From 2011.igem.org

Sugarfilm

Background

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.

Introduction

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, <a href="http://partsregistry.org/Part:BBa_K118022">BBa_K118022</a> , iGEM08_Edinburgh) together with CenA (Cellulomonas fimi endoglucanase A, <a href="http://partsregistry.org/Part:BBa_K118023">BBa_K118023</a> , 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, <a href="http://partsregistry.org/Part:BBa_K118028">BBa_K118028</a> , 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.

Design

In the Sugarfilm expression system, <a href="http://partsregistry.org/Part:BBa_K561004">BBa_K561004</a> is designed for bottom layer expression, conducting the first step of cellulose degradation. It is based on Rainbofilm bottom layer BioBrick <a href="http://partsregistry.org/Part:BBa_K561002">BBa_K561002</a> , with substitution of mRFP (<a href="http://partsregistry.org/Part:BBa_E1010">BBa_E1010</a> ) 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.

<img src="" width="600" /> <partinfo>BBa_K561004 SequenceAndFeatures</partinfo>

<a href="http://partsregistry.org/Part:BBa_K561005">BBa_K561005</a> is designed for medium layer expression, conducting the conversion of cellubiose into glucose. It is based on Rainbofilm bottom layer BioBrick <a href="http://partsregistry.org/Part:BBa_K561000">BBa_K561000</a> , with substitution of eYFP (<a href="http://partsregistry.org/Part:BBa_E0030">BBa_E0030</a> ) by 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.

<partinfo>BBa_K561005 SequenceAndFeatures</partinfo>

Coding sequence with regulation of Ptet would be suitable as the upper-layer device (such as <a href="http://partsregistry.org/Part:BBa_I13602">BBa_I13602</a> 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.

Over Sugarfilm and More

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.

Team:ZJU-China/Sugarfilm