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Team:Cambridge/Project
From 2011.igem.org
Contents |
Project Goals
We aimed to express reflectin in E. coli and to investigate its optical properties in order to build the groundwork for the manipulation of living structural colour. We also looked at the over-expression of reflectin in E. coli, in order to obtain relatively pure samples of the protein for making thin films.
Much of our work (particularly the in vivo work) simply hadn't been tried before, so, while we had high hopes, we could not be sure as to what would happen.
Achievements
In one short summer the 2011 Cambridge team has produced a set of BioBrick parts to allow future researchers to explore synthetic biology applications for structural colour.
In Vivo
Working with living cells we have;
- Imaged squid tissue using novel techniques to explore the in vivo properties of reflectins.
- Succesfully produced reflectins in E. coli.
- Characterised best practices for in vivo reflectin production.
In Vitro
By engineering E. coli to overexpress reflectins we have;
- Purified reflectin and documented best practice for high purity yields.
- Made thin films which show structural colours.
- Demonstrated the rapid colour changes possible with reflectin.
Software
We contributed to Gibthon to help create an intuitive set of tools for designing constructs, fully compatible with both BioBrick standards and newer assembly techniques.
- Greatly improved import and display of fragments.
- Added tools to allow management of uploaded parts.
Microscopy
Initially we looked at some squid tissue using a confocal microscope, to see the optical effect of reflectins in squid cells. We also imaged bacterial cells engineered to produce reflectins.
In Vitro
When we over-expressed reflectin in E.Coli, we found (by making a GFP fusion) that while reflectin is surprisingly non-toxic to E.Coli, it formed inclusion bodies. We then extracted these inclusion bodies, purified the protein using a number of different techniques, and made thin films by spin-coating and flow-coating.
In Vivo
In order to express reflectin at lower levels, we made an arabinose-inducible version, both with and without a GFP-fusion. We found that at lower levels of expression, the reflectin-GFP fusion would not form inclusion bodies, but appeared to be uniformly distributed throughout the cell (see the Microscopy page) . We made several attempts to image this – hoping to find some change in optical properties – but found that while the induced cells did appear to exhibit iridescence, so did the uninduced. This, we theorise, is due to thin film interference around the cell wall and membrane.
We also attempted to export both our reflectin and our reflectin-GFP to the periplasm, in the hope that this environment would be more similar to the environment in which reflectin naturally folds and that the small space will promote reflectin's membrane-associating properties. As of writing, this has yet to be successful.
Conclusion and Future Work
Our project attempts to lay some groundwork for future research in to reflectins. Reflectin has numerous possible future applications, from display technologies to rapid bio-reporters.
