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Team:UCL London/Project/Approach
From 2009.igem.org
After brain storming project ideas during the spring the team had come up with three ideas we considered not only very interesting but also manageable for a summer project. The three main ideas were:
1. Incorporation of some photosynthetic function into e.coli or characterisation of a small library of photosynthetic related biobricks for prokaryotes.
2. Tightly inducible and well controlled cell membrane disruption for drug release to replace mechanical homogenisation in large scale bioprocessing.
3. Traffic-light stress sensor for bio-processing application. For use within bioprocessing, process design or optimisation we would want examine stresses such as; low oxygen level, shear stress, acetate accumulation, high/low pH, high/low temperature, misfolded pharmaceutical proteins in cytoplasm/periplasm and growth phases.
We decided to make the Traffic-light stress sensor in ``E.coli`` and also to narrow our detection range to:
Low oxygen levels
Acetate accumulation
Misfolded proteins
The reason for calibrating our biosensor for the three stresses above is that these stresses seemed to be the most crucial ones for improving general bioprocessing. Literature review introduced us to a possible way to detect misfolded proteins in the periplasm via the two component signal transduction systems CpxAR or BaeSR and the Extracytoplasmic function Sigma-E. Low oxygen levels we thought we should be able to detect via activation of the FNR molecule in anaerobic conditions. Acetate seemed to be just too elusive to be detected in ``E.coli`` in a simple way. Long term exposure to acetate can increase transcription of certain flagellar subunits in ``E.coli`` but it is unknown how this is controlled. Acetate can also impact the rotation of a flagellum to make the bacteria tumble and change direction. However, we could not come up with a solution of how to translate this into something that can be detected more easily. A eukaryotic DNA binding protein; FacB from ``Aspergillus nidulance`` could possibly be used for acetate detection. At the end we decided that the quest for successfully incorporating a not very well characterised eukaryotic signalling system into a prokaryote would have added to many new layers of complexity into our project. We settled for making a biosensor that could detect low oxygen levels, misfolded proteins in the periplasm and possibly also shear stress.
