Our work so far began when considering the potential of reflectins in multiple different systems. We hoped to begin work that could lead to the following areas.

Contents 1 Reflectins as novel reporters 2 Reflectins as optical materials 3 Groundwork needed 3.1 Flexible thin films of reflectins 3.2 Living structural colour with recombinant reflectins 3.2.1 Export to the periplasm 3.2.2 Expressing multiple reflectins 3.2.3 Protein engineering 3.2.4 Eukaryotic cells

Reflectins as novel reporters

Spectrum spanning colour changes in Loligo pealeii tissue samples [http://rsif.royalsocietypublishing.org/content/7/44/549.long#F2 can occur in a few minutes]. This could provide a real-time response exceeding that of [http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0002351 superfast GFP] or previous attempts to create fast pigment production - a highly attractive feature in a biosensor, for example. Our in vitro work shows that structural colour can provide instantaneous colour changes, further demonstrating that structural colour is phenomenon that synthetic biology must exploit in the future.

In L. pealeii this is thought to be controlled by a tyrosine kinase, so a screen of predicted tyrosine kinases from a squid cDNA library or protein engineering to create kinase recognition sites could recreate this rapid colour change in vivo in responses to changes in a signal cascade.

Reflectins as optical materials

As a proteinaceous substance which self assembles into nanoscale structures, reflectins could be the future of nanophotonic devices. The protein Bragg reflector is flexible, rather than the rigid crystalline arrays which display similar optical properties.

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. In particular, most current display technologies require a constantly-on backlight, which drains power. In addition, a reflectin based display would be able to display the entire spectrum on one pixel rather than relying on three (red, green and blue) closely spaced pixels to give the illusion of any colour. This would allow an increase in resolution and reduced costs as you would in theory need a third of the number of connections for the same size screen.

Groundwork needed

iGEM is one short summer and the team had many potential areas of exploration that had to be abandoned due to lack of time. The above concepts still require further work to make them feasible, read below for our suggestions for what should be done next.

Flexible thin films of reflectins

We successfully made thin films with glass and silicon wafers as a base, but chose to focus on improving the evenness and the lifespan (which also requires further work) rather than trying multiple substrates. Some flexible substrates which we think would be suitable are PDMS, polyimide and block copolymers.

Living 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 in live cells.

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, [http://www.sciencemag.org/content/303/5655/235.full 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 (the paperwork was simplest too!). 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 [http://partsregistry.org/Yeast yeast] and plant cells - these may hold the key for living structural colour.