Improvement of the production of bacterial cellulose is the project's main objective. However, to be able to do this, several steps are necessary. Since the project as a whole is audacious, it was impossible to go through all the stages of the project during just one iGEM competition. However, the steps were chosen individually to be possible proof-of-concept of the proposed design.
With the use of the wild strain, it has already been possible to produce cellulose sheets in different formats. This demonstrates that the methods used for cultivation are valid and can be optimized in the future. The protocols for the cultivation of bacterial cellulose in 3D molds demonstrate promising results, and the results indicate it is possible to grow cellulose in different formats, opening new possibilities. This is an important achievement because if the cells can adapt any 2D different formats of the proposed molds, the necessary configuration for the consumer can be carried out.
>More information can be seen in the results section. link: https://2022.igem.wiki/unicamp-brazil/results
The laboratory tested the growth of mammal cells in the cellulose sheets, and the results demonstrated that bacterial cellulose is not cytotoxic for cells. This growth was performed on human skin NIH3T3 fibroblast cells with Dulbecco’s modified eagle medium (DMEM) high glucose culture medium. We used a 24-well plate, cutting pieces of cellulose equivalent to the size of each well individually and later depositing the prepared cells on top of this cellulose blanket.The results of this test demonstrated that human cells could attach themselves firmly to the cellulose blanket, in addition to maintaining their growth efficiently.
After first testing, we noticed that it was possible to grow but presented some differences in the phenotypes, the hypostasis the curvature in the sheets. After that, 3D models were printed for the growth in reduced sizes to fit in the 24-well plate.
For the production of cellulose, the design will use a light inducer promoter. In this way, it is necessary to grow under dark conditions. Besides that, Komagataeibacter rhaeticus is an obligate aerobic microorganism. The bioreactor prototype was tested with E. coli, as the first step, because of the elevated growth velocity. And there is already a list of new changes for optimization.
The principal cost of the production is the culture media to use a sustainable medium and reduce the total costs of production. The team tested different types of alternative media. Opening doors to the possibility of reducing production costs using media containing agroindustrial residues. The growth of Komagataeibacter was very efficient in the medium constructed from the waste left over from the production of fruit juice, having a good amount of sugar and allowing the normal development of the bacteria.
After several rounds of Design-Build-Test-Learn, the lineage transformation protocols were optimized. The K. raethicus AF1 strain will have reported a successful transformation process for the first time. Optimizing the protocols tests different parameters for thermal transformation or electroporation and drug resistance.
With the use of math modeling tools and based on data presented in the literature, it was possible to predict that when the complete system is assembled, it will be possible to obtain an increase in production and also in the speed of growth.
The last part of finalizing the project is still in progress. The delay in delivering the requested genes prevented the timely completion of the competition.
Taken together, all results indicate that our design can be applied to improve Bacterial cellulose production for the proposed implementation of our project, which is to use the agroindustrial waste to make BC more available to the industry.