References:

[1] EMISSÕES TOTAIS. Sistema de Estimativas de Emissões e Remoções de Gases de Efeito Estufa (SEEG), 2021. Available on: <https://plataforma.seeg.eco.br/total_emission>. Accessed: 18 sep. 2021.

[2] Ding, Y. et al. Nanorg Microbial Factories: Light-Driven Renewable Biochemical Synthesis Using Quantum Dot-Bacteria Nanobiohybrids. Journal of the American Chemical Society, v. 141, n. 26, p. 10272-10282, jun. 2019.

[3] Ninkuu, V. et al. Biochemistry of Terpenes and Recent Advances in Plant Protection. International Journal of Molecular Sciences, v. 22, n. 11, p. 5710, may 2021.

Figure 1. Rainbow Dots’ Infographic

References:

[1] Emissões totais. Sistema de Estimativas de Emissões e Remoções de Gases de Efeito Estufa (SEEG), 2021. Available on: <https://plataforma.seeg.eco.br/total_emission>. Accessed: 21 oct. 2021.

[2] Global CO2 emissions. Our World in Data, 2021. Available on: <https://ourworldindata.org/co2-emissions#global-co2-emissions>. Accessed: 21 oct. 2021.

[3] WOLOWSKI, M. et al. Relatório Temático sobre Polinização, Polinizadores e Produção de Alimentos no Brasil. Plataforma Brasileira de Biodiversidade e Serviços Ecossistêmicos (BPBES), 2019. Available on: <https://www.bpbes.net.br/produto/polinizacao-producao-de-alimentos/>. Accessed: 21 oct. 2021.

Figure 2. Project’s Design Roadmap.

Motivation

We’re already experiencing climate change, including more frequent and more extreme weather events.

IPCC co-author Prof. Ed. Hawkins

   The increased release of CO₂ and other greenhouse gases through anthropogenic actions has led to severe environmental changes felt throughout the entire planet Earth. Meetings between global leaders such as the “Earth Summit in Rio”, held in Rio de Janeiro in 1992, have already discussed for almost 30 years the need to control greenhouse gases (GHG) emissions. In 2015, by signing the Paris agreement, the world's nations pledged to act against the increase of 1.5°C in the planet's temperature by the end of the 21st century^[1].

   However, the report presented by the Intergovernmental Panel on Climate Change (IPCC) this year showed that this purpose will not be achieved in any of the forecasts made and will likely reach or exceed this number by 2040. This result is the most alarming of humankind's history because it predicts the irreversible effects of climate change on the planet^[2].

In Brazil and other countries in South and Central America, average temperatures have likely increased and will continue to increase at rates higher than the global average.

IPCC, 2021

   The major CO₂ emitters in the world are China, the United States, and India, with 27.92%, 14.5%, and 7.17% of global emissions. Brazil ranks 12th on this list, contributing 1.27% of global emissions, followed by Mexico with 1.2%. According to the IPCC document, Brazil and other countries in Latin America will suffer the effects of climate change through the increase in the period of droughts and fires and the relative rise in sea level contributing to coastal flooding, among others.

   In this context, there is an urgent demand for new technologies that allow greenhouse gases uptake, opening up new possibilities for converting abundant molecules such as CO₂ into several valuable products. To make this possible, new artificial photosynthesis technologies have been developed to use renewable sources such as sunlight, an unlimited source of energy, and CO₂ as a building block. This energy source, also denominated as "liquid sunlight" seeks to reach in the future the carbon neutralization values ​​decisive for the maintenance of life as we know it on the planet Earth, contributing directly to closing the carbon cycle^[3].

   Although nature presents several systems for CO₂ fixation through autotrophic organisms, these elegant systems deliver a low efficiency in capturing sunlight energy^[4]. In this way, creating a platform that allows an efficient solar-to-product conversion in a photosynthesis fashion is a crucial step toward net-zero carbon emissions in producing valuable products^[5]. In this way, the CNPEM.Brazil team proposes the combination of efficient semiconductor nanoparticles to capture light, and the power of synthetic biology (Synbio), through genetically engineered microorganisms to create a synergy between the abiotic and the biotic to develop a new generation of microorganisms: the nano-bio-hybrid bacteria. 

   Using nature as inspiration, the project developed in the iGEM Design League is called “Rainbow Dots: the nano-bio-hybrid platform for CO₂ uptake” (Figure 3). A versatile platform that allows the photosensitization of genetically modified microorganisms to consume CO₂ coupled to the biosynthesis of human interest products. The microbial chassis chosen to receive these modifications was the Escherichia coli bacteria due to the large number of Synbio tools already consolidated and also the great biotechnological interest in transforming this organism into autotrophic, demonstrated in several studies^[5,6].

Figure 3. Rainbow Dots platform.

   The Rainbow Dots microorganism photosensitization platform was developed to act as a third-generation biorefinery. As a proof of concept, we developed the Rainbow toolbox (see chapter 2.2.1) using the circuits and genetic devices used in the photosensitization of Escherichia coli. Its tools can be used according to the needs of each organism, as well as its semiconductor nanoparticle and corresponding wavelength. 

   The Rainbow toolbox uses quantum dots (QDs) nanoparticles to harvest electrons from light directly to our CO₂-consuming bacteria. QDs are semiconductor nanoparticles with optical and electronic properties that vary according to their composition. They have been applied to different devices and have attracted attention due to their unique quantum mechanical behavior, influenced by their specific size of energy band gaps and size-dependent optical properties^[5,7]. Toward this Design moment, we modeled the project using the Cadmium sulfide (CdS) quantum dots, especially for their ability to absorb light energy and increase NADH intracellular formation, improving the efficiency of the engineered routes. This increased formation is only possible due to the CdS band-gap for visible-light response, specifically under blue illumination (450 nm).

   Several natural and synthetic metabolic pathways for CO₂ ­fixation were also evaluated according to different criteria (e.g. number of enzymes, ATP requirements, and others) in order to optimize CO₂ uptake coupled to bioproducts synthesis. Next, the CNPEM.Brazil team evaluated several molecules that could meet our society's demands besides contributing to climate action through uptake of CO₂ molecules. In this way, we took into account side effects of climate change, such as the increase in crop pests^[8], and a decline in pollinators' population and performance in the yield of crops^[9]. Hence, as a proof of concept, the Rainbow dots platform proposes the production of terpenes, molecules with a wide range of applications, most essential for plant defense.

Rainbow dots’ solution: Terpenes - molecules for a sustainable and beekeeper agriculture

       Terpenes are a broad class of molecules that can be generated naturally by the secondary metabolism of several organisms, mainly plants, through the junction of isoprenes units (hydrocarbons with unsaturations). Monoterpenes are composed of two isoprene units, being found in abundance in herbs, spices, and fruits. As the main components of essential oils, these volatile molecules are known for their characteristic aromas, being used as precursors of different products in the industry, such as fragrances, pharmaceuticals, food, fuels, and others. Therefore, due to its high applicability, monoterpenes are molecules with high industrial demand and added value^[15,16]. Nonetheless, extracting them from their natural sources can be time-consuming, complex, and inefficient. In addition, these extractions require organic solvents that leave prohibitive residues for consumption, reducing monoterpenes' applicability^[16].

 In this way, the CNPEM.Brazil team decided to demonstrate the production of the monoterpene 1,8-cineole, also known as eucalyptol, through our nano-bio-hybrid microbial platform. The 1,8-cineole mainly attracts Euglossini bees (Hymenoptera: Apidae), found in neotropical areas with a hot and humid climate^[12]. Just in Brazil, more than 200 species are described. One of the main characteristics of these bees is the male's ability to visit more than 30 plant families, including more than 2000 species of orchids^[12], playing an important role in food security and biodiversity maintenance.

   Besides attracting pollinators, 1,8-cineole can also act in Caliroa cerasi L., an insect endemic to the Northern Hemisphere that mainly affects Prunus avium (cherries) trees. Their females are parthenogenetic and insert their eggs on the surface of the plants using a serrated ovipositor, also known as “sawflies”, drying a large area of ​​leaf mass and altering its structure^[14]. Besides, this monoterpene, being a volatile component, can be introduced in a beekeeping context and contribute to the attraction of parasites that control herbivore egg deposition^[14,11]. Therefore, 1,8- cineole was chosen as the platform's bioproduct by its pesticide and pollinator’s attraction properties, enabling the development of sustainable agriculture.

References:

[1] DELISI, C. et al. The Role of Synthetic Biology in Atmospheric Greenhouse Gas Reduction: Prospects and Challenges. BioDesign Research, 2020, p. 1–8, 2020.

[2] IPCC, 2021: Climate Change 2021: The Physical Science Basis. Contribution of Working Group I to the Sixth Assessment Report of the Intergovernmental Panel on Climate Change [Masson-Delmotte, V., P. Zhai, A. Pirani, S.L. Connors, C. Péan, S. Berger, N. Caud, Y. Chen, L. Goldfarb, M.I. Gomis, M. Huang, K. Leitzell, E. Lonnoy, J.B.R. Matthews, T.K. Maycock, T. Waterfield, O. Yelekçi, R. Yu, and B. Zhou (eds.)]. Cambridge University Press. In Press.

[3] YANG, P.. "Liquid Sunlight: The Evolution of Photosynthetic Biohybrids." Nano Lett, 21, p.5453-5456, 2021.

[4] LIU, C. et al. Water splitting–biosynthetic system with CO2 reduction efficiencies exceeding photosynthesis. Science, 352, p.1210-1213, 2016. 

[5] HU, G. et al. Light-driven CO2 sequestration in Escherichia coli to achieve theoretical yield of chemicals. Nature Catalysis 4, p. 395–406, 2021.

[6] GLEIZER, S. et al. Conversion of Escherichia coli to generate all biomass carbon from CO2. Cell, 179, p. 1255-1263.e12, 2019.

[7] CHENG, L. et al. CdS-Based photocatalysts. Energy and Environmental Science,,11, 1362-139, 2018.

[8] BARFORD, E. Crop pests advancing with global warming. Nature (2013). https://doi.org/10.1038/nature.2013.13644

[9] GOULSON, D. et al.  Bee declines driven by combined stress from parasites, pesticides, and lack of flowers. Science, 347, 2015. 

[10] YU, J. et al. Bio-based products from solar energy and carbon dioxide. Trends in Biotechnology, v. 32, p.5-10, 2014.

[11] WOLOWSKI, M. et al. Relatório Temático sobre Polinização, Polinizadores e Produção de Alimentos no Brasil, 2017. Rede Brasileira de Interações Planta- Polinizador. Available in: https://www.bpbes.net.br/wp-content/uploads/2019/03/BPBES_CompletoPolinizacao-2.pdf

[12] Rodrigues, Gleicy et al. ESTUDOS COM EUGLOSSINI (HYMENOPTERA: APIDAE) NO BRASIL: UMA ANÁLISE CIENCIOMÉTRICA DAS ÚLTIMAS CINCO DÉCADAS. Universidade Federal do Oeste do Pará, Santarém, Pará, Brasil, v.15 n.27; p.249, 2018. Available in: https://www.conhecer.org.br/enciclop/2018a/biol/estudos.pdf.

[13] Shmuel Gleizer, Roee Ben-Nissan, Yinon M. Bar-On, Niv Antonovsky, Elad Noor, Yehudit Zohar, Ghil Jona, Eyal Krieger, Melina Shamshoum, Arren Bar-Even, Ron Milo, Conversion of Escherichia coli to Generate All Biomass Carbon from CO2, Cell, Volume 179, Issue 6, p 1255-1263.e12, 2019. ISSN 0092-8674. Available in: https://doi.org/10.1016/j.cell.2019.11.009.

[14] Peschiutta, ML, Scholz, FG, Goldstein, G. et al. A oviposição por insetos herbívoros induz alterações nas propriedades ópticas e mecânicas das folhas de Prunus avium . Arthropod-Plant Interactions 12, 613–622, 2018. Available in:  https://doi.org/10.1007/s11829-018-9609-x.

[15] Bruneton J. Farmacognosia. Fitoquímica Plantas Medicinales. Segunda Edición. Editorial Acribia. 2001

[16] Simões Cláudia Maria. Farmacognosia: do produto natural ao medicamento. Porto Alegre, Artmed, 2017. Available in: https://books.google.com.br/books?hl=pt-BR&lr=&id=uo5vDQAAQBAJ&oi=fnd&pg=PT12&dq=farmacognosia+da+planta+ao+medicamento&ots=1D6Js-grjd&sig=TvUJXDeNdS-BeWqglAQWC6DJ20A#v=onepage&q=farmacognosia%20da%20planta%20ao%20medicamento&f=false.

In this chapter, the CNPEM.Brazil team demonstrates the rational development of the project "Rainbow Dots: the Nano-bio-hybrid platform for CO₂ uptake" through computational tools and the rational design of our proof of concept.

Rainbow dots is a project for everyone!

All software and databases used in our project are free to access.

All scripts and codes used are available in the document section.

• Softwares used: Benchling, Metabolic models: R (BacArena), GapSeq, python (Cobrapy), ESCHER, optflux, CRISPOR, antiSMASH
• Databases: Uniprot, iGEM Registry of standard parts, SynBio Hub, ModelSEED, BigModels, Metacyc

   All biological parts (biobricks) were standardized according to the iGEM Standards for biological parts, devices, or genetic circuits systems in order to ensure the compatibility between all the biobricks used in the Rainbow toolbox (Figure 4). For the standardization of the biobricks, the Benchling software was used to allocate the biobrick prefix and suffix^[1].

   All the biobrick sequences were codon-optimized to Escherichia coli (our chassi), removing EcoRI, XbaI, SpeI, and PstI restriction sites, and adjusting their GC content percentages to the average value (0.33 to 0.66), allowing the parts assembly for creating the composite parts of our circuits and devices.

Figure 4. The Rainbow toolbox.

   The registry's biological parts database^[2] was used to access parts already registered in previous designs. Uniprot^[3], SynBio Hub^[4] and KEGG pathways^[5] were used to obtain the nucleotide sequences of the new standardized biobricks used in the Rainbow toolbox!

References:

[1] Benchling [Biology Software].(2021) . Retrieved from https://benchling.com.

[2] Igem.org. (2021). parts.igem.org. [online] Available at: http://parts.igem.org/Main_Page [Accessed 24 Oct. 2021].

[3] The UniProt Consortium UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 49:D1 (2021)

[4] ACS Synth. Biol. 2018, 7, 2, 682–688. Publication Date: January 9, 2018. https://doi.org/10.1021/acssynbio.7b00403

[5] Genome.jp. (2021). KEGG: Kyoto Encyclopedia of Genes and Genomes. [online] Available at: https://www.genome.jp/kegg/ [Accessed 24 Oct. 2021].

2.2.2.1. Genome-scale metabolic models

   In order to simulate the growth of Escherichia coli in silico, we performed an automatic reconstruction of its genome-scale metabolic model (GEM), using Gapseq software^[1]. The genomic sequence in fasta of the bacteria Escherichia coli DH5α was used to construct the metabolic model. This strain was chosen for this design stage because it is already commonly used for genetic modification at the Brazilian National Center for Research in Energy and Materials (CNPEM).

   For the GEM reconstruction, the software uses a gap-filling algorithm to resolve gaps in the model in order to enable biomass formation on a given culture medium. The medium chosen for our simulations was the M9 medium. After the model reconstruction, the BacArena package in the R software was used to simulate a bacterial culture in silico. We simulated the growth for 13 hours in 10x10 grid cells randomly populated with 2 cells in each grid. The result of the prediction of the microbial growth of Escherichia coli DH5α under these cultivation conditions is shown in Figure 5.

Figure 5. In silico growth simulation of Escherichia coli DH5α in M9 medium consuming glucose as a carbon source for 13 hours using the BacArena R software package.

   Through the prediction by the computational tool, it was possible to obtain essential data on the behavior of our prospective chassi, such as the beginning of the exponential microbial growth phase (approximately 4 hours of cultivation) and the entry into the stationary phase (after 9 hours) under these conditions. Using the BacArena software was also possible to study the consumption and production of metabolites derived from cell growth. Thus, the consumption of the carbon source (D-glucose) and the production of CO₂ were analyzed using the graph in Figure 6.

Figure 6. In silico prediction of D-glucose consumption and CO₂ production during cell growth of the bacteria Escherichia coli during 13-hour cultivation using M9 medium as carbon source.

   Inspired by this tool, our team proposed to edit this metabolic model for the addition of CO₂ fixation pathways in E. coli. We used different tools in order to edit the metabolic model, including the adapt function in Gapseq to add new enzymatic reactions. We also worked with cobrapy in python, optflux, Escher (online software), and manual editing of our metabolic model^[3,4].

   Initially, we sought to add the CO₂ fixation pathways from the Calvin cycle, Wood-Ljundahl, Reverse TCA cycle, and half-Wood–Ljungdahl-formolase (HWLS). For this, we performed joint editing using the adapt function of Gapseq and cobrapy since the first one was not able to add all the necessary pathways, Thus, it was necessary to complete manually all the missing reactions based on the modelSEED, MetaCyc, and BigModels databases.

   Even after extensive efforts using the tools mentioned above, we were not successful in obtaining an E. coli GEM model with a complete functional CO₂ fixation pathway. GEMs models are quite complex, having more than 3000 reactions and 1000 genes. In order to try to overcome this challenge, we attempted to use an E. coli core model containing only main reactions^[5]. Volpers M. and colleagues who carried out these modifications in this type of model showed that the behavior of core models was similar to GEMs for this type of approach^[6]. By editing it with cobrapy and also manually, the addition of the reactions referring to the HWLS (the most efficient CO₂ uptake pathway as described in the next session) pathway was carried out.

   However, in this season we were not able to functionally add this pathway. Using the ESCHER online software, it was possible to plot our edited model with the non-functional HWLS pathway (Figure 7).

Figure 7. Graphic representation of the metabolism of the E.coli core model and the addition of the synthetic way of consumption of CO₂ HWLS (in red) through the ESCHER online software.

   Yet, even facing several issues during the metabolic model edition, the CNPEM.Brazil team emphasizes through these results that we have learned a lot about the applicability of these softwares, which none of the students in our team had the opportunity to work on before. Furthermore, in order to share our learning with the Synbio community, we make all the codes used by our models available on GitHub. The team CNPEM.Brazil is also available for possible suggestions for the construction of our metabolic model and contribution from the iGEM’s synthetic biology community.

References:

[1] Zimmermann, J., Kaleta, C. & Waschina, S. gapseq: informed prediction of bacterial metabolic pathways and reconstruction of accurate metabolic models. Genome Biol 22, 81 (2021). https://doi.org/10.1186/s13059-021-02295-1.

[2] BAUER, Eugen et al. BacArena: Individual-based metabolic modeling of heterogeneous microbes in complex communities. PLoS computational biology, v. 13, n. 5, p. e1005544, 2017.

[3] Isabel Rocha , Paulo Maia , Pedro Evangelista , Paulo Vilaça , Simão Soares , José P Pinto , Jens Nielsen , Kiran R Patil, Eugénio C Ferreira and Miguel Rocha. OptFlux: an open-source software platform for in silico metabolic engineering. BMC Systems Biology 2010, 4:45

[4] Rowe, E., Palsson, B.O. & King, Z.A. Escher-FBA: a web application for interactive flux balance analysis. BMC Syst Biol 12, 84 (2018). https://doi.org/10.1186/s12918-018-0607-5.

[5] Orth JD, Fleming RM, Palsson BØ. Reconstruction and Use of Microbial Metabolic Networks: the Core Escherichia coli Metabolic Model as an Educational Guide. EcoSal Plus. 2010 Sep;4(1). doi: 10.1128/ecosalplus.10.2.1. PMID: 26443778.

[6] VOLPERS, Michael et al. Integrated in silico analysis of pathway designs for synthetic photo-electro-autotrophy. PloS one, v. 11, n. 6, p. e0157851, 2016.

2.2.2.2. Choosing the best CO₂ fixation pathway

   For the engineering of an efficient autotrophic E. coli, several natural and synthetic pathways for CO₂ fixation were evaluated (Table 1). The ideal pathway should attend some principles, like containing a minimal number of enzymes, requiring the lowest amount of ATP and reducing equivalents (e.g. NADH, NADPH), being linear and disconnected from other metabolic pathways, and being thermodynamically favorable^{[1, 2]}. According to this criteria, the Reductive glycine (rGC) and HWLS (Half-Wood–Ljungdahl-formolase) pathways were the best options for composing the Rainbow Dots toolbox.

   Despite the rGC pathway requiring less ATP and cofactors, the engineering of a nano-bio-hybrid E. coli with the HWLS pathway was already experimentally validated^[3]. Furthermore, the previous engineering of an E. coli with the rGC pathway required the use of methanol or formate coupled to CO₂ uptake in the fermentation process^[4], while the association of quantum dots allows the versatile use of light, with adjustments of intensity and wavelengths in any time during the process^[5].

Table 1. Comparisons of natural and synthetic CO₂ fixation pathways.

*NADH = NADPH = FADH2

*3HP-4HB, 3-Hydroxypropionate/4-hydroxybutyrate cycle; DC/HB, dicarboxylate/4-hydroxybutyrate cycle; rGC, reductive glycine pathway; MOG, C4-PyrC-alanine malonyl-CoA-oxaloacetate-glyoxylate cycle; CETCH, crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA; HWLS, Half-Wood–Ljungdahl-formolase pathway.

   In order to improve the carbon flux to the production of 1,8-cineole, several deletions were addressed as proposed by Hu and colleagues^[3] (Figure 8). Also, the addition of carbonic anhydrase from Synechocystis sp. PCC 6803, an enzyme that catalyzes the interconversion between carbon dioxide and water and the dissociated ions of carbonic acid, can enhance the intracellular CO₂ fixation and contribute to 1,8-cineole productivity (Figure 8)^[1].

Figure 8. Schematic representation of Half-Wood–Ljungdahl-formolase (HWLS) pathway (light yellow box) that converges to glycolysis, generating precursors for 1,8-cineole biosynthesis (green box). Reactions with a red “X” indicate deleted reactions to optimize 1,8-cineole production. Co-factors were omitted to improve clarity.

Reference:

[1] CLAASSENS, N. J. et al. Harnessing the power of microbial autotrophy. Nature Reviews Microbiology, 14, p. 692–706, 2016.

[2] COTTON, C.A.R. et al. Renewable methanol and formate as microbial feedstocks. Current Opinion in Biotechnology, 62, p.168–180, 2020.

[3] HU, G. Light-driven CO2 sequestration in Escherichia coli to achieve theoretical yield of chemicals. Nature Catalysis, 4, p.395-406, 2021.

[4] KIM, S. Growth of E. coli on formate and methanol via the reductive glycine pathway. Nature Chemical Biology, 16,538-545, 2020.

[5] CLAASSENS, N. J. et al. Potential of proton-pumping rhodopsins: engineering photosystems into microorganisms. Trends in Biotechnology, 31, p.633-642, 2013.

[6] BASSHAM JA, B.A. and Calvin M. The path of carbon in photosynthesis. Journal of Biological Chemistry, 185, p.781-787, 1950.

[7] SCHUCHMANN, K. and MULLER, V. Autotrophy at the thermodynamic limit of life: a model for energy conservation in acetogenic bacteria. Nature Review Microbiology, 12, 809-821, 2014.

[8] NUNOURA, T. et al. A primordial and reversible TCA cycle in a facultatively chemolithoautotrophic thermophile. Science, 359, p.559-563, 2018.

[9] LODER, A.J. et al. Reaction kinetic analysis of the 3-hydroxypropionate/4-hydroxybutyrate CO2 fixation cycle in extremely thermoacidophilic archaea. Metabolic Engineering, 38, p.446-463, 2016.

[10] HUBER, H. et al. A dicarboxylate/4-hydroxybutyrate autotrophic carbon assimilation cycle in the hyperthermophilic. Archaeum Ignicoccus hospitalis. Proc Natl Acad Sci U S A, 105, p.7851-7856, 2008.

[11] HERTER, S. et al. A bicyclic autotrophic CO2 fixation pathway in Chloroflexus aurantiacus. Journal of Biological Chemistry, 277, p.20277-20283, 2002.

[12] VOLPERS, M. et al. Integrated in silico analysis of pathway designs supporting synthetic photo-electroautotrophy. PLoS ONE, 11, e0157851, 2016.

[13] BAR-EVEN, A. et al. A survey of carbon fixation pathways through a quantitative lens.J. Exp. Bot., 63, p.2325–2342, 2012.

[14] SCHWANDER, T. et al. A synthetic pathway for the fixation of carbon dioxide in vitro. Science, 18, p.900–904, 2016.

2.2.2.3. Optimization of the 1,8-Cineole production using computational tools

   Eucalyptol has antimicrobial activity that is reported in both gram-positive and gram-negative bacteria. This effect occurs due to the compound altering the permeability and function of the cell membrane, inducing intracellular filtration and morphological changes in the microbial cell. Due to this cytotoxicity effect, the E. coli ATCC 700728 strain was chosen as the chassis, which is reported by Martínez-Pabón and colleagues (2020) as a strain resistant to eucalyptol activity^[1].

   For the production of 1,8-cineole, nine enzymes organized in three operons expressed in a single plasmid were used. Among these enzymes, six were traditional in the mevalonate pathway, and contribute to increasing the availability of IPP (isoprenyl diphosphate), which is the substrate for the production of various monoterpenes. Then, the enzymes Isopentenyl diphosphate isomerase, geranyl pyrophosphate synthase, and cineole synthase were used to catalyze the subsequent reactions required to generate 1,8-cineole (Figure 9).

Figure 9. 1,8-Cineole production pathway illustrating the substrates used and the products generated in nine enzyme-catalyzed reactions with the abbreviated nomenclature above the arrows. ACAT: acetyl-CoA acetyltransferase. HGMS: hydroxymethylglutaryl-CoA synthase. HMGR: hydroxymethylglutaryl-CoA reductase. MVK: Mevalonate kinase. PMVK: phosphomevalonate kinase. PMVD: Phosphomevalonate decarboxylase. IDI: Isopentenyl diphosphate isomerase. GPPS: geranyl pyrophosphate synthase. CS: Cineole synthase.

   In E. coli, geranyl pyrophosphate (GPP), the last intermediate in the eucalyptol biosynthetic pathway, is also used for the production of farnesyl pyrophosphate (FPP), which is a precursor for the biosynthesis of undecaprenyl-phosphate (C55-P), an essential lipid that plays a role in transportation of the biosynthesis of peptidoglycan and other cell wall polysaccharide components. Taking this into account, the strategy designed to circumvent this problem was to adopt a Quorum Sensing (QS) based on the Lux system to separate the growth and production phases. With the proposed design, the entire bioproduct pathway will be auto-induced after the log phase onset, making the initial growth less affected by the competition for the intermediary GPP.

   The later production of eucalyptol is also advantageous due to its cytotoxicity effects, which would affect cellular growth at the beginning of cultivation. In addition, the induction of product formation by this system avoids the addition of a costly chemical inducer in the medium, as is the case with IPTG for the Lac promoter. Briefly, the functionality of this type of QS is mainly based on acyl-homoserine-lactone (AHL) molecules, produced by the LuxI protein, and their concentration increases as the population of bacteria grows, being diffusible to the membrane^[2]. When population density reaches the "quorum", these AHL molecules exceed the concentration threshold and are then recognized by proteins called R proteins, such as LuxR. LuxR, when binding to specific AHL molecules, forms a complex, and thus directly regulates the transcription of target genes through control with a promoter inducible by this complex^[2].

Initial design circuit

   The initial circuit design for eucalyptol production was based on the work of Mendez-Perez D. and colleagues, who performed numerous tests with the mevalonate pathway as the production pathway of 1,8-cineole^[3]. Different strategies, such as exchanging promoters, dividing genes into operon blocks, and testing the use of one or two plasmids with different copy numbers were evaluated. The strategy employing a second plasmid containing an additional copy of Cineole synthase (CS) improved terpene production by 33%^[3]. This two plasmids approach was organized in such a way that the upper and lower portions of the mevalonate pathway are in a half-copy plasmid (p15A), that carries one copy of the gene for CS and the Geranyl pyrophosphate synthase (GPPS). The other copy of CS is on the second plasmid containing a high copy origin of replication (ColE1) so that high levels of expression of GPPS and CS are achieved, making it less sensitive to small induction variations.

   Mendez-Perez D. and colleagues used inducible promoters to control expression. In its design, the team proposes the replacement of the inducible promoter by the QS system (Plux), maintaining the two plasmids system and the division of genes into operon blocks, which were shown to be more efficient to achieve higher monoterpene titers. The plasmids chosen for assembly of the pathway were pB1A3 (ori ColE1/pMB1) for high copy and pSB3K5 (ori p15A) for medium copy, with Ampicillin and Kanamycin as respective selection markers. The proposed circuit can be seen in Figure 10.

Figure 10. Initial circuit design for eucalyptol production based on two plasmids, pSB3K5 and pB1A3. 

   Later, we used iBioSim to build the circuit model (Figure 11), in which the production of 1,8-cineole (eucalyptol) is regulated by the bacterial quorum sensing (QS) LuxRI system.

Figure 11. Model of the circuit for 1,8-cineole production controlled by the quorum sensing (QS) system. Blue squares represent produced species, green arrows are activation, and dotted arrows represent complex formation. The LuxI and LuxR production modules have been separated in the model only to represent the function of the promoters.

   The construction was based on two blocks, the first responsible for controlling the QS reactions and the second for the metabolic pathway to produce 1,8-cineole. The LuxI protein is responsible for catalyzing the formation of acyl-homoserine-lactone (AHL), a signaling molecule that permeates the cell membrane and forms a complex with LuxR. The second part consists of the LuxR_AHL complex as an activator of the Plux promoter, which controls the expression of genes encoding enzymes responsible for 1,8-cineole synthesis (output). 

   The model simulation is shown in Figure 12, which depicts the operation of the circuit within a cell over time, demonstrating a positive result consistent with the strategy adopted. LuxR and LuxI are constitutively produced since the beginning of the simulation, with LuxI being accumulated rather than consumed over time, resulting in increasing AHL production over time. LuxR, unlike LuxI, is consumed throughout the process by complexation with AHL. At the beginning of the simulation, there is an accumulation of LuxR due to the delay in AHL production. The formation of the complex LuxR_AHL serves as a signaling molecule for eucalyptol production.

Figure 12. Graph of the simulation of the functioning of the built model.

References:

[1] MARTÍNEZ-PABÓN, María Cecilia; ORTEGA-CUADROS, Mailen. Thymol, menthol and eucalyptol as agents for microbiological control in the oral cavity: A scoping review. Revista Colombiana de Ciencias Químico-Farmacéuticas, [s. l.], v. 49, n. 1, p. 44–69, 2020. Disponível em: https://doi.org/10.15446/rcciquifa.v49n1.87006 

[2] MENDEZ-PEREZ, Daniel et al. Production of jet fuel precursor monoterpenoids from engineered Escherichia coli. Biotechnology and Bioengineering, [s. l.], v. 114, n. 8, p. 1703–1712, 2017. Disponível em: https://doi.org/10.1002/bit.26296

Math modeling of the genetic circuits in order to improve the design approach

   Semiconductor nanomaterials consist of a versatile platform for harvesting light energy and trans­ferring to biological systems. The complexation between these nanomaterials and biosystems (e.g.bacteria), called biohybrids, enables the use of light energy by commonly dark­-fermentative organisms, accelerating the development of sustainable and efficient technologies for H₂ production, N₂ fixation, and CO₂ reduction^{[1, 3]}. Their characteristics, such as tunable electronic structure, surface chemistry, and large surface area­-to-­volume ratios enable increased charge transfer rates and improved biopro­cesses’ efficiency^[3]. In this context, CdS quantum dots (CdS­-QD) have been widely applied in biohybrid platforms. They present robust biocompatibility, size­ dependent characteristics, high elec­trical conductivity and photostability, furthermore, their narrow band gap (i.e., 2.4 eV) made them suitable for visible light photocatalysis^[4].

   In the biohybrid system, the incident light promotes electronic excitation and charges separation at the QD surface. Then redox enzymes and molecules present in the cellular membrane or the medium act as electron acceptors and allow charge transfer from QD to organisms metabolism^[5]. The materials’ band gap determines the excitation light wavelength and can be controlled by QDs physical-­chemical properties, such as particle composition, size, and shape. In addition, different surface functionaliza­tion may improve the specificity and interaction between the material and the biological surface, also increasing charge transfer^{[5, 6]}.

   In this work, CdS­-QD interaction with a cellular membrane is maintained by the metalloprotein protein PbrR attached to the membrane by OmpA protein (See chapter 2.2.1 - Standardization and 3.1 - Improvement of an existing iGEM Design). The close interaction of the particle to the cell enables the photoelectron transferring, which mostly occurs by redox molecules mediators, such as methylene blue, methyl viologen, anthraquinone-­2, 6-­disulfonate, ferricyanide; or membrane redox enzymes, being cytochromes (e.g., MtrABC and OmcA) exposed to the surface of cell mem­branes the most common proteins^[7].

   As the main effect of light in the biohybrid system, several works report that photoelectrons can stimulate metabolic vias of generation of NADH, observing a significant increase in cellular con­centrations of this reducing equivalent^{[8, 9, 10]}. Raising NADH cellular concentration is essential to ensure the efficiency of enzymes catalyzing redox reactions, such as the synthetic CO₂ ­fixation pathway, half­-Wood–Ljungdahl-­formolase (HWLS)^[9].

   As the harvesting of light energy for NADH production is independent of the consumption of organic carbons, the carbon metabolism can be directed to the production of interesting compounds, which improve the biocatalysis efficiency and contribute to the economic feasibility of the process^[10]. In this sense, the effectiveness of the use of light in biocatalysis can be quantified by the increased yield of metabolic intermediaries or products in relation to the number of incident photons in the system, such a ratio is commonly termed as quantum efficiency^{[8, 11]}.

   The rate of NAD(P)H formation in CdS quantum dots can be quantified using the NAH(P)H quantification protocol found in the Build and test protocols file in the Document Section.

Modeling a Mevalonate Pathway

• Reactions of pyruvate to 1,8-cineole

   In Figure 13, we have the sequence of reactions considered for modeling the production of the bioproduct 1,8-­cineole. In the condition that light is off, pyruvate is normally consumed. However, when the light is turned on, we propose that pyruvate consumption is delayed due to an increase in its concentration coming from the CO₂ uptake pathway, which this model has not explored.

Figure 13. The sequence of modeled reactions, where Ei (i = 1, 2, ... and 10) represent the enzymes of each reaction. Adapted from Dalwadi et al.^[12]

   Modeling of all the reactions present in Figure 13 was made according to Michaelis­-Menten kinetics, a system of ordinary differential equations (available in supplementary material). The variable’s names and dimensional parameters are in Table 1 and 2 in Math Model Supplementary material, respectively.

   The objective of the model is to determine which reactions are more important for the production of 1,8-cineole. In addition, we aim to test if it is possible to reduce the accumulation of IPP due to its cytotoxicity^[12]. Following the methodology used by Dalwadi et al.^[12], we first assume that the system is well mixed and there is no spatial variation, the rate of enzyme formation is much faster than the rate of substrate consumption and therefore the rate of reaction is governed by Michaelis­-Menten. 

   The non­dimensionalization of the system allows better visualization of the concentration changes of each enzyme in the entire pathway. For this, it was necessary to place all the parame­ters in the same order of magnitude, therefore we applied a dimensionless parameter ε = 0.01 to place all in a range from 0.1 to 10^[12]. All the parameters are described in Table 3 of the Math Model Supplementary material. 

•  Optimization of enzymes expression

   For a primary analysis, we disregarded the presence of light in the system (I = 0) and tested the influence of each enzyme in the production of 1,8-­cineole, in order to determine which are the key enzymes of the metabolic pathway. Therefore, the ODEs (Ordinary differential equations) system was solved in Python (code available in GitHub) for the case in which each of the enzymes was doubled in concentration. In Figure 14, we can see the variation of IPP concentration and production of 1,8-cineole for each condition. It was possible to find which enzymes are more important to reduce the formation of IPP and increase the production of 1,8- ­cineole. The result of optimization shows that the overexpres­sion of “Cineole Synthase”, “Isopentenyl­diphosphate Delta­isomerase”, and “Hydroxymethylglutaryl­-CoA synthase” genes increases 13% of the production of 1,8-cineole and reduces 45% of the maximum concentration of IPP (Figure 15).

Figure 14. A) Accumulation of IPP for different concentrations of all enzymes present in the metabolic pathway. B) Production of 1,8-cineole for different concentrations of all enzymes present in the metabolic pathway.

Figure 15. Optimized system to improve 1,8-cineole production and reduce IPP. The genes "Cineole Synthase" (cs), "Isopentenyl-diphosphate Delta-isomerase" (idi), and "Hydroxymethylglutaryl-CoA synthase" (hmgcs), are in double concentration in relation of "Pyruvate Synthase Complex" (pdh), in this case.

• Influence of light on the production of 1,8-cineole

   To analyze the influence of light on the production of 1,8-­cineole, we solve the previously optimized system for I = 0, 50% and 100%, which is equivalent to 0, 30 and 60 mW cm⁻². In Figure 16, we can see an increase in the final concentration of 1,8­-cineole and a delay in the consumption of pyruvate.

Figure 16. Influence of intensity of the light used in the optimized system.

• Learn with modeling and Re-Design

   Using a dimensionless model, based on Michaelis-Menten kinetics for all enzymes responsible for the catalytic production of 1,8-Cineole, it was possible to find out which enzymes are more important to reduce the formation of IPP and increase the production of 1,8-Cineole. The result of optimization shows that the overexpression of genes encoding "Cineole Synthase'', "Isopentenyl-diphosphate Delta-isomerase" and "Hydroxymethylglutaryl-CoA synthase" has an increase of 13 % in the production of 1,8-Cineole and a reduction of 45 % in the max concentration of IPP (as described in the mathematical model's section). 

   This result corroborates the work of Mendez-Perez D et al. (2017) regarding CS enzyme overexpression, in which a second copy of this gene was added in a high copy plasmid, increasing 1,8-Cineole titers^[14]. As a result of this modeling, a Learn step was applied to lead to a redesign of the circuit strategy. Similar to the strategy employed by Mendez-Perez D et al. (2017) for CS enzyme, an extra copy of genes encoding the enzymes Hydroxymethylglutaryl-CoA synthase and Isopentenyl-diphosphate Delta-isomerase were also included in the high copy plasmid, seeking for a greater expression of these proteins towards lower accumulation of intermediates and greater accumulation of the product. This step allowed to close the DBTL (Design, Build, Test & Learn) cycle and to propose a new design for the production of the 1,8-cineole as shown in Figure 17.

Figure 17. New design for the production of the 1,8-cineole based on modeling results. 

References:

[1] Yuchen Ding, John R. Bertram, Carrie Eckert, Rajesh Reddy Bommareddy, Rajan Patel, Alex Conradie, Samantha Bryan, and Prashant Nagpal. Nanorg microbial factories: Light­-driven renewable biochemical synthesis using quantum dot ­bacteria nano-bio-hybrids. Journal of the American Chemical Society, 141(26):10272–10282, 2019. PMID: 31244185.

[2] 11. Boo A., Amaro R. L., Stan G. Quorum sensing in synthetic biology: A review. Current Opinion in Systems Biology, p. 1-14, 2021.

[3] James K. Utterback, Molly B. Wilker, David W. Mulder, Paul W. King, Joel D. Eaves, and

Gordana Dukovic. Quantum efficiency of charge transfer competing against nonexponential processes: The case of electron transfer from cds nanorods to hydrogenase. The Journal of Physical Chemistry C, 123(1):886–896, 2019.

[4] Guowen Dong, Honghui Wang, Zhiying Yan, Jing Zhang, Xiaoliang Ji, Maozi Lin, Randy A. Dahlgren, Xu Shang, Minghua Zhang, and Zheng Chen. Cadmium sulfide nanoparticles ­assisted intimate coupling of microbial and photoelectrochemical processes: Mechanisms and environ­mental applications. Science of The Total Environment, 740:140080, 2020.

[5] Sahng Ha Lee, Da Som Choi, Su Keun Kuk, and Chan Beum Park. Photobiocatalysis: Activating redox enzymes by direct or indirect transfer of photoinduced electrons. Angewandte Chemie International Edition, 57(27):7958–7985, 2018.

[6] Lei Cheng, Quanjun Xiang, Yulong Liao, and Huaiwu Zhang. Cds ­based photocatalysts. Energy Environ. Sci., 11:1362–1391, 2018.

[7] Bin Xu, Zhe Li, Yujia Jiang, Minjiao Chen, Boryann Chen, Fengxue Xin, Weiliang Dong, and Min Jiang. Recent advances in the improvement of bi­directional electron transfer between abiotic/biotic interfaces in electron ­assisted biosynthesis system. Biotechnology Advances, page 107810, 2021.

[8] Bo Wang, Cuiping Zeng, Ka Him Chu, Dan Wu, Ho Yin Yip, Liqun Ye, and Po Keung Wong. En­hanced biological hydrogen production from Escherichia coli with surface precipitated cadmium sulfide nanoparticles. Advanced Energy Materials, 7(20):1700611, 2017.

[9] Guipeng Hu, Zehong Li, Danlei Ma, Chao Ye, Linpei Zhang, Cong Gao, Liming Liu, and Xi­ulai Chen. Light-­driven CO2 sequestration in Escherichia coli to achieve theoretical yield of chemicals. 4(5):395–406, April 2021.

[10] Shuo Cui, Li­Jiao Tian, Jie Li, Xue­Meng Wang, Hou­Qi Liu, Xian­Zhong Fu, Ru­Li He, Paul K.S. Lam, Tian­Yin Huang, and Wen­Wei Li. Light­assisted fermentative hydrogen produc­tion in an intimately ­coupled inorganic ­bio hybrid with self ­assembled nanoparticles. Chemical Engineering Journal, 428:131254, 2022.

[11] J. M. Walter, D. Greenfield, C. Bustamante, and J. Liphardt. Light­powering Escherichia coli with proteorhodopsin. 104(7):2408–2412, February 2007.

[12] Mohit P. Dalwadi, Marco Garavaglia, Joseph P. Webb, John R. King, and Nigel P. Minton. Ap­plying asymptotic methods to synthetic biology: Modelling the reaction kinetics of the meval­onate pathway. 439:39–49, February 2018.

2.2.2.4. The need of boosting ROS resistance in our microbial platform

   Quantum dots (QDs) can be coupled with bacteria or other organisms to increase the yield of bioproducts. However, the coupling of QDs in live organisms presents the drawback of reactive oxygen species (ROS) production, which are toxic to the bacteria.

   Among ROS, the most common is the anion superoxide (O­₂^-) and the hydrogen peroxide (H₂O₂). O₂^- molecules are produced when O₂ captures an electron (Equation 1). ROS are toxic because they can oxidize proteins, lipids, and nucleic acids. To cope with the toxic effect of these substances produced endogenously through the metabolism, bacteria harbor an oxidative-stress response system. It is conserved among the bacterial group and is controlled by two main regulons in Escherichia coli, the Superoxide (SoxRS) and the OxyR regulons^[1].

O₂ + e^-  → O₂^-

Equation 1. Superoxide generation. Oxygen, in the presence of electrons, can generate a superoxide anion.

   The main regulator of intracellular O_­2^.- is the SoxRS. In this regulator, there are two adjacent genes, the soxR, and the soxS. They encode proteins involved in a two-stage regulatory system, in which the activated soxR induces the expression of soxS, regulating many genes involved in responses against oxidative stress^[1]. Superoxide dismutases convert the anion O­₂^.- into H₂O₂ and O₂ (Equation 2).

2O₂^- + 2H⁺  → H₂O_­2 + O₂

Equation 2. The conversion is performed by the superoxide enzymes. Two superoxides with two hydrogens are converted into one hydrogen peroxide and one oxygen.

   OxyR is the main regulator for H₂O₂ detoxification, and it controls a regulon with almost 40 genes. Among these genes, those codifying catalase/hydroperoxidase 1, glutathione oxidoreductase, and glutaredoxin are directly involved in the reduction of H₂O₂. OxyR is oxidized by H₂O₂ and it can be converted to its reduced form through the action of glutathione reductase and glutaredoxin^[1]. H₂O₂ can also be reduced by catalases, which convert them into H₂O and O₂ (Equation 3). 

2H₂O₂  → 2H₂O + O₂

Equation 3. The conversion is performed by the catalase enzymes. Two hydrogen peroxides are converted into two water molecules and one oxygen.

With the increased generation of reactive oxygen species caused by the activity of QDs and the fragility of E. coli against oxidative stress, it’s essential to optimize the endogenous ROS-defense responses to increase E. coli resistance into our culture system.

Increase E. coli resistance to intracellular ROS

   Under natural conditions, the activation mechanisms of soxR and oxyR genes are induced by the presence of intracellular ROS, anion superoxide, and hydrogen peroxide, respectively (Figure 18).

Figure 18. Circuit of resistance to natural intracellular reactive oxygen species (ROS). The soxR gene is transcribed and in the presence of superoxide, the activated protein works as a transcription factor for soxS, which acts as a transcription factor for superoxide dismutases. Superoxide dismutases are a class of proteins with a metal core (e.g. magnesium and iron) capable of converting two superoxides into one hydrogen peroxide. The oxyR gene is transcribed in the presence of hydrogen peroxide and is the transcription factor for several enzymes, related to the control of reactive oxygen species such as glutathione reductase and catalase. Catalase can convert hydrogen peroxide into water and oxygen.

   This ROS-dependent activation may become a limiting factor for adequate cell protection. Furthermore, the soxR gene is not directly responsible for encoding the protein that converts superoxide into hydrogen peroxide, but rather the soxS gene^[1]. 

To allow the use of quantum dots in bacteria that, like Escherichia coli, can suffer from the action of ROS, we developed a strategy to increase resistance to ROS.

   In our strategy Eschericia coli will constitutively overexpress the oxyR and soxS genes, breaking the natural time-consuming response cascade. In this context, a strategy to inhibit the expression of the endogenous oxyR and soxS genes through a CRISPR/Cas9-based system was designed. After that, the sequence of those endogenous genes of our wild-type strain will be amplified and assembled at a synthetic promoter library to provide a fine-tuning adjustment of expression levels, testing a combination of different promoters to increase E. coli native oxidative stress response. 

The detailed procedure for building our promoter library is described in detail in section 2.2.3. Build and Test.

Model efficiency prediction

   We simulated the efficiency of our proposed genetic modification circuit to increase the E.coli ROS resistance (2.2.3 Build and Test section) and compared it with the natural control of oxyR and soxS expression (Figure 19) using the iBioSim prediction software (3.0.0)^[2]. iBioSim uses the ordinary differential equation (ODE) model with Runge-Kutta-Fehllberg (Hierarchical) equation for prediction. For the predictions we selected a time limit of 1000 milliseconds and an absolute error of 1.0 E^-9.

A) 

B) 

Figure 19. A) Representation of oxyR and soxS genes controlled by natural E. coli promoters. B) Representation of the modified circuit through the addition of optimized promoters obtained by the promoter library construction strategy.

   We observed that by adding 200 mg of superoxide anion to the system at the time of 400 milliseconds (Figure 20), complete consumption of these reactive species under natural conditions occurred at 700 milliseconds, requiring 300 milliseconds for total consumption. Under optimized conditions, complete consumption of superoxide anion occurred at approximately 425 milliseconds, requiring 25 milliseconds for complete consumption. Our improvement has decreased the time required for superoxide anion consumption by approximately 91.66%.

   Also, by adding 200 mg of hydrogen peroxide into the system at time 400 milliseconds (Figure 20), it was observed that under natural conditions the consumption of the reactive species followed a logarithmic curve, reducing 50% of the peroxide in approximately 425 milliseconds, a balance of 25 milliseconds. However, when 200 mg of hydrogen peroxide was added to the system with the optimized circuit, the consumption profile changed from a logarithmic to a linear consumption, where 50% of the reactive species were consumed immediately of the addition in the system, and 100% of the species were consumed in about 100 milliseconds.

Figure 20. Prediction of circuit efficiency by ROS degradation. The blue color indicates the hydrogen peroxide present in the system. The purple color indicates the expression of oxyR. The moss green color indicates the expression of soxS. The light-green color indicates the expression of soxR. The red color indicates the presence of a superoxide anion in the system. (A) The natural response of E. coli to the presence of reactive oxygen species (superoxide and hydrogen peroxide), 200 mg of reactive species are added to the system in 400 milliseconds. Under natural conditions, the cell takes up to 700 milliseconds to consume all the superoxide and does not achieve complete elimination of the hydrogen peroxide present once the logarithmic curve is originated. (B) Response of E. coli to the presence of reactive oxygen species (superoxide and hydrogen peroxide), after optimization of the endogenous circuit. The cell eliminated all superoxide from the system in approximately 425 milliseconds and all hydrogen peroxide in approximately 525 milliseconds.

   Our production system will induce a notorious amount of ROS that could negatively affect the bacterial cell. Observing the problem that could arise the toxicity of these molecules, our team optimized the endogenous system of ROS resistance by inducing the constitutive expression of the oxyR and soxS genes, with the best promoters, directly in the bacterial genome, avoiding any loss of resistance due to the loss of the plasmid. In this way the bacterial cell will always be prepared to fight the reactive oxygen species, preventing any damage to the cell and favoring its growth. This enables our engineered E. coli to tolerate the stresses inherent in our cultivation process.

References:

[1] Chiang, S. M., Shellhorn, H. E. Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria. Archives of Biochemistry and Biophysics. 525, 2012, p.161-169.

[2] Leandro Watanabe, Tramy Nguyen, Michael Zhang, Zach Zundel, Zhen Zhang, Curtis Madsen, Nicholas Roehner, and Chris Myers. ACS Synthetic Biology. v. 8. P. 1560-1563. 2019. DOI: 10.1021/acssynbio.8b00078

2.2.3.1. Rainbow dots - Bench Step

If we have these very simple biological systems, it's time to (...) really understand how they work, and to go and build them.

Dr. Tom Knight

Figure 21. Lab Flowchart

Genetic manipulations

   The Rainbow toolbox genetic devices (See chapter 2.2.1 Standardization), with the exception of the 1,8-cineole synthesis pathway, will be added to the E. coli genome through genetic editing by CRISPR-Cas9 based technology including deleting or replacing genetic sequences of genes in E. coli (see more details in supplementary protocols and in the sections below).

Culture conditions

   Luria-Bertani (LB) medium will be used for plasmids and strains construction and M9 medium will be used for the bacterial culture. The strains will be cultivated following the protocol of HU, Guipeng (2021), in which the strain will grow aerobically for 24 h. After, the cultures will be transferred to a 100-ml serum bottle with a 50-ml working volume and the anaerobic conditions will be achieved by purging the headspace with H₂ gas and sealing the bottle with a rubber plug. 

References:

[1] HU, Guipeng; LI, Zehong; MA, Danlei; et al. Light-driven CO2 sequestration in Escherichia coli to achieve theoretical yield of chemicals. Nature Catalysis, v. 4, n. 5, p. 395–406, 2021.‌

Handling of CdS QDs in the laboratory

   To improve the security of the team members, we had chosen to use a commercial dispersion of cadmium sulfide (CdS) Quantum dots. This material will be purchased from Sigma-Aldrich (product: “Lumidot ™ CdS 440, core-type quantum dots, 5 mg / mL in toluene”) and its Safety Data Sheet (SDS) can be found on the Merk website (https://www.sigmaaldrich.com/BR/en/sds/aldrich/662380).

   Some precautions regarding handling and disposal of CdS Quantum Dots must be considered until stabilization is carried out to avoid risks to researchers and the environment. At the beginning of the process, the commercial solution is dispersed in Toluene, a toxic compound, which can be replaced after reaction with formamide and a solution of potassium hydroxide (KOH) in formamide, followed by a bond by adding acetonitrile. Finally, this precipitate can be dissolved in water.

   At the end of the eucalyptol production process, the Quantum Dots present in the solution can be precipitated with acetonitrile, solubilized in water, and collected for the next batch. All these reagents are available in the corresponding vials in the laboratory and then properly disposed of by the institution's specialized personnel. And for the operator's safety, this entire process will take place in a glove box, with an N₂ atmosphere. The correct disposal of all materials will be carried out in the CNPEM laboratories following the consolidated disposal procedures^[1].

References:

[1] Zhang, H., Dasbiswas, K., Ludwig, N. et al. Stable colloids in molten inorganic salts. Nature 542, 328–331 (2017).

1,8 - Cineole (Eucalyptol) biosynthesis and quantification

   Specific oligonucleotides will be designed for amplification of the parts of interest using genomic templates by polymerase chain reaction (PCR). Cloning will be done following the biobrick pattern, with restriction enzymes for the respective prefix and suffix sites inserted into the sequences. Circuit 1 will be inserted into the medium copy plasmid (pSB3K5) and circuit 2 into the high copy plasmid (pB1A3) (See chapter 2.2.1. Standardization). All the genetic transformations will be performed in chemically competent E. coli by heat shock protocol and subsequently verified by sequencing.

   After cloning, the transformant strain will be cultivated to analyze cell production and growth. The culture will be done for 12 h in Erlenmeyer flasks with M9 medium, in an incubator maintained at 37°C and 220 rpm. Samples will be taken periodically for the determination of the optical density at 600 nm by spectrophotometry.   

   To capture the terpenes produced, dodecane (10% by volume) will be added to the samples. This mixture will then be centrifuged to separate the phases and subsequently partitioned with 10 uL of the organic phase into 990 uL of ethyl acetate (Sigma -Aldrich, St. Louis, MO). Finally, 1,8-cineole will be quantified by gas chromatography coupled to mass spectrometry (GC-MS) according to a standard curve of Sigma Aldrich 1,8-cineole (470-82-6). The GC will be equipped with an Agilent 5973 mass selective (MS) detector and an Agilent J&W DB-5 ms capillary column (30 m 250 mm 0.25 mm thick, Agilent). The temperature program of the GC oven will be as follows: 80°C for 1 min, a 20°C/min ramp to 120°C, a 50°C/min ramp to 250C. The MS will be operated in selected ion monitoring mode (SIM) for scanning molecular ions at 93, 121, and 136 m/z.

Construction of promoter library to improve ROS resistance

   The promoter library to increase the E. coli ATCC 700728 strain resistance to intracellular ROS mentioned in the optimization section (See chapter 2.2.2) will be performed through the selection of the best combination of promoters in a two-step phase. Initially, a micro-scale screening will be made by testing the growth performance of 200 colonies in a 96-well plate with ROS-induced stress. After that, we will select 5 of the best colonies by kinetic parameters and do a bench-scale test with the same conditions, to ensure that we will select the best combinatorial mutant. The plasmids of the best-selected strain will be sequenced and an expression cassette with both optimized soxS and oxyR genes will be constructed and assembled with the light-responsive biocontainment circuit and the resultant cassette will be inserted at the SS9 region of the bacterial genome (Figure 22).

Figure 22. Flowchart for construction of intracellular ROS-resistant E. coli. The arrows indicate the sequence of steps to be performed to obtain a ROS-resistant cell.

Knockout of endogenous ROS resistance genes

   As previously stated, it will be necessary to inhibit the expression of the endogenous soxS and oxyR genes to avoid any influence on the results obtained. Initially, our idea was to perform a complete deletion of both genes from the bacterial genome. However, this strategy would be very harmful to the cell. A fragment of the 3' region of the oxyR gene is shared with the ORF of another gene, sthA, whose protein converts NADPH, originating from catabolic metabolism into NADH, which acts in the respiratory chain. Moreover, the natural promoters controlling both genes are bidirectional, controlling the transcription of genes at the complementary strand of the target genes (Figure 23).

Figure 23. Genomic regions of both target genes (A) oxyR and (B) soxS. oxyR gene shares its 3’ tail with the sthA gene. Both genes are controlled by bidirectional promoters. Genomic map obtained by EcoCyc^[2].

   Due to these factors, we chose to design a strategy to block the native transcription of the soxS and oxyR genes, keeping all other molecular mechanisms intact. As illustrated in Figure 24, the strategy developed by our team was to promote the partial truncation of this gene, deleting part of its 5' region. Then, we can ensure that both natural genes from E. coli will have their expression inhibited. We will use the CRISPR/Cas9 system to provide precise and markerless double-strand breaks (DSB) directed to the regions that will be deleted from the target genes.

Figure 24. Truncated genes. (A) Cleavage via endonuclease Cas9. (B) Pairing with a donor carrying the truncated oxyR gene and the intact sequences around the deletion. (C) Replacement of the active gene with the truncated oxyR gene. (D) Cleavage via Cas9. (E) Pairing with a donor with the truncated soxS gene and the intact sequences around the deletion. (F) Replacement of the active gene with the truncated soxS gene.

   The donor fragments, which will be used to induce DSB repair by homologous recombination, were assembled by joining 50bp of the upstream region and the downstream region of the fragment to be deleted. These fragments will be synthesized as linear double-stranded DNA fragments and inserted as linear donors, with the plasmid carrying the Cas9 endonuclease and the guide-RNA (Addgene: #42876), at the time of transformation. They were designed using the CRISPOR software, which helps in the search for protospacer sequence sites for the cleavage of Cas9, looking for PAM and mistargets domains in other regions of the genome (Figure 25)^[6].

Figure 25. Protospacer sequence prediction. Using CRISPOR protospacer sequence prediction software, we selected protospacers to guide the cleavage of Cas9 from each gene that will be truncated^[6]. (A) Selected protospacer sequence for the oxyR gene. (B) Selected protospacer sequence for the soxS gene.

   After deletion and confirmation by PCR of the target fragments, the library assembly step will be initiated to select the best promoter combination for overexpression of both genes.

Synthetic promoter library and bacteria transformation

   Using the Golden Gate-based Toolkit EcoFlex MoClo (Addgene®), we will select the best promoter combination for the soxS and oxyR genes (Figure 26). This kit has the advantage of having been built from parts coming from the iGEM's Registry of Standard Biological Parts. Therefore, the assembly and standardization of the parts were facilitated. The library will be constructed with a set of 10 constitutive promoters (J23100, J23108, J23114 SJM901, SJM902, SJM903, SJM905, SJM906, SJM910, SJM911) that will be assembled with each gene, generating a 102 combinatorial library.

Figure 26. Selection of the best promoters’ combination for oxyR and soxS expression. Under natural conditions, induction of the oxyR and soxS genes occurs in the presence of hydrogen peroxide and soxR, respectively. All promoters’ options will be tested. The combination of promoters that shows the best microbial growth under oxidative-stress conditions will be selected.

   Once the endogenous soxS and oxyR genes are inactivated and all promoter combinations determined, a library will be built for promoter testing. We will use the level 0 assemble pTU1 plasmid (MoClo EcoFlex kit (Addgene®) as a backbone to construct the promoters’ library (Figure 27). The assembly will occur following the Golden Gate Assembly protocol^[3]. No codon optimization was required since the gene sequences are already endogenous of E. coli. Bacterial transformations will occur by heat shock^[4].

   To select the best combination of promoters, we will perform micro-scale fermentation in a 96-well plate with 200 colonies carrying the promoter combinations under the same experimental conditions as determined at the Bioproduct session. The OD₆₀₀ reads will be performed in real-time with the Fluorimeter SpectraMax® M Microplates reader. We will calculate the growth curve for each transformed cell, in triplicate. The five cells with the fastest growth rate, shortest lag phase, longest exponential phase, and highest biomass yield (Yx/s) will be selected.

Figure 27. Promoters library. The assembled plasmids with different promoters’ combinations will be inserted into E. coli. After being selected and transformation-confirmed it will undergo micro fermentation under oxidative-stress conditions and be selected based on the growth kinetic parameters.

   After five colonies with the best promoter combinations have been chosen, a bench-scale, 1L fermentation will be performed under the same oxidative stress conditions. Samples will be collected every 4h. The freshly collected samples will be stained with bromophenol blue and live and dead cells will be counted in the Neubauer chamber. A growth curve will be calculated together with fermentation kinetic parameters.

   After sequencing, a cassette will be designed with both genes, oxyR, and soxS, with their respective selected promoters, and assembled with the light-responsive Biocontainment cassette (Figure 28). To insert the circuit into the genome of our bacterial strain, a set of three plasmids as designed by Bassalo and colleagues^[5] will be used.

   This set consists of:

(a) pX2-Cas9 (Addgene: #85811), 7907bp vector carrying the endonuclease Cas9 gene, inducible by arabinose with kanamycin selection;

(b) SS9_RNA (Addgene: #71656), 2584bp vector, carrying the guide-RNA sequence for direct Cas9 double-strand break at the SS9 locus with ampicillin selection^[5];

(c) pSS9 (Addgene: #71655) integration vector, carrying the optimized soxS and oxyR, and the color-responsive biocontainment cassette assembled with 600bp homology arms for integration at the SS9 genomic locus of our host E. coli cells.

   This CRISPR/Cas9-based strategy allows highly efficient, single-step integration of large pathways in E. coli and will be used in our project for the engineering of our bacteria without genomic scars or marker selections. The bacterial transformation to insert this cassette in its genome will be made by heat-shock transformation with our host bacteria, the pX2-Cas9, SS9_RNA, and pSS9 plasmids. The selection medium for the growth of transformant cells will have ampicillin, kanamycin, and tetracycline added.

Figure 28. oxyR, soxS, and the light-responsive Biocontainment circuit expression cassette. All fragments will be assembled in a single cassette and assembled in the integrative plasmid pSS9, which will integrate into the SS9 locus of the E. coli genome.

Reference:

[1] Chiang, S. M., Shellhorn, H. E. Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria. Archives of Biochemistry and Biophysics. 525, 2012, p.161-169.

[2] KESELER, Ingrid M.; MACKIE, Amanda; SANTOS-ZAVALETA, Alberto; BILLINGTON, Richard; BONAVIDES-MARTÍNEZ, César; CASPI, Ron; FULCHER, Carol; GAMA-CASTRO, Socorro; KOTHARI, Anamika; KRUMMENACKER, Markus. The EcoCyc database: reflecting new knowledge aboutescherichia colik-12. Nucleic Acids Research, [S.L.], v. 45, n. 1, p. 543-550, 28 nov. 2016. Oxford University Press (OUP). http://dx.doi.org/10.1093/nar/gkw1003.

[3] MOORE, Simon J.; LAI, Hung-En; KELWICK, Richard J. R.; CHEE, Soo Mei; BELL, David J.; POLIZZI, Karen Marie; FREEMONT, Paul S.. EcoFlex: a multifunctional moclo kit for E. coli synthetic biology. Acs Synthetic Biology, [S.L.], v. 5, n. 10, p. 1059-1069, 2 May 2016. American Chemical Society (ACS). http://dx.doi.org/10.1021/acssynbio.6b00031.

[4] Froger A, Hall JE. Transformation of plasmid DNA into E. coli using the heat shock method. J Vis Exp. 2007. v. 6 p. 253. DOI: 10.3791/253.

[5] BASSALO, Marcelo C.; GARST, Andrew D.; HALWEG-EDWARDS, Andrea L.; GRAU, William C.; DOMAILLE, Dylan W.; MUTALIK, Vivek K.; ARKIN, Adam P.; GILL, Ryan T.. Rapid and Efficient One-Step Metabolic Pathway Integration in E. coli. Acs Synthetic Biology, [S.L.], v. 5, n. 7, p. 561-568, 22 abr. 2016. American Chemical Society (ACS). http://dx.doi.org/10.1021/acssynbio.5b00187.

[6] Jean-Paul Concordet, Maximilian Haeussler, CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens, Nucleic Acids Research. v. 46, p 242–245, 2018. https://doi.org/10.1093/nar/gky354

[7] Leandro Watanabe, Tramy Nguyen, Michael Zhang, Zach Zundel, Zhen Zhang, Curtis Madsen, Nicholas Roehner, and Chris Myers. ACS Synthetic Biology. v. 8. P. 1560-1563. 2019. DOI: 10.1021/acssynbio.8b00078

2.2.3.2. Rainbow Dots - Scale Up

What I cannot create, I do not understand

Richard Feynman

The “RGB Bioreactor” Design

   Aiming to carry out the scale up of the genetically modified Eschericia coli ATCC 700728 bacteria with the insertion of biological parts of the Rainbow toolbox, the CNPEM.Brazil team performed the prototype of hardware design "RGB Bioreactor". The bioreactor was designed to respond to the different demands of the toolbox, having versatility of its components, adaptability to processes, and still has low cost.

   Experts, inside and outside the institution, were consulted to understand the basic principles of bioreactor and how it would work. One of them was Ph.D. Flavia Vischi Winck, where the team did a brief presentation of the Rainbow Dots project focusing on the possible challenges for E. coli nano-bio-hybrid platform cultivation (Figure 29).

Figure 29. Meeting with Flavia Vischi Winck.

   After the presentation, Flavia raised some concerns related to concepts that could be reformulated for better project delivery. Regarding the bioreactor (hardware), she also suggested some improvements to favor E. coli growth and the bioreactor's usefulness. One of the points that she suggested was the use of a cylindrical cultivation environment to reduce the impact of bacteria on the bioreactor extremities, and the implementation of an airlift process and a pH control sensor.

   Through the combination of microorganisms and LED’s, the team suggests the use of three different wavelength frequencies (Red, Green and Blue). Each one would have a different purpose in the light-activated circuits designed, as described below:

🔴 RED (λ = 650 nm): biocontainment strategy with CRISPR/Cas9 system;

🟢 GREEN (λ = 525 nm): ATP pump with the green light-induced proteorhodopsin in the photosensitizing device;

🔵 BLUE (λ = 450 nm): excitation wavelength of quantum dots Cadmium Sulfide QDs.

   The mechanical and electronic components can be seen in Figure 30. While their detailed descriptions can be followed in the following topics.

Figure 30. Components of RGB Bioreactor.

Mechanical Components

   For the design of the mechanical structure, the team used Autodesk Inventor software for 3D modeling and choosed to use low-cost components and models for democratization and easy replication of the design by those who want to replicate it in the future. The green components are from the group's development and are designed to run on 3D printers.

Container

   The main part of the structure used in the RGB bioreactor is an airtight bottle, a translucent container with an insulated pressure, a cylindrical shape, easy to find to build, and cheap. The team-designed structural parts are about 300 mm high and 100 mm in diameter, which results in a total volume of 2.2 L and a usable volume of 1.5 L, considering the volume occupied by the air transport and control devices.

Supply CO₂

   For the bacteria supply of CO₂, the design brings a rechargeable commercial cylinder, which can be injected through an airlift system, which allows the agitation of the system. The gas will be feeding the bioreactor through a flow valve, fixed at the cylinder outlets. There's also another entry in case of necessity to implement O₂ gas, in a higher level of purity than that found in the atmosphere.

Heating System

   For the heating system, the water bath was chosen as it is a good way to promote the heat exchange of the water to the vessel. This is a way to distribute heat more evenly and with this method, we could easily control the temperature. For this, we designed a 145 mm high and 120 mm diameter container. The vessel is mounted inside this container, the water used in its interior has a volume of 360 ​​mL.

Entries

   To use sensors and electrodes inside the container, it was decided to redesign the vessel top creating entrance for the components. So far, the prototype has six inputs to couple the sensors (which control temperature, pressure and CO₂ level), air entrance and the electrodes.

   The design requires incident light at different wavelengths, so the prototype has an addressable RGB LED strip to provide this light which is being supported by a cylindrical structure that can be printed on 3D printers to provide better uniformity of light distribution for E. coli.

Airlift

   The bioreactor is a gas-liquid type, which was based on the draft tube principle. In this reactor, compressed air was used for aeration and agitation. In our case, instead of using a normal airlift, where the gas connection is placed in the down part, the team chose to design it on the vessel's cover. The decision was made based on the problems with the pierce and seal, that is why there are no holes on the device's body.

   The airlift system can be built with a perforated silicone hose that circulates inside the bioreactor. Also, to prevent the hose from floating and to promote a better distribution of bubbles inside the device, there will be a stainless steel wire inside the hose to rigidly mold it.

Electrode

   As mentioned in item 2.2, the project is based on a light-driven system with CdS quantum dots and one of the main issues is that reactive oxygen species (ROS) can be generated outside and inside the microorganisms. In general, the conventional ROS sensing tests are time-consuming, requiring reactive compounds and complex techniques, such as chemiluminescence and liquid scintillation counting. Recently, the urgency for fast, simple and effective routes to monitor ROS has driven the development of electrochemical sensors. Electrochemical sensors can detect several analytes using low cost equipment with high sensitivity.^[1]. Because of that, the design includes an electrochemical sensor to monitor some of these molecules.

   The proposed sensing method to this section is a three-electrode cell to consume hydrogen peroxide (H₂O₂). The electrochemical cell is composed of 3 electrodes, the working electrode (WE), the reference (RE), and a counter electrode (CE). The WE is the electrode of interest, where the electrochemical reactions of interest. The CE is made of a bare material that does not produce electrolytes that may interfere with the working electrode, and it is used to close the circuit between WE and CE. The current that is flowing through the internal system between WE and CE is measured by the equipment. The reference does not interact with the reaction, which makes it remain with the same electrical potential throughout the reaction process, and will be used as the name says, as a reference to analyze the behavior of the system over time^[2].

   In this arrangement, when an electric potential difference is applied, the electron exchange semi-reactions occur, generating an electric current, formed by the transition of the electrons or ions. This electron transition usually occurs at the electrode interface, where there is a high electric field, and its available quantity is directly linked to the reaction stoichiometry. So, to monitor the formation or consumption of the molecules, it is needed to monitor these interface reactions, as well as cheaper materials and easy to work with are necessary to this application^[3].

   In the designed cell, the electrodes have to be separated, the WE and RE are placed on the bioreactor, while CE is on the auxiliary reservoir, and to connect both, there's a salt bridge. This configuration was used to avoid any by-products that can be generated on the CE. The CE is made of a steel mesh covered by carbon black^[4]. In the bioreactor, we have the RE, a silver chloride (Ag/AgCl) commercial electrode, and close to it, the WE, also a steel mesh, recovered with an ink composed of carbon black and molybdenum sulfide (MoS₂) particles to promote the consumption of ROS molecules^[4].

   Molybdenum sulfide has a layered structure, which has covalent bonds Mo-S in the layer, and van der Walls forces between the layers. This material is one of the Transition-Metal dichalcogenides (TMDs), which is abundant, flexible, and can be applied as an antioxidative agent. Due to its chemical surface composition, with Sulfur atoms, the MoS₂ has a great adsorption ability and an amazing catalytic activity, because of electron transfer and valence change. These MoS₂ characteristics have attracted attention from different fields, like biomedicine, bioremediation, catalysis, environmental, and sensing.

   In the case of ROS sensing, more specifically the H₂O₂ degradation, Chen et al. reported in 2018, the oxidative stress caused by H₂O₂ was reduced by MoS₂ nanosheets with CAT-like activity. It was possible because of the higher Mo6+ oxidation capacity over the H₂O₂. The report also proposed the 2 possible reactions (equations 1 and 2).

Mo⁶⁺  + H₂O₂ + 2OH^- → Mo⁴⁺ + O₂  + H₂O (1)

Mo⁴⁺  + H₂O₂ → Mo⁶⁺ + 2OH^-  (2)

   And also showed SOD-like activity, due to the positive charge of Mo⁴⁺ attracting O₂^.-, promoting the electron transfer between the ions (equations 3 and 4).

Mo⁴⁺  + H⁺ + O₂^.-  → Mo⁶⁺ + H₂O  (3)

Mo⁶⁺  + O₂^.- → Mo⁴⁺ + O₂  + H⁺  (4)

   For future perspectives this arrangement can also work for biosafety and biosecurity, which is possible due to its versatility, so after certain adjustments, it can also produce ROS with enough concentration to kill the bacteria. Specially because the material has, under visible light, absorption capacity, great stability against photo corrosion, and band gap adjustable. 

References:

[1] Yao Yao, Xiaoxue Liu, Yuzhou Shao, Yibin Ying, Jianfeng Ping, Noble metal alloy nanoparticles coated flexible MoS2 paper for the determination of reactive oxygen species, Biosensors and Bioelectronics, Volume 166, 2020, 112463.

[2] Bard, Allen J.; Faulkner, Larry R. Electrochemical methods: Fundamentals and applications. 2nd edition. 

[3] Kissinger, P. T, Heineman, W. R. Cyclic Voltammetry. Journal of Chemical Education. 1983, 60:702-706.

[4] Hen, Y. H., et al. Carbon coated stainless steel mesh as a low-cost and corrosion-resistant current collector for aqueous rechargeable batteries. J. Mater. Chem. A, 2017,5, 15752-15758.

Electronic components

   One of the biggest challenges was the development of a system with the changing monitoring and correction requirements for bacterial survival at a low cost. Thus, the team decided to propose as a controller device the Arduino microcontroller in this prototype. Furthermore, the sensors and devices that work with this class of devices are also low cost and easy to install, use, and, if necessary, exchange for another model.

   In Figure 31, it is possible to see all the electronic components proposed to the Bioreactor and a brief description of their respective functions for testing the Rainbow Dots project below.

Figure 31. Electronics components of RGB Bioreactor.

1. Arduino Nano: microcontroller from the Arduino family, characterized by its small size and its main function in this project is to receive the values from the sensors and make the necessary decisions for the cultivation of E. coli.

2. Addressable LED Strip: Its function in the project is to maintain E. coli alive and activate the Quantum Dots (Respectively RED and BLUE light.)

3. Gas Sensor: shows the level of CO₂ inside the RGB Bioreactor. It is using a sensor with a module that will measure the concentration of CO₂ in PPM available in the top reservatory.

4. Heated Table: Due to the necessity of E. coli to survive at 37.5°C, it is necessary for a device to keep the temperature at the same level all the time. In this case, a heated table for the 3D printer is being used, as it can heat a larger surface while keeping the temperature evenly distributed.

5. Temperature Sensor (NTC): This sensor allows the visualization of the temperature inside the reservoir. For the project, an NTC probe model is used because it is waterproof, it can easily be placed inside the bioreactor and measure the temperature directly with the purpose of controlling the temperature more accurately.

6. LCD Display: This component will allow the user to see the value of the variables provided by the sensors such as temperature, CO₂ level, or the color being emitted in the bioreactor.

7. Relay Module: The relay module has the function of controlling the current coming from the source to power devices such as the heated table, as this current is greater than the value bearable for the Arduino operation. Thus, this Module is very necessary for the use of these components without damaging the microcontroller.

8. Electrodes: The electrodes have their function linked to the reaction of consuming the ROS to allow the bacteria to survive. Its detailed explanation is in the subtopic "Electrode”.

   To organize some of these components (1, 6 and 7), an electronic module 43 mm high, 89 mm wide and 74 mm long was designed. The idea was to make this part separated from the main body to facilitate the exchange of the module for different projects and to minimize its possible contact with water, since this could cause a short circuit and later an accident with the user.

Next steps

   After prototyping and studying the best configuration for the bioreactor the team is figuring out ways to optimize its design and as a consequence to better evaluate parameters relating to both agitation/mixing mechanisms and numerical methods.

   The next step is to use software that provides qualitative and quantitative parameters such as the Ansys software oriented to a CFD (computational fluid dynamics) approach, among them CFX and Fluent, both inside Ansys, allowing students to study many aspects of optimization: design, fluid flow, responses to model disturbances (related to the optimal temperature, pressure, pH, etc). Also, it is possible to simulate fluid flow with the OpenFOAM (Field Operation And Manipulation) library using C++ programming language without having issues related to commercial software such as Ansys. The same methodology (consult specialists) and a hands-on approach would be applied. In Figure 32, there are two sensors (9 and 10) that can be implemented in the next steps of the project. 

Figure 32. Future sensors for RGB Bioreactor. To buy and assemble this bioreactor with the current electronic components we are using, we would spend about R$1030,84 or U$194,50.

9. Pressure Sensor Module: This sensor will measure the pressure inside our bioreactor, and it can also help us to know how much CO₂ and O₂ we have.

10. PH Module Sensor: To control the PH of our bioprocess, we will use this module which will allow our microorganism to survive in the environment.

   In the “Bioreactor - Table of Costs Supplementary” it has 3 different tables with components costs, divided into Mechanical, Electronic and Next Components for the prototype. In the overall budget to buy and assemble this bioreactor with the current electronic components using, the team would spend around R$ 1,030.84 (U$ 194.50).

   The main goal of human practices in the competition was to engage the participants to observe society’s problems and propose innovative solutions. To achieve this goal the team had brainstorm meetings about local population requirements. Through this, three pillars were idealized to build up the project based on the transformations sought:

• First Pillar - Education: The team realized that a lot of students in Campinas didn’t have social opportunities and high-quality education. Consequently, they rarely had access to a good scientific repertoire. In this context, the team aimed to contribute to the education of young students, focusing on students aged from 14 to 22. For this proposal, the team established a network with local high schools and preparatory schools and planned educational classes to teach basic concepts of molecular and synthetic biology to these students.

See chapters: 3.4.1 (teaching students) and 3.4.5 (teaching material)

• Second Pillar - Science outreach: The team realized that the Campinas' students rarely have access to more specific scientific content; very few participants were aware of the diversity of science competitions, including iGEM. In this context, one goal of the project was to make scientific outreach activities. For this proposal, the team created didactic materials to explain bioeconomy and biotechnology concepts aiming to distribute and raise awareness about this theme. Besides that, the use of social networks to disseminate scientific information and events with this theme, for example, live interviews with renowned scientists to engage the population to consume scientific content. 

See chapters: 3.4.1 (teaching students), 3.4.2 (lives), 3.4.3 (podcast), 3.4.4 (ebook and comic book), 3.4.5 (teaching material), 3.4.6 (scientific events), 3.4.7 (web series)

• Third Pillar - Inclusion: Another goal for the project was to impact the entire local community, including various social groups. The team realized that the elderly public was constantly in social vulnerability. In this context, the team focused on the inclusion of these social groups. The proposal to develop this pillar of the project was to discuss the social exclusion of the elderly to raise awareness of this issue to the general population and consequently impact their attitudes

See chapters: 3.4.7 (web series “Já diziam meus avôs”) and 3.6 (Diversity and inclusion)

   Over the months, the team CNPEM.Brazil met with different professionals from local industries involved in the development and application of biotechnological processes. The main purpose of these meetings was to validate the proposal of the Rainbow Dots project from the perspective of experts and to understand the potential difficulties and challenges expected on implementing the project's ideas on large scales. In addition, the meetings enabled the team to recognize which problems society and local companies constantly confront. Using the insights from these meetings the team focused on modulating and adapting the project ideas to address local demands.

Meeting with Felipe Guastelli (Natura)

Figure 33. Meeting with Felipe Guastelli.

   Team members met with Felipe Guastelli, a specialist on sustainable product packaging from Natura. In this meeting, the team was in an early phase of the project doing a lot of research and investigation to define the proof of concept bioproduct. One possibility discussed in this meeting was bioplastics, which could be interesting in packaging production. For this reason, the contact with Felipe was an excellent opportunity to understand the market and technologies in bioplastics.  

   Felipe highlighted essential points to evaluate. For example, if the bioplastic is biodegradable, it can become inert even if the raw material is biodegradable before polymerization. Furthermore, he clarified the various categories of biodegradable products and thus different respective disposal systems. Biocompostables plastics, for example, are desired materials.  

   The team also consulted with Felipe about the company’s position on the use of genetically modified organisms. And he replied that the implementation of a product by GMOs would require an extensive analysis by the company because the public has a rash opinion on GMOs, which would significantly affect the marketing of the product.   

   Reflecting on Felipe's considerations, the team realized that the community's negative opinion about GMOs stems from the lack of available information on the subject. The group of human practices devised an action based on creating dissemination materials about GMOs and introduced the topic in classes for high school students to combat this device.

Meeting with Paula Delgado and Marina Pessoa (Rhodia - Solvay)

   The team met with professionals Paula Delgado and Marina Pessoa from the Industrial Biotechnology Laboratory (IBL) of the company Rhodia (Solvay Group), installed in Paulínia (SP). The lab focuses on innovation and sustainable chemistry via biomass, with bioeconomy (To learn more visit: https://www.prp.unicamp.br/es/node/567).

   Among the discussed points, the professionals highlighted that the general population prefers food and agricultural products of natural origin, prioritizing biological routes instead of chemical ones. On the other hand, genetically modified organisms' use in processes may not be so accepted, requiring more significant investment in downstream steps and bioproduct isolation. Another challenge pointed out is the industry's structures, which often need major adaptations to satisfy fermentation conditions, raising the price compared to purely chemical processes.

   The team considered the points discussed to create a production designer that would not require a complicated extraction or fermentation process. In addition, the team had dealt with the acceptance of GMOs by planning the activities of human practices mentioned above.

Meeting with Carolina Delgado and Daniel Cozetto (Braskem)

Figure 34. Meeting with Carolina Delgado and Daniel Cozetto.

   Another consulted group of experts was Carolina Delgado and Daniel Cozetto from Braskem's research and development (R&D) team. Braskem is a Brazilian petrochemical company located in Campinas (SP), producing thermoplastic resins and investing in innovative projects that propose sustainable products.   

   In this meeting, the members of Braskem indicated using the criteria for evaluating projects by the industry, such as market analysis and manufacturing costs. Furthermore, it was necessary to choose an easy-to-engineer metabolic pathway and the most promising host for producing the product searched. Regarding the issue of using GMOs, the professionals consider that there is no problem in accepting inputs for application in materials since they didn't contain engineered organisms in the final product. 

   After analyzing the advice offered by Braskem representatives, the team members held an internal meeting to think of ways to make the metabolic pathway designer less complex without giving up on the ideas proposed until then.

Meeting with Murilo Morais (Tradecorp Microquímica)

Figure 35. Meeting with Murilo Morais.

   In this meeting, team members gathered with professional Murilo Morais from Tradecorp Microquímica, an enormous agricultural market company working with fertilizers and biofertilizers (To know more: https://www.linkedin.com/company/tradecorp-do-brasil/).  

   CNPEM’s team did a brief presentation about the project and objectives of the final product. Murilo was very interested in the Quantum Dots platform to produce terpenoids to the agricultural market as biopesticides or hormonal supplementation of plant growth. The same subject of other meetings had talked in this meeting: the high demand for products with a non-synthetic biological background, with lower to consumer’s body and external environment.  

   Murilo also added, explaining that there is a high search for products against biotic stress and plagues, especially weevil and Fusarium, because they are very impactful in agriculture. In this context, Tradecorp is willing to invest in new projects to develop biomolecules and metabolites and even develop biotechnology labs to produce them. 

   The feedback received from Murilo and his comments on the agricultural sector reinforced the perception that the team chose a suitable bioproduct. Increasing crop yields is of utmost importance since the agricultural sector is only growing, and the population of Brazil is experiencing a situation of food insecurity aggravated by the pandemic and the economic crisis.

Meeting with Beatriz Pacheco (Ginkgo Bioworks)

Figure 36. Meeting with Beatriz Pacheco.

   The team gathered with Beatriz Pacheco, an employee of Ginkgo Bioworks and a participant and judge of iGEM, in order to get some feedback on the progress of the project. After the project presentation, Beatriz highlighted some points on her experience. She praised the project’s ideas, so outlined the team’s storytelling and communication. Beatriz called attention to the importance of modulating the project with insights from both advisors and representatives of society and seeking feedback to measure the project’s impact on society.

   Reflecting on Beatriz's advice on seeking feedback from society on the project and biotechnology techniques such as genetically modified organisms, the team thought of approaching other audiences on these issues. The team aimed to assess high school students' knowledge and opinions about GMOs and their uses. For this proposal, the team created question forms distributed before and after classes presented by team members to high school students. 

   Through the 17 Sustainable Development Goals (SDGs), the United Nations (UN) makes an urgent call to all developed and developing countries, asking them to act in partnership towards the mitigation of the world’s major challenges. Responding to this call, the CNPEM.Brazil team wanted a project versatile enough to address multiple SDGs, coming up with the Rainbow Dots nano-bio-hybrid platform for CO₂ uptake.

   The idea behind Rainbow Dots is to propose a toolbox for the production of different molecules desired by society through several possible associations of microorganisms and nanoparticles, adopting CO₂ as a carbon source. Thus, the CO₂ consumed by the platform avoids the emission of greenhouse gas (GHG) to the atmosphere, tackling the 13th SDG (“Climate action”). Controlling GHG emissions and the negative effects of climate changes also helps to enhance quality of life, assisting the 3rd SDG (“Good health and well-being”).

   Furthermore, for each configuration of the platform, its products may contribute to distinct SDGs. As a proof of concept, the team designed a system to generate eucalyptol, a terpene which can be used in agriculture, attracting pollinators and parasites to caterpillar eggs. In this way, it can promote sustainable use of terrestrial ecosystems and avoid the use of chemicals in plantations, being a bio-alternative that attends to the 15th SDG (“Life on land”). In addition, this system can be associated with the manufacturing of energy products and make this process cleaner, contributing to the 7th SDG (“Affordable and clean energy”) as it will be explained further ahead. Still, the platform may generate other bioproducts and contemplate SDGs such as the 6th (“Clean water and sanitation”) in the case of microorganisms that help treating effluents, the 7th in the case of fuel production and the 3rd in the case of medicaments.

   Moreover, Rainbow Dots platform alludes to the principles of a biorefinery. In other words, it involves the use of a renewable carbon source (specifically CO₂ instead of biomass as the substitute to crude oil) for the production of bio-based products and bioenergy. Besides bringing this innovative approach, the team proposes a bioreactor of 1.5 L as a hardware that would be built when the project reaches its practical step. Hence, the 9th SDG (“Industry, innovation and infrastructure”) is covered by this project. The sustainability of the production process can be highlighted as well and, considering that the platform is able to deliver substances applicable in cities and human settlements, the 11th and 12th SDGs would be addressed too (“Sustainable Cities and Communities” and “Responsible Consumption and Production”, respectively). The circular economy is also promoted by the reuse of residues from other processes, as it will be explained further ahead. Finally, the 4th (“Quality Education”) and the 17th (“Partnerships to achieve the Goal”) SDGs are encouraged to all iGEM Design League teams and more information concerning these topics can be found in the Human Practices sections. As the team has a very large female representation, the 5th goal is also included.

Figure 37. SDGs that can be addressed with Rainbow Dots.

   To be sure that the team was on the right track and that its contributions were being effective, the CNPEM.Brazil implemented some strategies. One of them was to collaborate with other teams, focusing on the use of CO₂. As CO₂ Snatchers was also studying this thematic, both teams worked together in order to better understand current CO₂ mitigations or capture technologies and their complex context in Latin America. After comprehending the big picture, the teams desired to know the public’s opinions on this matter and elaborated a questionnaire sent through social media. More information on this subject can be found in the Section 3.2 and in the document “CO₂ Emissions and Current State of Capture Technologies”. Besides, CO₂ Snatchers provided contact with Dr. Yadira Itzel Vega-Cantú, who assisted CNPEM.Brazil regarding the use of quantum dots and their toxicity, an important concern for the safety of researchers and the environment. More information about it can be read in Section 3.5.

   Furthermore, CNPEM.Brazil consulted stakeholders in different areas covered by the project, such as members of the Research and Development of Braskem. This company is deeply concerned with the impact they may cause on the environment, having already implemented the production of green plastic. As the employees consulted were working on the emerging sector of biotechnology inside Braskem, they have shown great interest in Rainbow Dots platform due to its versatility and sustainability. In addition, CNPEM.Brazil consulted a company in the terpene business, called Tradecorp. Their representative was very interested in the platform too, because of its new ecological way of producing a biomolecule to be used in agriculture. These meetings’ descriptions can be found in Section 2.4.

Figure 38. Meeting with Matheus Ferreira Chagas.

   Finally, the team consulted Mateus Ferreira Chagas, a researcher from the Brazilian Biorenewables National Laboratory (LNBR) inside CNPEM, regarding Rainbow Dots’ role to promote the SDGs and the complexity of measuring the sustainability contributions of the technology. In this way, Mateus proposed a solution based on the Life Cycle Assessment (LCA), which is an internationally standardized methodology (ISO 14040) that quantifies the environmental performance of a product, process or service throughout its life cycle (from production to disposal). This technique consists in compiling an inventory of the inputs and outputs and calculating the potential environmental impacts in terms of several impact categories, such as climate change, fossil depletion, acidification, human health, etc.

   Unfortunately, as in this step of the project the team does not have quantitative information about all the inputs/outputs involved in the platform, it was not possible to carry out an assessment yet. However, it will be held in future stages of the project thanks to its numerous benefits. One of them is the possibility to compare our results with the conventional products with similar functions. For example, it would be possible to compare the differences between the use of Rainbow Dots’ eucalyptol and the usual chemicals applied in plantations to attract pollinators or decrease the proliferation of caterpillars, allowing the interpretation of results based on a reference process. Eucalyptol would probably show better biocompatibility with ecosystems and less toxicity.

   Besides, LCA also permits the assessment of the integration between processes. For example, it would be possible to see if an integration of Rainbow Dots with the ethanol production process would be advantageous, as the latter produces a residual flow of biogenic CO₂ with elevated purity. In addition, LCA helps to compare different configurations of the system in order to help improve the design, becoming a valuable decision-support tool. As an example, when performing a LCA of the project’s proof of concept, some impacts (such as toxicity) would rise due to the presence of cadmium in the quantum dots. Thus, the team would already have feedback on how to improve the platform in practical steps and obtain results using carbon dots. Additionally, other scenarios of the platform could be evaluated to identify the best inputs, because the wrong inputs can carry several negative impacts from their production or application process. This would decrease the overall benefits of the process even considering the carbon capture by the microorganisms. The balance between yield and environmental impacts could be compared for the use of sunlight versus lamps, for use of glucose versus residual carbon sources in bacteria medium, etc. Residual carbon sources (as vinasse, filter cake or ash from ethanol production, for example) are considered free of impact and their use promotes a circular economy, because they would be discarded.

Improving the function of existing parts and projects developed in the iGEM competition is not only important for the maintenance of the part registry, but essential for the development of more complex projects and more robust genetic circuits. 

   The essence of the Rainbow Dots platform comes from the photosensitization of microorganisms through the synergy between genetically engineered bacteria, through synthetic biology approach, and semiconductors nanoparticles induced by light. To construct and design our light-driven system, the process will be induced by adding Cadmium sulfide (CdS) quantum dots on the E. coli cell surface. One key element is fuse protein ompA-PbrR, in which OmpA (Outer membrane protein A) fixes the protein complex on the outer cell membrane while PbrR (lead-specific binding protein) adsorbs the CdS on the cell surface. 

   For the development of the Rainbow dots platform, an indispensable part is the photosensitization device. This composite part was created by the improvement of the 2016 Nanjing China team (http://2016.igem.org/Team:Nanjing-China/Design) which used these 2 biobricks:

• BBa_J36836 coding for the outer membrane protein (OmpA) of E. coli
• BBa_K1958007 encodes to a natural metal-binding protein PbrR from the bacterium Cupriavidus metallidurans

   The 2016 Nanjing used these 2 proteins to fix CdS nanoparticles and provide electrons by light irradiation to hydrogen production by Escherichia coli bacteria. The CNPEM.Brazil team optimized the Nanjing team's project to develop the photosensitization composite part adding a green light-induced proteorhodopsin gene (GPR) responsible for performing ATP pumping when excited by green light.

   The Nanjing team's design has been enhanced not only to allow the regeneration of NAD through the absorption of electrons but also the availability of ATP which allows even more complex biological circuits such as the HWLS carbon uptake synthetic pathway used by our autotrophic bacteria (see chapter 2.2.2.2).  

   This new composite part is expected to form part of the Rainbow toolbox and be used on the Rainbow dots platform for the construction of light-induced nanobiohybrid microorganisms.

3.2.1 Ararinhas UFF

   The CNPEM.Brazil joined Ararinhas UFF team in a multi collaboration involving different areas like bioinformatics, modeling and human practices. The first meeting consisted of a workshop hosted by Gustavo Gimenis, from CNPEM’s team. Gustavo teached some methodologies involving DFT calculations to solve questions in Ararinhas’ project using the ORCA and APBS softwares. The ORCA program package was one of the main themes discussed in the meeting. ORCA is an ab initio quantum chemistry software capable of solving electronic structure questions.  

   Ararinhas team hosted an event too, starring Ana Carolina Ramos Guimarães, a researcher at FIOCRUZ. Ana introduced her project and coordinated a question and answer session to contribute to CNPEM’s project and problems involved. 

   In the human practices area CNPEM.Brazil participated in a series of videos from Ararinhas team. The videos consisted of CNPEM’s participants communicating important words of the synthetic biology field in sign language to encourage the public to know the topic and to be concerned about its importance on society.

Figure 39. Meeting with Ararinhas UFF iGEM Design League team.

3.2.2 UnB|Embrapa

   The collaboration with the UnB|Embrapa team also involved several pillars. In the area of ​​human practices, the two teams collaborated to create a children's e-book/audio book with the theme of climate change, from the arts to the dubbing of characters. For more details see the section 3.4.4.

   For the modeling, members of the CNPEM.Brazil team were responsible for simulating two metabolic models on a genomic scale using Gapseq, contributing to the continuity of their project. In addition, a tutorial video of the iBioSim platform recorded by CNPEM.Brazil served as material for collaborators to apply this tool in their project too.

Figure 40. Meeting with UnB|Embrapa iGEM Design League team.

3.2.3 UTPrimers

   The collaboration with UTPrimers was very similar. The CNPEM.Brazil team was responsible for searching their host's secondary metabolism products through the antiSMASH platform. In addition, insights were given at a meeting on how to use iBioSim to develop their project, in addition to the recorded video tutorial. In the area of ​​human practices, the UTPrimers team was responsible for publicizing our project in a children's magazine that will be published on their page.

Figure 41. Meeting with UTPrimers iGEM Design League team.

3.2.4. CO₂ Snatchers

   Brazil and Mexico are the major players for CO₂ emissions in Latin America. This concerning fact motivated CNPEM.Brazil and CO₂ Snatchers to work on the consumption of CO₂ and promote collaborations to better understand this thematic. Initially, the teams looked up for information on current strategies and technologies for CO₂ capture or mitigation, besides investigating how they are applied in their countries nowadays. Detailed descriptions were compiled in the document “Collaboration CO₂ Snatchers - CO₂ Emissions and Capture Technologies”, which can be found in the Documents Section. 

   Seven major technologies were identified: Forestation, reforestation and silviculture; Carbon Capture and Storage (CCS); Bioenergy with CO₂ capture and storage (BECCS); Direct Atmospheric Carbon Capture and Storage (DACCS); Improving carbon content in soil with Biochar; Improved weathering of rocks or ocean alkalinization; Ocean fertilization. Examples of these initiatives are the artificial trees of microalgae from Biomitech in Mexico and the Tupi Field CO₂ capture and storage facility in Brazil. However, initiatives that actually put into practice these technologies are rare in Latin America and the existing ones are still in early stages of development due to their high cost and the lack of financial incentive from government or companies, for example. In order to overcome these challenges, mitigation strategies may be preferred, because more research has been done concerning them. For carbon capture strategies, the development of more partnerships is suggested, both within the countries and globally.

   After understanding this context, CNPEM.Brazil and CO₂ Snatchers desired to find out how much people from Latin America countries know about this matter. With this in mind, the teams elaborated a questionnaire and published it in social media. The version in portuguese can be accessed in the following link: “Emissão de CO₂ na américa latina”. The results can be analyzed in Figure 42.

Figure 42. Graphs of the results obtained in the Portuguese form.

   It’s possible to notice that the Brazilians participating in this research identified land use as the major sector of CO₂ emissions and energy as one of the largest as well, although agriculture doesn’t impact as much as they imagined. In addition, they were familiar with some mitigation strategies, such as “Forestation, reforestation and silviculture”, “Bioenergy” and “Carbon Capture and Storage (CCS)”. However, they generally don’t know the country’s legislation concerning the decrease of these emissions. This last fact is concerning due to the large amount of CO₂ emissions in Brazil.

   Finally, CO₂ Snatchers organized a meeting between Dr. Yadira and CNPEM.Brazil, seeking information regarding nanoparticles. More details about this conversation can be found in the 3.5 section.

   Throughout the development of our project, there was concern with essential points for the implementation of a business model as a startup in the future, from the cost of reagents and affordable hardware to the industry's interest. Rainbow Dots was created as a versatile, multidisciplinary, and innovative platform, all qualities desired for a successful business. The team strongly believes in the need to validate and polish the entire project before its implementation as a business, valuing security, reliability, and optimization.

   Science is an important tool for the development of society. Through science, it is possible to understand natural phenomena, create new knowledge and novel technologies in order to improve the quality of life. Thus, humanity is constantly seeking ways to improve such a valuable tool. It is important that all members of civil society be able to access and understand what is produced within the scientific environment, as it directly interferes in their lives. Furthermore, it is necessary for science to produce results consistent with the needs of each reality. 

   In this context, the team developed and participated in several initiatives in order to find ways for effective scientific communication aiming to facilitate the understanding of science concepts. They are briefly illustrated in Figure 43.

Figure 43. Initiatives are undertaken by the group for scientific communication. 

Initiatives are undertaken by the team for science communication.

3.4.1. Teaching students

   Genetically modified organisms (GMOs) are part of the daily lives of all people, but not all of them are aware of it. With that in mind, our team held online classes in public and private high schools and popular pre-university courses. The students that attended these virtual classes were 15-51 years old.

Figure 44. Meeting with the “João Manuel” high school.

   These lectures were focused on defining what genetically modified and transgenic organisms are, the possible applications of this technology and their positive and negative impacts, so that students can critically analyze their reality. We also presented the iGEM Design League competition and our project Rainbow Dots and highlighted how students can participate in such international events. 

   The team asked students to answer a questionnaire before and after classes in order to obtain information about the students and their knowledge about the problematic CO₂ emission in Brazil in addition to be able to assess the impact of our initiative. In Figure 45 are some graphs of the data compiled by the questionnaire.

Figure 45. Graphs of the data collected by the questionnaire in the schools.

   These results made it possible to verify that more students answered "Yes" to the question “Do you know the Genetically modified organisms (GMOs)?” and “Can you identify if a product is produced from a genetically modified organism?" than before attending the class. In addition, after class, the iGEM became better known among students.

3.4.2. Lives

   The profession of scientist faces several stereotypes and a distanced relationship with the general public. The team promoted spaces on Instagram where scientists are free to talk about their personal and professional lives, which is an effective way of bringing society closer together through better communication in the field of science. Therefore, we invited two influential and inspirational Brazilian scientists to talk to us about their professional and personal experiences. We put together a script that included the community's doubts and curiosities, through questions that were sent through our social network

   The first guest was the physicist Eduardo Couto. We talked about how multidisciplinarity affects our lives and professional decisions. You can watch it here: https://www.instagram.com/p/CUJD1eeJo-d/.

   The second guest was the astronomer Duilia de Mello. We talked about her life and what it is like to be a woman in science. You can watch it here: https://www.instagram.com/p/CUlY4dihYV6/.

3.4.3. Podcast

   We partnered with the podcast Biologia In Situ in order to introduce the CNPEM.Brazil team and make science popular. The podcast is an independent group initiative that explores scientific knowledge about biology-related to day to day subjects. In order to do so, we attended a meeting with the podcast crew and wrote the episode’s script. The recording was made remotely with four members of the CNPEM.Brazil’s team and one member of Biologia In Situ’s team (Figure 46).

   The podcast episode discussed the following themes: what is synthetic biology, what is the iGEM Design League and what is the Rainbow Dots project, developed by the team. With science popularization in mind, the episode was recorded with colloquialism, in order to make it accessible to a broad audience. The team was challenged to think about how to explain the project and the other subjects with clarity and precision. The episode was published on all of Biologia In Situ’s platforms on September 23rd entitled as “Bio na Prática 017 – Biologia Sintética – com CNPEM.Brazil” (https://biologiainsitu.com.br/2021/09/23/bio-na-pratica-017-biologia-sintetica-com-cnpem/).

Figure 46. Recording for “Biologia in Situ” podcast.

3.4.4. E-book and Comic Book

   The team was invited by iGEM UnB|Embrapa to develop a chapter for the children’s book “As crônicas ambientais” (The environmental chronicles), and to make an audio recording for it as well. This ebook was produced by iGEM UnB|Embrapa to explain some concepts about environmental education to children.

   We partnered with them to make chapter II of the ebook, talking about carbon dioxide emissions. Through the chapter, it was possible to discuss climate change, the greenhouse effect and the emission of greenhouse gases by human activities and how we can mitigate it. As it is developed for kids, the language and content were adapted for a better understanding by this public.  

   The team's mascots, Dottinhos and Gigi are present in the chapter. Besides that, other characters were inspired by environmental activists. The character Professor Rao is inspired by the cacique Raoni Metuktire, leader of the indigenous people in the fight for the preservation of the Amazon rainforest and for indigenous people’s rights. The character Raquel is inspired by Rachel Carson. Environmentalist and marine biologist, Rachel was the first researcher to warn the population about the use of DDT insecticide for the environment.  

 The ebook (Figure 47) and the link to the audiobook were shared freely at Issuu Inc (https://issuu.com/igemunbembrapa/docs/capitulo_ii_dotinhos_4_) and are also available for download freely at iGEM UnB|Embrapa’s linktree.

Figure 47. Cover of the e-book built in collaboration with the UnB team.

   Additionally, we were invited by UTFPR_PG BRAZIL (UTPrimers) to develop a comic book (Figure 48) about our project. This comic book was produced by UTPrimers with the objective to promote synthetic biology to children and teenagers that have some interest in the area. In the comic book, we use our team’s mascots to illustrate our project and we adapted the language for a better understanding by the public. The comic book was shared freely at Flipsnack (https://www.flipsnack.com/5B9B7CBBDC9/hq-cnpem.html). Also, UTPrimers will post on their social media and use it in their classes in the schools.

Figure 48. Comic book page built in collaboration with the UTPrimers team.

3.4.5. Teaching material

   Booklets and posters are great strategies for disseminating information in a visual way, with a very illustrative structure, which leads the reader to learn about a specific topic with direct information, and with more accessible language. The purpose of developing posters is to be able to fix them in public places with a greater flow of people seeking greater impact, while the booklet is a compilation of the content displayed in a clean, dynamic and more compact way, with distribution more focused on a target audience. With this in mind, the team developed two posters and a booklet, with themes of great relevance to society and that the project addresses, directly or indirectly. The booklet entitled: iGEM for all was developed with the aim of informing high school, technical and graduate students about the existence of the iGEM competition, along with what synthetic biology is. It was inspired by the CNPEM.Brazil team itself, since many members, when faced with the opportunity to join the team, did not know what the competition or synthetic biology was. Furthermore, the team realized that schools and universities did not publicize these events. The two posters, one on Bioeconomy and the other on transgenics, emerged from a reflection within the team regarding some aspects addressed by our project, that society is not well informed about. In Brazil, transgenics suffer many prejudices and are difficult to penetrate in the market, because many people do not know what they are and fear them for the unknown. With this poster, we seek to inform society more about transgenics and break some myths. Regarding bioeconomy, many people do not even know its definition nor its importance for the future. So this poster brings important aspects of current problems of an economy based on fossil resources, emphasizing the importance and advantages of transitioning to a renewable and sustainable bioeconomy. 

 The booklets and posters can be found in the team's Google Drive (https://drive.google.com/drive/folders/1bWx0htbz5j44ct2WLSkC_0avhwZU4_W5). The public is redirected to the download page via the linktree (https://linktr.ee/igemcnpem.brazil), which received an exclusive post on Instagram, for publicity, inviting all audiences to download them and help in the disclosures. In addition, all material was also sent directly to the target audience during educational actions in high-schools and pre-university courses.

3.4.6. Scientific events

   Scientific events are spaces where students and scientists can share their knowledge and projects. This rich exchange provides opportunities for discussion that add to the project's development. With this objective, the CNPEM.Brazil team participated in scientific events presenting the Rainbow Dots project.

Presentations 1st Brazilian Symposium on Synthetic Biology

   The team was present at the event “1st Brazilian Symposium on Synthetic Biology”, which was organized by the Brazilian Association of Synthetic Biology from 07/24/2021 to 08/01/2021. It was a meeting focused on scientific dissemination and networking, bringing together students, researchers, companies, and professionals from Brazil who work or are interested in the area of Synthetic Biology.

   On July 27th we participated in the event, presenting our team and project to the event's spectators. Also, we had the opportunity to get to know other project ideas from iGEM world and Design league, being a moment of learning exchange and team integration.

Synthetic Biology of Microorganisms Applied to Industrial Biotechnology Course

   The “Synthetic Biology of microorganisms applied to industrial biotechnology” course was organized by Brazilian Biorenewables National Laboratory (LNBR) from 08/03/2021 to 08/04/2021 and, as a way to spread the competition and our team to the internal public of our institution, the team made a brief presentation at the opening of the synthetic biology course.

   This event was the first time that "Rainbow Dots: the nano-bio-hybrid platform for CO₂ uptake" was presented. The team also exposed some success stories and start-ups that emerged from previous editions of iGEM, demonstrating how the field of synthetic biology is broad, comprehensive, and multidisciplinary.

Figure 49. Presentation in Synthetic Biology of Microorganisms Applied to Industrial Biotechnology Course in Brazilian Laboratory of Biorenewables.

Conociendo iGEM LATAM

   On September 9th, the team participated in a live of the series “Conociendo iGEM LATAM” at the invitation of Biotec Latina, on Instagram, to talk a little about the team, how it was formed, what it is like to be a group that is not linked to a university, present the project, talk about the importance of a competition focused on Latin America on synthetic biology, discuss aspects of Brazilian science and, finally, send a message of motivation to those who want to enter this field. It was a great opportunity to show everyone, including people outside the competition and from other countries, what the CNPEM is, what was being developed by the team and get to know the cause of Biotec Latina.  

   But what is Biotec Latina after all? Biotec Latina is self-defined as a community of biotechnology communities and initiatives in Latin América. It exists to embrace discussions on shared issues amid Latin countries, thus generating an atmosphere of union and belonging. The first plan to achieve this goal is to work the local language barrier to construct bridges in communication. Therefore, all activities, meetings, and publications on social media are performed in Portuguese or Spanish, banishing English for most communications. In the first year, they created: a WhatsApp group comprising communities from 12 Latin American countries, an open Slack channel, and a profile on Instagram and Facebook. Also, they organized scientific debates, appreciation of local projects, and political and cultural activities. The video series "Meeting iGEM LATAM" (translation of Conociendo iGEM LATAM) on Instagram aimed to promote and value the Latin force in developing competitive synthetic biology work while sharing common difficulties among the Latin teams.

3.4.7. Web series “Já diziam meus avôs" (as my grandparents used to say)

   Most of the time, scientific knowledge does not reach the elderly, a group that makes a lot of use of this knowledge. Thus, as presented in section 3.6, our team created a web series "Já diziam meu avôs" that seeks to create scientific communication with this group of people, seeking to bring their knowledge closer to scientific knowledge.

3.5.1. Quantum Dots

   Considering the team's concern regarding the use of CdS QDs, we sought a specialist in the area, and through the CO₂ Snatchers team, it was possible to schedule a meeting with Dr. Yadira Itzel Vega-Cantú. Dr. Yadira is a professor at the School of Engineering and Sciences, Technological in Monterrey, and has extensive experience in the nanotechnology field.

Figure 50. Meeting with Dr. Yadira Itzel Vega-Cantú and CO₂ Snatchers.

   At this meeting, we talked about the environmental problem caused by the quantum dots made by toxic materials such as Cadmium, the iGEM China team Nanjing route to synthesis the CdS on E. coli by using ionic solution, how to improve the project due to CdS QDs, eve they being expensive, and not so good to up-scale it.

   Whereas manipulating ionic Cadmium or Sulfur solution, there are high risks for both operators and bacterias. Paesano et al. demonstrated that ionic Cadmium has a bigger impact on human cells by reducing in 70% the mitochondrial membrane function meanwhile, the CdS had a 30% reduction^[1]. So, after the meeting and due to the observed impact, the team decided to use a commercial dispersion of CdS QDs, because of the risks of using a Cadmium or Sulfur solution for both operators and bacterias. Even using the commercial one, there are some issues to deal with, like the toluene as a dispersant, so, it is necessary to stabilize the QDs to change it. As proposed by Zhang et. al, there's a solvent change method to make a stable CdS colloid, an important step to avoid the risks^[2]. In this method, there are also a few steps to recover the QDs and it was described in section 2.2.1.

   The recovery of CdS is extremely necessary, because even at low concentrations, the CD-based QDs have nano-specific ecotoxicity. If released in an uncontrolled environment, like in the aquatic environment, organisms that lack protein photosensitizers end up suffering oxidative stress and considerably lose their biomass^[3]. Taking into account the problems of cadmium sulfide quantum dots, the team has researched other types of materials. One of them that has gained attention, it's the carbon dots, due to their biocompatibility with E. coli, low toxicity, easy synthesis, low cost, and excellent stability^[4].

References:

[1] Paesano, L. et. al. Differences in toxicity, mitochondrial function and miRNome in human cells exposed in vitro to Cd as CdS quantum dots or ionic Cd. Journal of Hazardous Materials. 393, 2020,122430.

[2] Zhang, H., Dasbiswas, K., Ludwig, N. et al. Stable colloids in molten inorganic salts. Nature 542, 328–331 (2017).

[3] Yu, Z., et al. Effects of TiO2, SiO2, Ag and CdTe/CdS quantum dots nanoparticles on toxicity of cadmium towards Chlamydomonas reinhardtii. Ecology and environmental Safety. 156. 75-86. (2018).

[4] Zhang, Z; et al. Progress of Carbon Quantum Dots in Photocatalysis Applications. Particle & Particle system characterization. 33, 457-472 (2016).

3.5.2. Biocontainment

   Genetically modified organisms (GMOs) have been used on large scales in many fields, such as agricultural, bioenergetic, bioremediation and therapeutics. GMOs can produce substances on a large scale, like value-added isoprenoids^[1]. Some isoprenoids can play a role as insecticides, being useful in the agriculture field. Although a broader range of applications of GMOs can be found, there is a concern about their release in the natural environment^{[2, 3]}. The leakage of GMOs in the environment and their proliferation can impact the equilibrium of the ecosystem. Therefore, efficient strategies to avoid the unintended proliferation of GMOs must be employed.

   Some engineering tools can be used to avoid this problem. Among them, the biocontainment strategy is a synthetic circuit that blocks the leakage of engineered microorganisms to the environment^[4] and there are several strategies used in the biocontainment systems, such as the CRISPR-Cas, which is based on the degradation of target DNA by CRISPR nuclease. Since this strategy is highly specific to the target sequence, a minimal side-effect and impact on the cell growth occur. In addition, this strategy is robust, and it can operate even after two months^[3].

   The biosafety and biosecurity of the projects involving GMOs must be considered to avoid any impact on the environment and in people’s health. These are the main reasons why these issues are commonly addressed in iGEM projects. Taking this into consideration, the Rainbow Dots Project designed a light-responsive biocontainment circuit which is explained in detail in the following topics.

Light-responsive Biocontainment circuit

   Thinking about a robust biocontainment, we idealized the insertion of the endonuclease Cas9 gene and a sequence-specific guide-RNA in the genome of our modified E. coli, to cleave the sequence that encodes the subunit A of the DNA-Gyrase enzyme. This genetic circuit will not be expressed in the presence of red light, due to the nature of our light-mediated cultivation project. However, in the absence of light, both Cas9 and GyrA guide-RNA will be expressed, leading to the double-strand break (DSB) of the sequence. Thus, we ensure that any cell that accidentally escapes from the production system will not survive long term, because sometimes the cell will find a dark phase, and in this condition, a cut in the genome will lead to cell death.

• About the circuit

   To reach our biosafety goals, we chose to use a light-responsive biocontainment circuit. The transcription factor Cph8 is a chimeric light receptor engineered by Levskaya et al.^[5]. It is a fusion of the photoreceptor Cph1 and the EnvZ histidine kinase. The light-sensing unit is connected to transcription via the EnvZ-OmpR signaling pathway, phosphorylated OmpR acts as a light-responsive transcription factor. Cph8 is inactive in the presence of red light (λ = 650nm)^[5]. In the presence of this wavelength, Cph8 does not bind to the pOMP promoter and, consequently, does not express Cas9 and GyrA guide-RNA, which are under its translational control. If the cell encounters an absence of this wavelength, Cph8 will be catalytically activated and will be able to bind with pOMP, which will lead to express Cas9 and GyrA guide-RNA. The Cas9 will bind with the guide-RNA and will be guided to provide a DSB at the DNA sequence of A subunit of DNA-Gyrase, which is an essential gene for E. coli survival (Figure 51). Even if the cut is directed to an essential gene, a simple cleavage in both strands of DNA is enough to cause cell death, as E. coli does not have a mechanism to promote repair by Non-Homologous End Joining (NHEJ)^[6].

Figure 51. Biocontainment circuit using CRISPR.

   In the presence of red light (650nm), the transcription factor Cph8 is inhibited and blocks the expression of Cas9. In the absence of red light, the catalytic activation of Cph8 occurs, which will bind with the pOMP promoter and induce Cas9 expression. It is important to note that the guide-RNA will be expressed under the control of the same promoter, so Cas9 and its guide-RNA will be transcribed together. They will promote the cleavage of a particular region: the subunit A of the essential DNA-Gyrase enzyme.

   The expression cassette (Figure 52) will be synthesized as a linear double-stranded DNA fragment. To insert the circuit into the genome of our bacterial strain, we will use the strategy explained at the ROS resistance session, inserting a unique cassette containing the ROS resistance improved genes together with the entire Biocontainment circuit of 6913 bp, ensuring genetic stability to our circuits. To guarantee the survival of the bacteria in the production system, in the Hardware session we have designed a bioreactor with a light filter and LED lamps that will ensure the presence of this wavelength (650nm) during the entire cultivation period.

Figure 52. Biocontainment cassette. Cassette sequence of 6913bp designed for biocontainment using the Benchling software.

• Model efficiency prediction

   We checked the efficiency of the genetic circuit using the University of Utah's iBioSim prediction software (3.0.0). The software uses the ordinary differential equation (ODE) model using the Runge-Kutta-Fehlberg (Hierarchical) equation for prediction. We selected a time limit of 1000 milliseconds and an absolute error of 1.0 E-9 (Figure 53).

   We observe that in the presence of red light (650nm), the Cph8 cofactor, constitutively expressed, is full-time inactivated. Then, neither Cas9 nor guide-RNA are expressed. When we remove the red light from the system, in 400 milliseconds, the transcription factor Cph8 changes to its activated conformation and, consequently, provides the expression of Cas9 and the GyrA guide-RNA. With the expression of Cas9 and the guide-RNA, the cleavage of the E. coli genome will occur at the DNA-Gyrase A subunit gene, leading to cell death.

Figure 53. Biocontainment system.

   Under red light (650nm) until 400 milliseconds and without red light after that. In blue, the transcription factor Cph8 is constitutively expressed, but inactivated under red light. In green, the GyrA guide-RNA, and in purple is the endonuclease Cas9 expression. In the presence of red light, the transcription factor is not activated, and consequently, the Cas9 and guide-RNA are not expressed. When the light is removed from the system, they start to be expressed and provide a DSB in the E. coli genome.

   A robust biocontainment system is essential to ensure the safety of our project. Our light-responsive biocontainment system ensures that if the bacteria accidentally escape our cultivation system, they will die because at some point in their life they will encounter the absence of red light (650nm). To die in the absence of light, we will insert in its genomic DNA the endonuclease Cas9 and guide-RNA expression cassette, targeting the A subunit of the DNA-Gyrase enzyme, an essential enzyme for cell survival. So, if the bacteria survive the DNA DSB, which is unlikely because it does not perform Non-Homologous End Joining repair (NHEJ), it will still die by the absence of this essential enzyme. To ensure death only in the absence of light we use the transcription factor Cph8, whose catalytic activation is repressed by red light (650 nm). When in the dark, Cph8 is activated and binds to the pOMP promoter, leading to the transcription of Cas9 and the guide-RNA.

   For bench-scale proof-of-concept of our biocontainment circuit, we will perform a simple growth test comparing the WT strain and the cassette-containing strain under and without red-light culture. Both strains will be cultured until OD₆₀₀ = 1.0, and, sequentially, pass through a tenfold serial dilution with an automatic multichannel pipette. Before that, 5 μL of each dilution will be spotted on Petri dishes with M9 medium. If the biocontainment strain grows under red light and is inhibited without red light, we will have our proof-of-concept that our biocontainment circuit works well. Thus, we obtained a robust and stable light-responsive Biocontainment system for the safety of our project.

References:

[1] NAVALE, G. R., DHAME, M. S., SHINDE, S. S. Metabolic engineering and synthetic biology for isoprenoid production in Escherichia coli and Saccharomyces cerevisiae. Applied Microbiology and Biotechnology. 105, 2021. p. 457-475.

[2] MANDELL, D. J., LAJOIE, M. J., MEE, M. T., TAKEUCHI, R., KUZNETSOV, G., NORVILLE, J. E., GREGG. C. J., STODDARD, B. L., CHURCH, G. M. Biocontainment of genetically modified organisms by synthetic protein design. Nature. 518. 2015. p. 518-527.

[3] KIM, D., LEE, J. W. Genetic Biocontainment Systems for the Safe Use of Engineered Microorganisms. Biotechnology and Bioprocess Engineering. 25, 2021. p. 974-984.

[4] YANG, D., PRABOWO, C. P. S., EUN, H., PARK, S. Y., CHO, I. J., JIAO, S., LEE. S. Y. Escherichia coli as a platform microbial host for systems metabolic engineering. Essays in Biochemistry. 65, 2021. p. 225-246.

[5] LEVSKAYA, Anselm; CHEVALIER, Aaron A.; TABOR, Jeffrey J.; SIMPSON, Zachary Booth; LAVERY, Laura A.; LEVY, Matthew; DAVIDSON, Eric A.; SCOURAS, Alexander; ELLINGTON, Andrew D.; MARCOTTE, Edward M.. Engineering Escherichia coli to see light. Nature, [S.L.], v. 438, n. 7067, p. 441-442, 23 nov. 2005. Springer Science and Business Media LLC.

[6] ZHENG, Xuan; LI, Shi-Yuan; ZHAO, Guo-Ping; WANG, Jin. An efficient system for deletion of large DNA fragments in Escherichia coli via introduction of both Cas9 and the non-homologous end joining system from Mycobacterium smegmatis. Biochemical And Biophysical Research Communications, [S.L.], v. 485, n. 4, p. 768-774, abr. 2017. Elsevier BV.

KUCHO, Ken-Ichi; OHYAMA, Kanji; FUKUZAWA, Hideya. CO2-Responsive Transcriptional Regulation ofCAH1 Encoding Carbonic Anhydrase Is Mediated by Enhancer and Silencer Regions in Chlamydomonas reinhardtii. Plant Physiology, [S.L.], v. 121, n. 4, p. 1329-1337, 1 dez. 1999. Oxford University Press (OUP). http://dx.doi.org/10.1104/pp.121.4.1329.

3.5.3. Internal Biosafety Commission (CIBio) of CNPEM

   CNPEM.Brazil had the opportunity to consult Dr. Marcio Chaim Bajgelman, president of the Internal Biosafety Commission (CIBio) of CNPEM, regarding the biological risks of our project. Through this conversation, the team was able to answer the “Form for the submission of research projects with GMOs for analysis by CIBio” and understand which safety aspects should be considered before carrying out any activity involving GMOs. The purpose of this questionnaire is to help researchers better understand their initiatives and guide them about the risks involved in their activities. Therefore, this questionnaire and the answers will be available in the Documents Section, in the attempt to assist other teams that want to clearly comprehend their designs and biosafety issues.

Figure 54. Meeting with Dr. Marcio Chaim Bajgelman.

   During the meeting, the GMOs involved in the project were discussed. The team will use Escherichia coli ATCC_700728 bacterium, which belongs to level 1 of the biosafety risk groups. Besides, the team designed parts from other organisms, including Staphylococcus aureus and Streptococcus pyogenes (biosafety risk 2 bacteria). Nonetheless, these genes will be entirely synthesized based on sequences published in the NCBI database. Hence, it will not be necessary to use cells from this organism. It is noteworthy that the genes of interest from this bacterium are not associated with the production of toxins, oncogenic proteins or with the increase of virulence. Therefore, the engineered E. coli strain will not become more hazardous. Dr. Bajgelman commented that the project could be carried out in a level 1 risk laboratory and adopt procedures and equipment for individual or collective protection corresponding to this level. More information can be found in the Safety Form (Documents Section).

   The CNPEM.Brazil teams selected the elderly public as our target to build ideas upon diversity and also inclusion. It’s important to highlight that they're currently at constant risk, in a manner above all others, since they’re more affected by the pandemic. Having this in mind, we tried to capture their difficulties before the COVID-19 pandemic and after it. Before the COVID outbreak, the scenario could be illustrated as if there was no space for them to work or to live inside society, meaning that it was just as hard to be included in back in the days as it is now, getting even worse as the pandemic reached its peak much time all along the year. The project’s primary issue was investigating how the pandemic affected this, how the government handles public lives, the impact of reimbursement, society/family acceptance, immersion, etc. 

   The team’s initial intention was to perform integrated practices with the elderly public, aiming for their inclusion in society and looking at their point of view of the world. Although the covid-19 pandemic frustrated these plans, it influenced the team to find ways to interact online with the target public. In this way, CNPEM.Brazil introduced the mini web series “Já diziam meus avôs”. The show consisted of Instagram videos including older people of participants' families. The video hosts taught scientific knowledge through family traditions and empirical costumes, utilizing a simple language to target diverse people. You can watch through the following links:

• https://www.instagram.com/tv/CT9szSMhEFY/?utm_source=ig_web_copy_link
• https://www.instagram.com/tv/CU-J1o1ghzh/?utm_source=ig_web_copy_link

   Science is a space that reflects social differences in society, such as gender inequality in prizes and recognition of outstanding achievements. It is essential to create events and other initiatives to debate this to increase the inclusion of women in science. In this way, as described in section 3.4.2, the team organized a live on Instagram to discuss this issue and hear the opinion of a female researcher.

   Art is a way for humanity to express their emotions, stories and also communicate ideas to promote their understanding. There are several languages in which art can be performed, and the team sought to simplify scientific concepts through drawings and illustrated schemes, aiming to unite two areas that for many may seem distant, art and synthetic biology. 

   Therefore, as mentioned in the topic 3.4.5. we attempted to disseminate information about GMOs and bioeconomy in poster format that was made available for free to the public and can be accessed at (https://drive.google.com/drive/folders/1bWx0htbz5j44ct2WLSkC_0avhwZU4_W5?usp=sharing). And through a banner, we also seek to publicize the iGEM and iGEM Design League competition so that more people have the opportunity to get to know and be a part of this international community of synthetic biology. 

   In our series “Já diziam meus avôs" (topic 3.4.7), we also aimed to communicate complex biology concepts in a simple way, validating the empirical knowledge of the elderly through science. Figure 5 illustrates the cover, our character, and the subject of one of the episodes.

Figure 55. Episode of the web series "já diziam meus avôs".

   In addition, the team's mascot, “Dotinho” (“Little Dot” in free translation), was present throughout our path as a team and its various versions were used to permeate the challenges proposed by iGEM Design League. In one of our collaborations (3.4.4), they were protagonists in the promotional material on environmental awareness for children. 

   We believe that science should be shared and accessible to the widest possible audience and art was one of the ways in which we carried out this exchange between the team and the population of all ages, aiming for more and more people to be able to design with biology.

The following documents are attached to this project page. Just click on the "Documents" window instead of "About" or click on the links below:

• Parts Information - Supplementary

• Designed Biological Parts - Fasta

• Metabolic Models - Code (also on GitHub: https://github.com/levandoskije/RainbowDotsCodes)

• Math Model - Supplementary

• Math Model - Code (also on GitHub: https://github.com/levandoskije/RainbowDotsCodes)

• Bioreactor - Table of Costs Supplementary

• Collaboration CO₂ Snatchers - CO₂ Emissions and Capture Technologies

• Safety Form

• "Formulário CIBio para projeto com OGM"

• Protocols

• JOGL Template