As a naturally formed immobilizing structure of bacteria, biofilm outstands in many ways such as the ability to withstand considerable degree of environmental fluctuation, the possibility for more efficient cell-cell communication as well as cooperation, and the stratified structure which may function greatly in phase-related synthetic processing. With this understanding and beyond, 2011 ZJU-China iGEM team tried to explore more about the potential of biofilm in applications.

Cellulose utilization for renewable bio-material production (especially bio-fuel production) has been a heated research interest for quite a long time. Due to the structural complexity and poor permeability of cellulose, a big obstacle for enzymatic break-down process, progress in promoting the utilization efficiency is slow.

Considering this problem with the advantage of biofilm as a reactor system, we believe the employment of biofilm and its stratified feature would help to surmount such obstacle.

Firstly, Sugarfilm, a three-layer biofilm-based expression system, is designed to directly attach to the surface of substrate (for instance, a sheet of cellulose debris). In this way, the distance between our “degradation engine” (E.coli cells) and the targeted substrate would be reduced greatly, laying the foundation of a more efficient system.

Two main cellulases involved in the first step of degradation, Cex (Cellulomonas fimi exoglucanase, BBa_K118022 , iGEM08_Edinburgh) together with CenA (Cellulomonas fimi endoglucanase A, BBa_K118023 , iGEM08_Edinburgh) will be expressed in the bottom layer of biofilm (as described in the “Design” part below in detail). Therefore, the secreted enzymes would get in touch with the substrate instantly.

In the next step, the preliminary products cellubiose from the first layer would diffuse to the middle layer of Sugarfilm due to concentration gradient. Beta-glucosidase BglX (from Cytophaga hutchinsonii, BBa_K118028 , iGEM08_Edinburgh) expressed in the middle layer would now work to degrade the disaccharide into glucoses. As the simplest saccharide and the starter substrate of many synthesizing pathways, glucose can diffuse to the upper layer of Sugarfilm and set off the last step of the reaction system.

The upper layer of Sugarfilm is capable of being designed to host any synthesizing pathway starting from glucose. Except for the versatility, such design can protect cells from being harmed by synthesized products detrimental to the cell. Since the procedure happens in the top layer of Sugarfilm, the undesired by-products in the system could be got rid of quickly, which would lead to death of only a small amount of cells in the biofilm, ensuring the integrity and functionality of other regions of the system.

Generally, with proper medium flow outside the biofilm, final products and wastes would leave the system immediately, and the continuous nutrient input provides good conditions for upper layer renewal. In ideal conditions, Sugarfilm could stick tightly to the substrate sheet surface until the whole sheet was degraded thoroughly.