences traditionally works in eukaryotic cells, some studies suggest the viability to use this strategy to produce polyproteins in prokaryotic cells (Dechamma, 2007). In addition, the team UESTC Life/Project - iGEM 2013 demonstrated the capacity of P2A and F2A to produce chimeric and cleavage proteins respectively in E. coli.

• T1 terminator Part:BBa_J61048

T1 terminator from E. coli is a transcriptional terminator that causes approximately 98% of termination. This sequence is confirmed by a 64 bp stem-loop and was evaluated by 2018 Hawaii iGEM.

• Plasmid: IDLBB_001751

The pET Flag TEV LIC cloning vector (1L) (Plasmid #29662) is an empty cloning vector for E. coli expression. The backbone has a size of 5340 bp,and has the Kanamycin bacterial resistance with growth temperature of 37°C and FLAG-TEV (N terminal on backbone). It runs under the UBMTA terms and license and was a gift from Scott Gradia.

Map of the backbone plasmid used to create the engineered machine

• Phytase (PhyCB): Part BBa_K912000

The enzyme used in our bioengineered machine was cloned from Citrobacter braakii, a gram-negative bacterium. It breaks down phytic acid into inositol, which results in the release of the phosphate groups on the phytate. In Guatemala, the main source of phosphate in soul is the phytic acid, so the use of this enzyme is convenient for the development of the modified bacteria and its application to improve production yield and promote faster growth (Nicholas, 2012).

(Coban, & Demirci, 2017)

• Glucose dehydrogenase: Part IDLBB_001752

The secretion of gluconic acid is one of the most common mechanisms used by phosphate solubilizing microorganisms. Gluconic acid contributes to mineral phosphate solubilization by acidification of the medium and functions as a chelating agent. Glucose dehydrogenase (GDH) enzymes catalyse the oxidation of glucose to gluconic acid. This process depends on the reduction of NADP. The activity, stability, and ability to regenerate the cofactor (NADPH) of GDH are variable between bacterial species. GDH from Bacillus amyloliquefaciens (GDHBA) constitutes a promising alternative for GDH overexpression in heterologous systems because it has a higher specificity activity than GDH from other Bacillus species, and Kinetic studies indicate that it has a higher affinity toward glucose and NADP. (Pongtharangkul, et al., 2015).

• Regulation system:

Our partners and advisors were concerned about the implications of using a genetically engineered microorganism directly on soils, especially because of the risk of over acidification due to the production of gluconic acid, a condition that would be detrimental for the sugarcane growth and development. For this reason, we devised a regulation system that knocks down the production of gluconic acid. The system is dependent on pH and comprises four molecular components that work collectively to interfere the translation of GDH: a pH-sensitive promoter, dCas13, the adenosine deaminase RNA specific (ADAR), and guide RNA (gRNA). ADAR protein can catalyze the conversion of adenosine (A) to inosine (I), a non-canonical nucleoside that is interpreted by the ribosome as guanosine. A variant mutant of ADAR (ADAR2) is capable of catalyzing the conversion of uridine (U) to cytidine (C), For this reason, once ADAR2 is coupled with a guiding system (such as a CRISPR-Cas system), it can be harnessed to interfere with translation by targeting the introduction of stop codons in mRNA by means of the conversion of C to U. ADAR2 has been coupled with inactive Cas 13 (dCas13) to develop base editors (Abudayyeh & Gootenberg, 2017; Abudayyeh & Gootenberg, 2019). Here, we coupled ADAR2 and a gRNA/dCas13 system to introduce a stop codon in GDH mRNA; this system is regulated by the ASR promoter which is only active under acidic conditions with pHs from 4.5 - 5.0.

The gRNA was designed according to the guidelines of Abudayyeh and Gootenberg, the developers of the base editing systems REPAIR and RESCUE which use ADAR and ADAR1 resp The site of edition (U to C) has a mismatch for the optimum activity ADAR2 and is 22 nucleosides downstream of the gRNA loop that makes the RNA couple to Cas13. The spacer length has a total length of 30 nucleosides.

References

• Coban, H. B., & Demirci, A. (2017). Phytase as a Diet Ingredient: From Microbial Production to Its Applications in Food and Feed Industry. Microbial Production of Food Ingredients and Additives, 33–55. doi:10.1016/b978-0-12-811520-6.00002-7 
• Gradia, S (n. d.) pET Flag TEV LIC cloning vector (1L). Extracted from: https://www.addgene.org/29662/
• Nicholas, G. (2012) Citrobacter braakii phytase. Extracted from: http://parts.igem.org/Part:BBa_K912000. 
• Pongtharangkul, T., Chuekitkumchorn, P., Suwanampa, N. et al. (2015). Kinetic properties and stability of glucose dehydrogenase from Bacillus amyloliquefaciens SB5 and its potential for cofactor regeneration. AMB Expr 5, 68. 
• Li, C., Gao, X., Peng, X., Li, J., Bai, W., Zhong, J., ... & Li, C. (2020). Intelligent microbial cell factory with genetic pH shooting (GPS) for cell self-responsive base/acid regulation. Microbial cell factories, 19(1), 1-13.
• Sužiede˙ liene˙, E., Sužiede˙ lis, K. S., Garbenčiute, V., & Normark, S. (1999). The acid-inducible asr gene in Escherichia coli: transcriptional control by the phoBR operon.

• Provost, E., Rhee, J., & Leach, S. D. (2007). Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos.
• genesis
• ,
• 45(10), 625-629.
• H J Dechamma (2007) Processing of multimer FMD virus VP1-2A protein expressed in E.coli into monomers. Indian Journal of Experimental Biology.



2.2.2 Optimization.

We decided to use GDHs from Bacillus species since our final goal is to use a species from the genus tas our final model. We screened the affinity to phytic acid of two Bacillus GDH variant and we noticed that, although the overall structure of GDH1 from Bacillus amyloliquefaciens and GDH2 from Bacillus megaterium remains the same (video 1), there are key differences between them (video 2).

Genetic circuit of our engineered machine. Solubilization of phosphates starts with phytic acid and produces inositol and phosphates.

2.2.3 Build and Test 

Process to evaluate the feasibility of the design.

The built and test process is well described in "SOP IGEM" document, Documents section. 

2.3 Human Practices 

In order to assess the value, importance and relevance our project will have in the world in which it’ll exist, we met with Ingenio Magdalena two times to discuss several aspects of our design and proposal. Ingenio Magdalena is committed to switch to a biotechnology-based business model, so they’re not only interested in using biotechnology and synthetic biology to solve their issue with phosphate solubilization (which they rank as their first or second first most important problem to solve), but also many other projects (see diagram 1 of this section in the website). In our first meeting, the board of directors of Ingenio Magdalena listed several problems within their plant that could be targeted with synbio (diagram 1.1). 

In order to assess the value, importance and relevance our project will have in the world in which it’ll exist, we met with Ingenio Magdalena two times to discuss several aspects of our design and proposal. Ingenio Magdalena is committed to switch to a biotechnology-based business model, so they’re not only interested in using biotechnology and synthetic biology to solve their issue with phosphate solubilization (which they rank as their first or second first most important problem to solve), but also many other projects (see diagram 1 of this section in the website). In our first meeting, the board of directors of Ingenio Magdalena listed several problems withing their plant that could be targeted with synbio (diagram 1.1).

They also listed their social responsibility (responsabilidad social empresarial) in this exercise (diagram 1.2). Based on this, we decided to approach the problem of phosphate solubilization. 

Stormboard of the first meeting of igroBio and Ingenio Magdalena

2.4. Integrated Human Practices. 

2.5. Impact on the Sustainable Development Goals (SDGs). 

igroBio is working with Ingenio Magalena, which is committed to business sustainability, social and environmental development, so our interest is to benefit the Guatemala’s population. Our project main objective is to develop a tool to aid the part of the population that lives in poverty and extreme poverty and grows their own food or use crops as a means of income. The engineer E. coli can potentially help to grow crops faster and generate a higher production yield. At the same time, our project could reduce the impact of agricultural practices on the environment educating the population about responsible agricultural practices and reducing the contamination of the soil and water sources with excess phosphates from fertilizers. The Sustainable Development Goals of the United Nations’ Agenda 2030 that we are hoping to lend a helping hand are zero hunger (2), quality education (4), responsible consumption and production (12) and climate action (13). We believe that we could greatly improve the quality of life of a great portion of the population. To do this, igroBio is working to develop tools to generate forms to generate quality education in topics regarding sustainable production of food and correct soil management with the use of microorganisms. As we have talked about these issues with Ingenio Magdalena, we are hoping to work together to reach neighboring communities, as a start point, in the near future.

3.1 Improvement of an existing iGEM design. 

3.2 Collaboration.

The activities that were carried out with other teams consisted of meeting virtually to maintain all the biosafety protocols due to Covid-19. In this discussion, both teams presented the progress they had of their proposals and their main ideas for the competition. The team with which this was done was Iktan Kaan´EK (Figure 1). The objective of this collaboration was to give each other feedback about the projects to have new perspectives on the solutions we were generating. It was important for us to hear the opinion of people outside of our team. After listening to each other's project proposals, we move on to the comments. We first wrote them on the Stormboard web application (Figure 2), which is a digital board where multiple persons can write at the same time in sticky notes. Then we talked to each other and gave our opinions about how we thought the other team's project could be improved. The other team in turn did the same to us. Both teams came out very satisfied with new perspectives and new ideas to continue their own project.

Members of both teams in the Zoom call.

The Stormboard web application with the sticky notes while we give each other feedback.

Consent Form to share the screenshots and other materials generated in the meeting for Iktan Kaan´Ek team.

3.3 Entrepreneurship and Innovation. 

For iGEM Design League, we design a genetic circuit to optimize the phosphorus solubilization capacity of soil microorganisms. First, we will evaluate its effectiveness at the laboratory level using E. coli as the expression system. Then, we will analyze the role of the circuit in Bacillus subtilis and microorganisms isolated from agricultural soils in laboratory and greenhouse conditions. Our vision is to use synthetic biology to develop and formulate a safe, effective, and accessible biofertilizer. We will collaborate with a project about the genomic identification of microorganisms isolated from agricultural soils. This information will be relevant to selecting capable microorganisms for the biofertilizer formulation. Using microorganisms as a biofertilizer has potential not only for large producers but also for small farmers, being these two sectors the potential market. So we consider that the idea can be transformed into an entrepreneurial project in the short and long term. 

Stages of the project: turning the idea into an entrepreneurial project

Simplified business model

3.4 Education and science communication. 

One of the biggest challenges today in science education is to beat reluctance to scientific knowledge and false information. People are convinced that money is the key to everything, discarding the needs of the many to satisfy the needs of the few. Improving the general knowledge in topics concerning bioinformatics and microorganisms to aid in the sustainable production of food and correct soil management is of upmost importance to meet sustainable development goals number 4, 2, 12, and 13. To do this, our team participated in the Congress of Biochemistry, Microbiology and Biotechnology of del Valle Guatemala University. To make sure the information we communicated was correct and up to date, our team´s PI and mentor, PhD. Dalia Lau and MsC. Augusto Franco oversaw the conferences. 

PhD. Dalia Lau congress´ conference was titled “Bioinoculants phosphorus solubilizers, keys in sustainable agriculture” and she discussed the importance of bioinoculants and the role that the plant´s microenvironment play in its development. As we are in partnership with Ingenio Magdalena, she also talked about the relationship between our project and how it fits with their interests. Magdalena develops and markets food, agricultural and energy products, committed to business sustainability, social and environmental development, so they´ve been invested in igroBio since the begging. On the other hand, M.Sc. Augusto Franco spoke about his team´s bioentrepreneurship, Balam Noj. He talked about the importance of bioinformatics tools, molecular techniques in the developments of research projects in agrogenesis, metagenomics, bioinformatics and more. Since the igroBio´s project is almost exclusively done vía bioinformatics tools, Augusto talked about the use of said tools in research about synthetic biology. The results were surprinting, close to one hundred participants were counted in both days of the congress for each conference. Giving, ninety-two individual contestants for PhD. Dalia Lau and eighty-three for M.Sc. Augusto Franco. 

Additionally, to congress conferences, igroBio went on 9.49´s radio show “Concepto de Mente” (Mind Concept). 9.49 is the most tuned radio on Guatemala´s younger audience (<35 years old). The two and a half hours talk show hosted topics about educating the general audience in synthetic biology, its uses, and advances on agriculture, what is IGEM and why are we participating, as well as our objectives in the competition and emphasized about being the first team in Guatemala to compete in IGEM. To create a stronger connection with the community, we challenged the talk radio show host to recreate our initial reel´s choreography (with accumulated about 140K views and 4K likes), which he shared it to his almost 50K followers on Instagram. 

3.5 Policy, biosafety and/or biosecurity.

We propose the expression of the circuit in E. coli to evaluate the functionality of the design at the laboratory level. We are aware of the risks that the introduction of exogenous or potentially pathogenic species can cause to the chemical and biological balance in soils. Therefore, in the next phase of the project, we will adapt the circuit to a Bacillus subtilis strain, a bacterium commonly found in soils and considered GRAS. We will make the necessary modifications to avoid the introduction of antibiotic resistance markers or any other gene with a potential pathogenicity risk. As discussed above, we incorporated a mechanism to regulate gluconic acid production into the circuit to reduce the risk associated with soil acidification. Then, the optimized circuit will be expressed in an autochthonous bacterium isolated and identified from sugarcane fields before its greenhouse evaluation. Considering that in Guatemala there is no legislation for the use of biofertilizers in the agricultural industry, our work can serve as a basis for the relevant regulatory institutions to initiate a process of building a regulatory framework for the application of biofertilizers.

3.6 Diversity and inclusion. 

Our team is made up by a variety of professions, giving us the opportunity to look at a problem from various perspectives. In this way we have a distinct set of minds working together for a single objective, create an engineered machine that is capable of solubilizing phosphate.  Our team is characterized by an open mind, we listen and contemplate each of our members ideas and we are proud to announce that three of our members are openly part of the LGBT+ community. We also have a strict no hate policy, so no matter the beliefs and way of life that our members partake, we give them the option to express themselves freely. igroBio also hosts a member who is not currently active in the academic division, and we hope to help and guide her in any way that she might need.

3.7 Arts and creativity activities and results

In the Documents section you will be able to find: 

• Designed Biological Parts
• Safety Form
• Web site Filled Template
• SOP IGEM