The Unicamp_Brazil team was established based on the premise of employing synthetic biology to solve local environmental challenges, providing a significant contribution to society. We began with visits to waste management facilities and initially identified polystyrene accumulation in landfill as a pressing matter that demanded attention. We designed a project for the bio-conversion of polystyrene into bioplastics such as PHA or PHB, however, upon calculating the negative environmental impact of polystyrene transport, we realized that the environmental costs would outweight the potential benefits of the proposed bioconversion. Thus, we returned to the planning stage and identified another massive source of pollution in our region: agroindustry residues.
Searching for ways to up-cycle this biomass, we came across Dr. Hernane Barud´s research group using agroindustry waste as a substrate for bacterial cellulose (BC) production by the bacteria Komagataeibacter rhaeticus AF1. BC is chemically very similar to its plant counterpart but synthesized in a purer state, which allows many downstream applications in areas as diverse as packaging or treatment of wounds. The strain K. rhaeticus AF1 was isolated from kombucha tea. Due to its high capacity of producing BC from unconventional sources, its genome was sequenced¹, but the strain (owned by BioPolMat (UNIARA), Biosmart Nanotechnology LLC, and HB Biotec consortium) had never been genetically manipulated.
In June we decided to take on the challenge of domesticating K. rhaeticus AF1 and developing a pipeline spanning the cultivation of the strain to creating new biomedical applications for BC. Following the synthetic biology principles of design-build-test-learn, we designed, executed, and evaluated experiments from pulp to new BC applications. As Unicamp_Brazil is a diverse and multidisciplinary team, we worked on the optimization of culturing conditions, metabolic modeling as a guide for genetic manipulations, development of transfection protocols, design and construction of a toolbox with plasmid backbones suitable for K. rhaeticus AF1 engineering, design of low-cost induction system, construction of a low-cost dual-stage bioreactor, development of molds for custom BC sheet production, successful tests of adhesion of human cell lines to BC membranes, and, last but not least, dissemination of synthetic biology to the wider community.
The world demands sustainable technologies in order to prosper, and one of science's means of achieving this is by using synthetic biology techniques. Cellulopolis is a project designed to meet demands from several markets, with the production of pure bacterial cellulose (BC) obtained from genetically-modified strains. Cellulose (CL) is a naturally occurring biodegradable polymer that has properties that allow its applicability as a fibrous scaffold for skin, cartilage, and vein, among others, as well as being an excellent alternative in the treatment of burns. Obtaining CL from plant sources by traditional methods is cumbersome due to difficulties in purification, genetically modified organisms have been the most viable alternative in this regard. Some microorganisms produce CL naturally, as is the case with Komagataeibacter rhaeticus AF1 strain, found in a fermented beverage popularly known as Kombucha.
Genetic engineering can optimize the metabolic pathways of various microorganisms. In our project, we performed computational simulations of key features in the BC synthesis pathways and identified potential targets for modification. Thus, we are genetically engineering, for the first time, K. rhaeticus AF1 by using Modular Cloning (MOCLO) compatible transcription units designed to increase BC production with minimal impact in biomass accumulation, so as to not give a selective advantage to non-BC producing strains. This will be achieved through a bimodal growth/production strategy, as will be discussed below.
Brazilian agroindustry produces over 290 million tons of waste each year, which could lead to social and environmental risks (Siqueira et al. 2022). We decided to work on a bacteria strain capable of producing the BC in a culture media derived from agroindustrial residues. Our project aims to sustainably increase the production of BC, aided by strain engineering employing a genetic toolkit we are adapting for Komagataeibacter, thus making BC more accessible for the treatment of burns, other injuries, or further applications.
The availability of natural resources is limited, which has a great impact on the social, scientific, and economic aspects of our daily lives. Since the industrial revolution, the world has witnessed the unbridled exploitation of the environment. This has been shaping people's way of life; we have had many scientific advances, but we witness the negative consequences of climate change and environmental and socio-economic imbalances. Underdeveloped countries suffer most from this exploitation, and communities are often unaware of the catastrophic structure behind policies that exclude sustainability from the mainstream agenda.
Our generation has the duty to circumvent all these issues by working in a sustainable and interdisciplinary fashion. Moreover, it is imperative to disseminate knowledge, so that with access to information, we can all unite for a better world. With this in mind, our Cellulopolis project is based on sustainability guidelines, striving for the production of pure BC in high yields at low cost, to maximize benefits to the medical field and further areas.
Following the principles of green chemistry, we aim to up-cycle biomass generated in the massive agroindustrial production in Brazil, by giving it a useful destination, in contrast to discarding in the environment. We disseminate our work in schools so that new generations are inspired by science and its various contributions. With support, we are sure that Cellulopolis will achieve significant improvements in the medical treatment of burns in people regardless of their socioeconomic status and will enable the advancement of medical research using BC as a scaffold.
Cellulose is a natural polymer present in the structure of cell walls throughout the plant, formed by a complex chain of sugars, including glucose. It is a very abundant material (33% of all plant biomass on the planet) and is intrinsically linked to various elements of everyday life, such as paper, diapers, fabrics, pills and even food. However, the processing of wood into cellulose requires large amounts of water and energy, in addition to the use of polluting chemicals, such as nitrogen and sulfur, which are released into the air. It is also worth noting that the purification of cellulose from plants is expensive and laborious, leading to the need to replace the production of this polymer with a more sustainable alternative.
A good alternative to plant cellulose is bacterial cellulose, which has the same chemical structure as the plant counterpart, but without “contaminants” such as hemicellulose, lignin, and pectin. This easily moldable material has a three-dimensional network structure of cellulose nanofibrils in the form of a ribbon, which is capable of efficiently absorbing and retaining water.
This BC is produced by bacteria in a natural and purer way than by plants, being a very versatile natural biomaterial that can have its production optimized through bacterial engineering techniques. Bacteria can convert about 50% of carbon into cellulose, varying according to growth conditions, and naturally produce it for various purposes, such as protection and preservation of nutrients. The properties of cellulose vary according to the bacterium that produces it and the substrates available in the cultivation medium.
There are several possible applications for bacterial cellulose, including in the medical field, where it can be widely used in the treatment of wounds and burns, as artificial skin during healing. However, this process has a high cost and pulp production is not very efficient, making it necessary to seek measures for the development of modified strains in order to increase pulp production, and also to lower the costs involved in this process. Bacterial cellulose can also have applications in food, cosmetics, and bioethanol areas, not forgetting its use as a scaffold for the growth of cells, tissues, and organs.
The standard medium for cultivation of Komagataeibacter is HS (Hestrin; Schramm, 1954), which we employed during most of our work.
The idea for a waste management project arose from research and brainstorming within the team, which started its journey in 2021, with an intense interest in making a real impact on the paths of synthetic biology in Brazil and the desire to transform waste into high-value products. Our goal until May 2021 was to engineer bacteria to digest polystyrene and convert it into PHA or PHB, in a project named Styropolis.
We designed a strategy whereby that goal could technically be achieved, however, through diverse insights obtained by stakeholders we learned that, even though polystyrene is recyclable and could potentially be used as a carbon source for bioplastic production, its low density leads to high transportation costs per kg of raw material, creating a negative environmental impact that would detract from our “green” goals. This is discussed in greater detail in the Human Practices section.
Inspired by the Imperial_College iGEM team (K. rhaeticus iGEM strain sequencing and toolkit development) and by collaborators from BioSmart Nanotechnology, we decided to develop a strain capable of producing cellulose on a large scale utilizing based on Komagataeibacter rhaeticus AF1, through synthetic biology. The Cellulopolis project was born!
BioSmart Nanotechnology is a research and development company that, in collaboration with JBT Corporation, works on the Food Waste project, which consists of the reuse of agroindustry residues to produce BC (a technology proprietary of the consortium Uniara, BioSmart Nanotechnology, and HB Biotec).
We received permission to work with the industrial K. rhaeticus AF1 strain and proceeded with its characterization. As we were working with the same species as the Imperial_College iGEM team (K. rhaeticus iGEM), we based our initial strain domestication attempts on protocols optimized by the London-based team, however, as will be discussed in other sessions, K. rhaeticus AF1 strain behaves very differently from the K. rhaeticus iGEM strain, therefore culturing and engineering conditions had to be dramatically changed. The scientific literature presents a number of plasmid origin of replication, promotors, RBS, terminators, and antibiotic selection markers that should work for Komagataeibacter, however, even though the iGEM distribution kit supplied many of the individual components we required, they were not in a combination that would allow K. rhaeticus engineering, therefore we had to develop a custom toolbox for K. rhaeticus AF1 genetic manipulations.
Most of our technical challenges were not encountered in the lab, but on accessing the iGEM part collection, primers and custom genes, all of which were retained by customs for prolong periods (IDT primers and parts were delivered less than 3 weeks prior to the wiki freeze). Taking our maiden journey into the iGEM world, we are also learning how to navigate the intricacies of uploading parts into the part collection (NOT intuitive and impossible to correct) and competition requirements and expectations.
Our goal with Cellulopolis is to provide a draft of the multiple steps required in the process of taking waste into a high-value product with multiple biomedical applications.
We began by cultivating wild-type K. rhaeticus AF1 in HS medium, established conditions for genetic manipulation and performed metabolic modeling (based on the genome-scale metabolic model available for the closely related Komagataeibacter xylinus) to suggest genetic changes that can improve BC production, developed and tested a low-cost bioreactor, created a series of GoldenGate K. rhaeticus compatible plasmid backbones, designed and constructed multiple basic parts to drive BC synthesis in a bimodal fashion (low-cost light-inducible expression - not characterized due to time constraints), produced BC sheets in custom formats, and confirmed the viability of utilizing BC as a substrate for culturing cells, which can ultimately be employed in human tissue engineering.
In order to consistently work with accessible tools, we only made use of free online resources and also made available to the community the notebooks used for the computer simulations. Their respective explanations are reachable on our model page, which may be useful to future teams using genomic scale models (GEM) in their research. Using Python on the Google Collaboratory platform with open-source libraries (Cobrapy), the results previously reported in the literature were successfully reproduced. Our results can predict critical reactions in BC production pathways and the approach used to estimate parameters for kinetic models, using flow balance analysis, brings insights into the possibilities of using GEM. Furthermore, the simplified kinetic model can be adapted for different processes and conditions. We also designed a simplified interactive pathway (Flow Mechanism Kinetics game), in which the user can vary the concentration of different enzymes involved in directing cellular energy towards the production of BC, with the main objective of balancing cellular metabolism in order to achieve maximum BC production by the bacterial population.
Bacterial cellulose is produced by several groups of bacteria and among them is Komagataeibacter, with one of the most efficient productions of this polymer. K. xylinus is the most studied bacterium of the genus and its metabolism has been well studied and described, being used as a model for the study of cellulose production. In the present work, we focused our efforts on the related species Komagataeibacter rhaeticus AF1, a producer of the fermented tea (kombucha), which was isolated similarly to K. xylinus, as a producer of cellulose. The iGEM project was hosted by a yeast laboratory, therefore we carried out several experiments to gain experience with bacterial growth and BC production, aiming at an optimization of cellulose synthesis through the cultivation conditions, including the composition of the culture medium itself. The literature describes genetic engineering efforts to develop Komagataeibacter with improved BC synthesis, however, it is unclear if engineered strains are stable in large-scale production conditions. Thus, we would like to contribute to the BC research community by building new parts and adapting methods and protocols, aiming at a more efficient cellulose synthesis.
As K. rhaeticus AF1 is not a standard model organism, there are no protocols adapted for this specific bacterial strain, so the Unicamp_Brasil team worked with experimental conditions and protocols to allow the cultivation and genetic manipulation of the locally isolated strain. We performed experiments to identify optimal incubation temperatures for K. rhaeticus AF1, looking for the most suitable temperature to maximize its growth. In addition to the media commonly used in laboratories, such as Hestrin-Schramm (HS). In addition, titrated the concentration of different antibiotics in the growth media in order to determine the minimal concentration required to discriminate between wild-type and transformed strains. This required extensive research into optimized protocols for closely related species and a long period of testing with many variations in parameters, which culminated in an adapted set of instructions for handling and genetically manipulated Komagataeibacter rhaeticus AF1.
Based on metabolic modeling, we designed a pipeline for the bimodal production of cellulose, in which the first phase focuses on bacterial growth by deactivating the cellulose production operon, while the second focuses on the activation of genes that involve the synthesis of cellulose by light induction. Aiming at optimizing and increasing the effectiveness of the growth of aerobic obligate bacteria, we designed and built a low-cost and easy-to-handle bioreactor, being an efficient tool for other research groups and future iGEM teams working with aerobic organisms or stimulus-dependent species. lights to achieve the expected results.
From the idea of using BC as a surface for tissue culture and using a 3D printer, we built molds that allow the formation of this in well-defined shapes, representing the surface of a complex object, capable of entering tissue or organ engineering with BC. We also design and build "cookie cutter" models for 3D surfaces such as a sphere and create multi-hex scaffolding that allows BC to be produced in dimensions compatible with 24-well tissue culture plates. Furthermore, we were able to purify BC, successfully with cultured fibroblasts and melanoma cells on the surface of our polymer.
We cultured human skin MIH3T3 fibroblast cells and human melanoma cell lines (data not shown) on a bacterial cellulose blanket we produced in our laboratory. We observed that both cell types adhere well to BC, which paves the way for employing bacterial cellulose sheets as scaffolds for tissue engineering. We cultured C2C12 myoblasts cells plated on bacterial cellulose that was produced in large sheets, and bacterial cellulose produce in a 3D 24-well plate model both produced in our laboratory (Figure 2). We observed no difference in the adherence of the C2C12 cells cultured in bacterial cellulose and the control cells. Bright-field images were captured in confocal microscopy. This paves the way for employing bacterial cellulose sheets as scaffolds for tissue engineering.
