Bacterial cellulose (BC) is a biopolymer that has been explored by numerous iGEM teams over the years. This is due to its wide range of applications, versatility and purity: great qualities that demonstrate its importance as an alternative to plant cellulose. In this context, Cellulopolis addresses this hunger for development and innovations in BC production, establishing new tools and approaches, and demonstrating new uses. Furthermore, during the course of the project, actions in collaboration with other iGEM teams exemplify some of Unicamp_Brazil's contributions to future teams.
Bacterial cellulose biosynthesis is carried out by different groups of bacteria, which are the research focus of different groups aiming to optimize production. The genus Komagataeibacter is known to be one of the most efficient cellulose producers, capable of using carbon and nitrogen from different sources. Within the group, K. xylinus (Corrêa dos Santos et al. 2014) was the first to have its cellulose production mechanism described and studied, facilitating its use as a model for studying the biopolymer production process.
More recently Komagataeibacter rhaeticus AF1 was isolated in Brazil from fermented tea, kombucha, and identified as a major producer of cellulose, in conditions similar to K. xylinus. The Komagataeibacter AF1 strain was donated from BioPolMat (UNIARA), Biosmart Nanotechnology LLC, and HB Biotec consortium. Since then, some analysis have been carried out on the productivity and growth of this strain, leaving a gap in its genetic studies.
For a long time, most studies aimed at optimizing cellulose synthesis through changes in cultivation conditions, including the composition of the growth medium itself. Only in recent years, new techniques of genetic engineering and synthetic biology have been applied to the development of microorganisms more efficiently in the synthesis of cellulose.
We've secured permission to use the industrial strain K. rhaeticus AF1 from BioPolMat (UNIARA-Araraquara - São Paulo/Brazil), Biosmart Nanotechnology LLC (Araraquara - São Paulo/Brazil), and HB Biotec (Araraquara - São Paulo/Brazil) consortium and, by contributing to the construction of new parts and the adaptation of methods and protocols, we were allowed to use it in new studies aiming at higher cellulose synthesis. Therefore, Unicamp_Brazil is dedicated to researching the conditions and tools which make K. rhaeticus AF1 more amenable to manipulation.
Given that K. rhaeticus AF1 is not a model organism, there are no protocols adapted for this particular bacterial strain. The team performed intense troubleshooting with experimental conditions and protocols to allow cultivation and genetic manipulation of the locally isolated strain.
Different temperatures were tested in order to identify the optimal growth temperature for K. rhaeticus AF1. From a common initial inoculum, the experiment was developed at three different temperatures, 30℃, 37℃ and room temperature. We observed that K. rhaeticus AF1 is not able to grow at 37℃ and that its growth at room temperature is less efficient than at 30℃; at such temperature, it presents a significant increase in its doubling time. Therefore, 30℃ was defined as the ideal temperature for the cultivation of the strain, maximizing its growth.
The standard culture medium used for the growth of bacteria of the genus Komagataeibacter is Hestrin-Schramm (HS), supplemented with of a significant amount of glucose (2%). For competent cell preparation, strain transformation and quantification of cellulose production, we adopted some small changes in this medium. Furthermore, Cellumi-natti (the partnership with the Ui_Oslo team) utilized an optimized medium for the growth of K. xyllinus, which we tested for K. rhaeticus AF1.
From the onset, a concern of the Unicamp_Brazil team has been to upcycle waste products. Brazilian agroindustry produces over 290 million tons of waste each year, which could lead to social and environmental risks (Siqueira et al. 2022). To deal with this issue, we propose the use of agroindustrial residues as a more sustainable and low-cost alternative for the growth medium for K. rhaeticus (Akintude et al. 2022, Gomes et al. 2013, Pacheco et al. 2017, Urbina et al. 2021).
In order to obtain a better understanding of K. rhaeticus AF1, we studied the efficiency of different antibiotics in inhibiting the growth of the bacteria. We were surprised to notice that K. rhaeticus AF1 strain is manyfold more sensitive to the antibiotics kanamycin, chloramphenicol, ampicillin and spectinocymycin than reported in the literature (Goosens V. J. et al., 2021) for the closely related strain K. rhaeticus iGEM. This preliminary characterization of our strain is essential for the development of transformation protocols and the tuning of antibiotic concentrations that could allow the selection of transformants without impinging their growth. This knowledge can contribute to its manipulation by future teams.
Since the strain adopted as the chassis in Cellulopolis had never been transformed, this experiment wasn't trivial. It demanded extensive protocol research optimized for closely related species and a long testing period with many variations in parameters, which culminated in an adapted set of instructions for the manipulation of Komagataeibacter rhaeticus AF1. Unicamp_Brazil was able to efficiently transform the bacterium, through the development of a customized protocol, helping future teams interested in working with the same organism or similar species.
Following the idea of empowering the community, the team chose to only use free tools to expand access for all. With this in mind, we made available all the notebooks used for the simulations, and the explanations on the model page, which can instigate and help future teams use genomic-scale models (GEM) in their research. The modeling was entirely run using Python in the Google Collaboratory platform with open-source libraries (Cobrapy), and successfully reproduced the results previously reported in the literature. Our results could predict critical reactions in the pathways to produce cellulose. Also, the approach used to estimate parameters for kinetic models using flux balance analysis brings insights into the possibilities of using GEM. Moreover, the simplified kinetic model can be adapted for different processes and conditions, and can be used to predict the best timing for inducing cellulose production.
With the goal of making metabolic models more comprehensible to non-mathematicians, we designed a very simplified interactive pathway (the Bacterial Cellulose game), where the user can vary the concentration of different enzymes involved in directing the cell energy towards BC production. As the GEM used predicted a strong competition between biomass accumulation and BC production, the main goal of the game is to balance the cell´s metabolism in order to achieve maximum BC production by the bacterial population.
Inspired by the ease and reproducibility of toolkits developed for different model organisms, and identifying the gap in specific tools used with Komagataeibacter rhaeticus AF1, Cellulopolis planned and is successfully building a customized toolkit for the strain. The toolkit is based on the cloning strategy of MoClo - Modular Cloning, which is applied through the hierarchical Golden Gate (GG) method, which benefits from the qualities of Type IIS restriction enzymes and allows other research groups to work efficiently with the same species. This toolkit includes level 1 and level 2 plasmids with replication origins and antibiotic resistance markers functional in K. rhaeticus, as well as basic parts highlighted in metabolic models as possible engineering targets. We also planned and partially constructed a light-inducible system to allow the transfer of our platform to large-scale BC production.
Following predictions and suggestions from metabolic modeling, we designed a pipeline for the bimodal production of cellulose: in the first phase, the focus is the growth of the bacteria by deactivating the cellulose production operon, while the second phase consists on the activation of the genes involved cellulose synthesis through light induction. As a way of optimizing this process and increasing the growth efficiency of the obligate aerobic bacteria, Unicamp_Brazil designed and built a low-cost bioreactor that is easy to handle in any laboratory. This hardware is an efficient tool for other research groups and future iGEM teams that work with aerobic organisms or species dependent on light stimuli to achieve the expected results.
Research groups had proposed the use of bacterial cellulose as surface for tissue culture. Inspired by this idea, we used a 3D printer to build molds that allow the formation of BC in well defined shapes, which could represent the surface of a complex object, paving the way for tissue or organ engineering with BC as a scaffold. We designed and built these “cookie-cutter" models for 3D surfaces such as that of an sphere. We also created scaffolds for multiple hexagons that allow BC to be produced in dimensions compatible with 24 well tissue culture plates. We purified the BC and successfully cultivated fibroblasts and melanoma cells on the surface of our polymer.
Reproducibility and reliability of measurements between different laboratories is a challenge in the research universe, including the area of synthetic biology, which makes it difficult to share data and interpret results. In this context and through the International InterLaboratory (Interlab), iGEM proposes studies capable of developing more robust measurement procedures capable of minimizing these difficulties. Our team participated in the Sixth International InterLaboratory Measurement Study performing all the three proposed experiments and was therefore able to contribute to future teams.
The experiment uses three-color calibration dyes prior to measuring emissions from cells expressing a single fluorescent proteins or 2 simultaneous fluorescent proteins. The experiment proposes the evaluation of the reproducibility between laboratories of the three-color calibration protocol.
The experiment is based on the measurements of the expression of two fluorescent proteins in the same cell, following a three-color calibration protocol. It encourages the analysis of the effects of the order of transcriptional units on the expression levels of the two different proteins.
With this experiment, we evaluated the reproducibility of the fluorescence measurement in 96-well plates over time.
Participation of developing country teams in iGEM is hampered due to the high registration fees and high costs for transport, food, and accommodation in the city chosen to host the Jamboree. We also struggle to receive kits and products provided by competition sponsors, which often have long delays, or aren't even delivered. Faced with these issues, our team contacted other LATAM teams that registered for iGEM 2022, seeking to understand all the demands and discuss possibilities, as well as talking to participants from previous editions of the competition. Finally, our team elaborated a letter to formally communicate iGEM headquartes about the main difficulties faced during our journey, in an attempt to incite changes that could help the next teams, make the competition more inclusive, and expand synthetic biology through the world.
Our society is facing the threat of a modern dark age with anti-science movements (such as antivaxxers, flat-earthers, anti-GMOs, etc) gaining strength and popularity. We believe that it is imperative to dedicate ourselves to change this scenario with a bottom-up aproach. While there are many talented groups at Unicamp working on synthetic biology, the university lacked a hub to disseminate and educate society on the potential of sinthetic biology. With this in mind, Unicamp_Brazil iGEM team members started a Synthetic Biology club that focus on taking Synbio to the wider community.