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Team:Edinburgh
From 2011.igem.org
A biorefinery is a special type of refinery in which biomass, such as plant cellulose, is converted by microorganisms into useful products. Edinburgh's 2011 iGEM project is a feasibility study into the creation of biorefineries using E. coli, the workhorse of synthetic biology; and whether biorefineries can be improved by arranging for the different enzymes involved to be in close proximity to each other, so as to create synergy between them. We investigated two methods of bringing the enzymes close together: cell surface display via Ice Nucleation Protein, and phage display via M13's major coat protein. We attempted a new DNA assembly protocol, provisionally named "BioSandwich". We constructed computer models of synergy to assess whether it is a feasible option. Finally, we considered the broader economic and social questions surrounding the construction of a biorefinery: can it be done, and should it be done?
Synergy
In many applications, several enzymes are needed to produce the desired product. And it is often the case that these enzymes work synergistically; meaning their efficiency is increased if they are in close proximity.
Last year, Slovenia found a way to achieve synergy in the cytoplasm. This year, Edinburgh is investigating whether such synergy can be achieved outside the cell.
We attempted to create microscopic reaction scaffolds holding various enzymes that carry out reactions in the extracellular environment. Our hope is that, by combining the activity of multiple enzymes in a small space, high efficiency will be achieved. Two different systems are being investigated.
Cell surface display
The simplest system uses E. coli bacteria as the scaffold. Each bacterium generates several enzymes and displays them on its outer membrane. If sufficiently high numbers are present on each cell, the synergystic effect should be achieved.
To achieve a high expression level, we will attempt to use Ice Nucleation Protein as a carrier for enzymes. See the cell surface display page for more details.
Phage display
A more radical proposal involves use M13 phage as the scaffold, and attaching enzymes by phage-display techniques to the pVIII coat protein.
These modified phage will be produced by modified E. coli. If multiple copies of the enzymes could be attached to a single phage, then synergy should be achieved. See the phage display page for more details.
Modelling
Synergy can be assessed in silico, using computer models. We created and compared multiple models of cellulase action and we showed that by just bringing enzymes close together we can increase their efficiency in our potential systems. See our modelling page for more details.
DNA assembly
DNA assembly is a difficult problem, and some teams have almost abandoned the BioBrick approach in favour of homology-based methods like Gibson Assembly. During our project, we attempted to use a hybrid system, BioSandwich, which has some of the advantages of both approaches.
Biorefineries in society
We believe that it is insufficient to ask whether the low-level biological challenges can be overcome. There are also engineering and economic problems to consider, and so we have worked on an actual design for a large-scale physical biorefinery.
More than this, political and social implications of biorefineries demand our attention. We must ask not only whether we can do something, but also whether we should. Answering this question is one of the most important parts of our feasibility study, and so we conducted a number of interviews with participants in the debate around synthetic biology.
Questions
Our feasibility study therefore seeks to answer the following questions:
- Would the efficiency of cellulases be increased by having different types close together?
- Could this be done by displaying them on a cell outer membrane?
- Could this be done by displaying them on a phage?
- Does the BioSandwich DNA assembly method work properly?
- How would a biorefinery involving either system actually be constructed?
- Would such a biorefinery be economically viable?
- What are the social implications of creating such a biorefinery?
- What are people's thoughts and feelings regarding this project?
- Should we (meaning society) actually build such a biorefinery?
Our answers, or best attempts at answers, are found throughout this wiki.
