The Cellulopolis project involves multiple stages in the development of a Synthetic biology-based approach to solving real-life problems. In the course of our work, we dedicated ourselves engineer solutions at multiple levels, from problem identification to the final product. In the table below we enumerate key steps in our journey as examples of our engineering success.
| Task | Intermediate steps | Current status |
|---|---|---|
| Define project that can have a positive outcome for our environment and society | Design of the Styropolis project. Abandoned in May as would have a negative environmental impact when pollution due to transport was accounted for. Switch efforts to Cellulopolis. | All results obtained thus far are consistent with the potential of positive impact of Cellulopolis. |
| Acquire suitable chassis and learn how to cultivate it (growth conditions and BC production) | Invited to UNESP in Araraquara for hands on training | We established good cultivation conditions for K. rhaeticus AF1 and K. medellinensis and produce BC in the lab. |
| Define antibiotic concentrations and establish electroporation protocols | Standard protocols for closely related K. rhaeticus iGEM strain was not applicable to AF1 | We established new growth conditions and electroporation protocol, achieving good transformation efficiency. We confirmed that AmpR and KanR can be used for selection in AF1, but have no conclusive results on CmR or SpecR. |
| Identify candidate plasmid backbones | Literature searches point towards the use of RK2, pBBR1 and RSF1010 replication origins | Thus far we validated the efficiency of pBBR1 and RSF1010 as ori in AF1, but have no conclusive results on RK2. |
| Identify candidate promotor, RBS, reporter, selection marker and terminator | Order synthesis or employ parts from iGEM toolkit | Subcloning into level 0 plasmids apparently successful (colony numbers and gel profile). Minipreps awaiting sequencing results. |
| Model metabolism to establish targets for engineering | Suggestions of major changes (bimodal control of bcs) and of individual genes which overexpression might favor BC production yields | Awaiting cloning into level 1 and 2 plasmids, as well as genomic integration, for testing |
| Design primers for cloning | All PCRs successful | Transformations into level 0 plasmids successful. Minipreps awaiting sequencing results. |
| Design genes for synthesis | Genes recently arrived | Transformations into level 0 plasmids successful. Minipreps awaiting sequencing results. |
| Design new level 1 and level 2 backbones with selection markers compatible with Komagataeibacter | Level 2 plasmids with selected ori encoded SpecR, not used for Komagataeibacter |
PCRs successful
Validation in E. coli successful
Transformation into Komagataeibacter successful
|
| Design constructs for genomic integration | PCRs from genomic DNAs from K. rhaeticus AF1 and K. medellinensis ID13488 successful | Integration cassette under construction |
| Design new level 2 backbones with adapters compatible with assembly of 4 TUs derived from pOdd1-4 | Adapters PCRs successful | E. coli colony phenotype consistent with successful level 2 adaptor constructs! |
| Design of level 1 composite parts | Most level 0 parts required are ready | Under construction |
| Design of level 2 composite parts | Under construction | Under construction |
| Design of a low cost bioreactor | Constructed | Functional but requires optimization (current tubing not resistant to autoclaving) |
| Design of a container for light induction | Constructed | Awaiting light responsive candidate strains for testing |
| Design of molds for custom BC production | GCode generated and 3D printing done | Molds efficient in limiting BC shapes allowing the formation of membranes suitable for coating spheres or tests in 24 well plates |
| Cultivate of human cells using BC as substrate | Fibroblasts and melanoma cell lines | Cells adhere and grow well on BC |
Our engineering started with the conception of the project, which focused on dealing with environmental problems and providing a positive outcome for society. We began constructing it from the information we collected with research on waste management and visits to recycling centers. This culminated with the idea of working with an enzyme capable of degrading polystyrene, the main component of styrofoam, which has almost no destination other than landfills, because of the lack of profitability in its recycling process. Thus, we began to run the tests for pre-treatment of the material and encountered some barriers due to the approach’s inefficiency, such as a negative environmental impact when pollution due to transport was accounted for. With this scenario, we learned that our first idea could be unfeasible and re-set the design project design for the production of BC from agroindustrial residues, a project that actually shows consistent results regarding positive social and environmental impact.
An important step in engineering our chassis is the transformation of Komagataeibacter with the plasmids of interest. We planned this step from extensive literature research and protocol analysis in order to tailor efficient instructions for the strain of interest. We tested the original transformation protocols published for K. rhaeticus iGEM, using our Komagataeibacter rhaeticus AF1 strain. Unfortunately, our first attempts were not successful, since it was not possible to observe the growth of transformed strains on the antibiotic plates. This scenario led us to question the antibiotic concentrations adopted in the published protocol and perform experiments to test the resistance of K. rhaeticus AF1 to various concentrations of the antibiotics Kanamycin, Ampicilin and Chloramphenicol. With the results of this experiment, we interpreted the bacterial response to different antibiotics and learned to adapt the protocol efficiently. From there, we rethought the design for the transformation, establishing optimized protocols for retesting, which allowed us to successfully transform K. rhaeticus AF1 for the first time. It is important to note that we successfully transformed K. rhaeticus AF1 with novel backbones constructed in this project.
Level 2 plasmids from iGEM’s kit that have compatible origin of replication for Komagataeibacter encode for the spectinomycin resistence marker which has not being reported as functional for K. rhaeticus. Furthermore all level 1 plasmids with ori compatible with K. rhaeticus encoded KanR marker, limiting the options of plasmid combinations. Therefore we designed and constructed level 1 and level 2 plasmids with AmpR and CmR, markers compatible with Komagataeibacter.
Our strategy for the assembly of multiple transcriptional units for concomitant overexpression of all genes encoding key enzymes involved in BC synthesis, requires the level 1 assembly of the promotor, RBS, CDS and terminator in pOdd1-4. Inserts from pOdd plasmids can then be assembled in compatible level 2 plasmids. Unfortunately, the iGEM distribution kit did not provide level 2 plasmids that could combine pOdd1-4 with a backbone encoding K. rhaeticus compatible ori. Thus, we designed adaptors for the replacement of prefix and suffix from BBa_J428366, BBa_J428367, BBa_J428369, BBa_K4435304, BBa_K4435305, BBa_K4435311, and BBa_K4435312 with an adapter suitable for GoldenGate assembly of pOdd1-4 using the type IIs enzyme SapI. Thus far we confirmed the correct replacement of cloning regions of 6 out of 8 plasmids. It is important to note that 5 of the newly engineered plasmids were derived from marker swap plasmids we constructed in stage 9.
An astonishing application of bacterial cellulose is its potential as a scaffold for cell culture. To better evaluate BC suitability for this purpose we designed an assay to grow fibroblasts over BC sheets. The results showed good adhesion of human cells to the novel substrate but exposed the possibility that BC membranes might interfere with cell development and differentiation. After some modifications in the protocol BC production protocol and using human melanoma cells, we still identify steps that could be improved, such as the production of BC with more consistent thickness. This process drove us to design 3D molds for BC production in the adequate format and thickness for cell cultivation. We built Gcode, 3D printed the molds, and tested the production of BC hexagons of defined dimensions. Those were used in a new assay for cell culture, showing improved results, but suggesting new aspects that can be further improved.