Team:Cambridge/Project/Future

Ideas which deserve more time

Structural colour with recombinant reflectins

As of the time of writing we were unable to produce convincing evidence of a change in the optical properties of E. coli. However, we had a number of ideas to try and promote self-assembly of the reflectin ultrastructure to give thin film interference.

Export to the periplasm

We attempted to export both our reflectin and our reflectin-GFP fusion 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 we were unable to achieve succesful export, but we hope that this may result in reflectin membrane association.

Expressing multiple reflectins

Squid reflector cells contain more than just one reflectin - for example, the original study in E. scolopes identified at least 6 different genes for reflectins. Little is known about the interactions between these homologues - it may be possible to promote reflectin assembly by expressing a suite of different proteins.

Protein engineering

Rational engineering of reflectins to add membrane-binding domains may give an approximation of native reflectin membrane association.

Eukaryotic cells

We made the decision to focus on expression in E. coli due to their short replication time and because our advisors have a great deal of experience working with bacteria. However, reflectin is a eukaryotic protein and may require chaperones or a specialised lipid composition to associate with membranes. In addition, the iridophore platelets in squid cells are considerably longer than a bacterial cell - size constraints may be limiting the assembly process. Synthetic biology is developing toolkits for many eukaryotic systems including yeast and plant cells - these may hold the key for living structural colour.

Reflectins as optical materials -Flexible substrates - did we have ideas for this? -Different methods for controlling colour change -Pixel

Reflectins as novel biosensors

Spectrum spanning colour changes in Loligo pealeii tissue samples can occur in a few minutes. This could provide a real-time response exceeding that of superfast GFP or previous attempts to create fast pigment production.

Why this is cool - colour change is rapid (compare to imperial project and other reporters)

What needs to be done - identifying a kinase/engineering kinase recognition sites engineering a signal transduction pathway

Reflectin screens Link paper - reflectin inspired No need for backlight

Further Work

No research group has yet induced exogenously-introduced reflectin to give colour in-vivo. It is unlikely that it is folding correctly, whether over-expressed or induced at low levels. Aiding in-vivo folding, e.g. through protein engineering could restore some of the optical effects seen in the squid; it should be borne in mind however that there is excellent evidence that the protein requires an associated membrane complex for its optical function (Tao et al. Biomaterials 5, pp. 793-801).

A number of research groups are interested in developing reflectin as a novel bio-reporter. Within the squid the colour of the protein structure is dynamically altered through phosphorylation on specific residues. If this effect could be recreated in-vivo a coloured reporter could be made to result that continually reports on changes in signal.

The team have demonstrated that thin films of reflectin have interesting in-vitro properties, not least the ability to display colour from across the entire visible spectrum. Should the films be made to change colour reliably in response to e.g. an applied charge, novel displays could be formed without some of the disadvantages of current technology, such as the need for a continual backlight.