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.

As an example, we designed a down-stream system to transform biomass into bio-fuel, producing iso-butanol with glucose. The non-fermentative metabolic pathway to do the job has been studied and optimized before, so we designed a very simple device to try it within our biofilm. Three enzyme coding sequences each with a RBS line down-stream the PtetR promoter, thus these bacteria can further transform the products of sugarfilm into biofuel.

Excitingly, we found this design opens a bright future for biofilm-reactors. In natural situations, biofilms can be formed by multi-species with cooperation, thus heavy burden can be resolved by adding ‘cooperators’. For example, type-A degrades cellulose into glucose while type-B make them into fuels.

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.

BBa_K561004 Sequence And Features

In the Sugarfilm expression system, BBa_K561004 is designed for bottom layer expression, conducting the first step of cellulose degradation. It is based on Rainbofilm bottom layer BioBrick BBa_K561002 , with substitution of mRFP (BBa_E1010 ) by Cex and CenA. The sequence is under regulation of anaerobic promoter fdhF, and the TetR expression would repress upper-layer device (Ptet regulated) in anaerobic condition, i.e. in the bottom-layer condition.

BBa_K561005 SequenceAndFeatures

BBa_K561005 is designed for middle layer expression, conducting the conversion of cellubiose into glucose. It is based on Rainbofilm bottom layer BioBrick BBa_K561000 , with substitution of eYFP (BBa_E0030 ) by BglX. To get the beta-glucosidase exported from cytoplasm, PelB tag(BBa_J32015 ) is added in front of the coding sequence of BglX.The sequence is under regulation of micro-aerobic promoter vgb, and the TetR expression would repress upper-layer device (Ptet regulated) in micro-anaerobic condition, which mostly exists in the middle part of the biofilm.

Coding sequence with regulation of Ptet would be suitable as the upper-layer device (such as BBa_I13602 used in Rainbofilm system). Expression of the device would be repressed either in the bottom or medium layer, and would do its job faithfully in the upper-layer region.

This device is an expression cassette of alsS, Kivd and Adh2 each with a RBS. alsS gene is from B.subtilis, encoding alpha-acetolactate synthase protein, which is a substitution of E.coli ilvHCD to optimize the production of 2-Ketoisovalerate. Kivd gene from L. lactis and Adh2 gene from S.cerevisiae encodes 2-keto-acid decarboxylases and alcohol dehydrogenases, respectively. These two enzymes catalyze the last two reactions in the pathway producing isobutanol from 2-Ketoisovalerate.

Three kinds of enzymes are needed to turn cellulose into glucose. Enzymes Exoglucanase and Endoglucanase are placed in bottom layer of bioflim, and β-glucosidase are in layer 2. layer 3 could be used to further degrade glucose to further degraded molecule such as alcohols. Our application is a combination of stratified bioflim and bio-conversion of celluloses which is a part of the project of Team Edinburgh of igem 2011. Therefore their model (with matlab) could be used to describe our system with some adjustment.

Based on the results of former sections, the expression of proteins in the bottom layer and middle layer are determined by only factor of time, and they would become a constant after the system reached a steady-state. Thus we could set the concentration of these three enzymes to be two constants E_1 and E_2, where Exoglucanase and Endoglucanase are of same concentration C_1 in bottom layer, and concentration of β-glucosidase is C_2 in layer 2. Team Edinburgh has already built a matlab based model in their igem 2011 project for free floating enzyme approach in simulating conversion of celluloses. Their method could be applied in our model. Assumptions of their models are listed below.

1 Underlying assumption: cellulose, cellobiose, and glucose concentrations change continuously with time.
2 Rate equations assume enzyme adsorption follows the Langmuir isotherm model.
3 Glucose and cellobiose, which are the products of cellulose hydrolysis, are assumed to "competitively inhibit enzyme hydrolysis".
4 All reactions are assumed to follow the same temperature dependency Arrhenius relationship (shown below). However, in reality it should be different for every enzyme component, "because of their varying degrees of thermostability, with β-glucocidase being the most thermostable. Hence the assumption is a simplification of reality".
5 Conversion of cellobiose to glucose follows the Michaelis-Menten enzyme kinetic model.
6 Concentration of xylose is assumed to be zero all the time for simplify.
7 Diffusion is assumed to be linear. That is, the concentration of cellobiose in layer 2 is assumed to be a constant times c (assumed to be 0.6) its concentration in layer 1.
8 The temperature of the system is a constant. (30℃)
9 Concentration of celluloses in middle layer is assumed to be zero (no diffusion).

Cellulose to cellobiose reaction with competitive glucose and cellobiose inhibition in bottom layer.

Cellulose to glucose reaction with competitive glucose and cellobiose inhibition in bottom layer.

Cellobiose to glucose reaction with competitive glucose and cellibiose inhibition in bottom layer.

Cellobiose to glucose reaction with competitive glucose and cellibiose inhibition in middle layer.

The Langmuir Isotherm model mathematically describes enzyme adsorption onto solid cellulose substrates.

Cellulose mass balance in bottom layer

Cellobiose mass balance in bottom layer

Glucose mass balance in bottom layer

Glucose production in middle layer

Overall glucose production is equal to g+G.

Overall glucose production is equal to g+G.

In bottom layer, we set E_1 to be 1g/kg, E_2 to be 0.01g/kg, and concentration of cellulose to be 100g/kg. Without considering the reaction in middle layer, the concentrations in bottom layer are shown in following graph.

• 1
• 2
• 3

 

Taking most of the advantages of biofilm as a reactor, Sugarfilm makes phase-related synthesizing procedure (in this case, 3 phases in total) more suitable in biological ways:
(1)Each cell only carries out one reaction step in the system, which greatly relieves stress to the cell when compared with cells carrying the whole process;
(2)The efficiency would not be weakened due to the separation of each step, since the formation of biofilm links all the cells together to get the work done in a nice flow.

What Sugarfilm illustrates here is more than just a simple application. It's a symbol of infinite possibilities coming with our stratified expression biofilm system (Rainbofilm). For similar synthesizing pathways involving a lot of extracellular reaction and intermediate product flow, and relatively clear phase division in the pathway, Sugarfilm would be an ideal model. Only by substituting the corresponding coding sequence parts in the device with the desired coding sequences (enzymes or regulating molecules, etc.) would the system get off to a quick and neat reaction state.

In future development, Sugarfilm should aim to further improve the versatility of the system. First, the targeted substrate of the reactor system should not be limited to saccharide. In fact, Sugarfilm system has the potential to apply to any phase-related synthesizing pathway, as long as the intermediate and final products would not have strong interactions with the biofilm structure (either causing damage to biofilm or be damaged by the extracellular materials within biofilm). Second, to better fit the synthesizing procedure, tweaks about the bacterium strain could be done. Better secretion efficiency and preservation of enzyme function are all vital for whole process. Also, with improvement of the basic Rainbofilm model, especially in accelerating biofilm formation, controlling of biofilm thickness and density, and choosing promoters of higher efficiency in each device (anaerobic promoters particularly), Sugarfilm would benefit a lot.

Improvements listed above may be subtle, yet they all mean a lot to the whole system. Sugarfilm, with its versatility and feasibility, has great vista in application in synthesizing work flow. By every small step towards the perfect, we believe Sugarfilm would outstand in the end, as a truly beneficial and powerful method in industry.