Team:Cambridge/Project/Future

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Our [[Team:Cambridge/Project | 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.
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==In Vivo==
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==Reflectins as novel reporters==
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==In Vitro==
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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 [https://2010.igem.org/Team:Imperial_College_London/Modules/Fast_Response fast pigment production] - a highly attractive feature in a biosensor, for example. Our [[Team:Cambridge/Project/In_Vitro | in vitro work]] shows that structural colour can provide [[Team:Cambridge/Media#Dynamic_Iridescence | instantaneous colour changes]], further demonstrating that structural colour is phenomenon that synthetic biology must exploit in the future.
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==Microscopy==
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In ''L. pealeii'' this is thought to be [[Team:Cambridge/Project/Background | 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.
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Under 'reflection confocal microscopy' (illuminating the sample from above, collecting light at the same frequency as the illlumination), all bacteria give interesting optical effects, putatively due to thin-film effects around the membrane layers. This makes this particular tool less useful than hoped for investigating the optical effects of reflectin in-vivo.
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==Software==
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==Reflectins as optical materials==
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==Further Work==
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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.
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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).
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A number of research groups are interested in developing reflectin as a novel bio-reporter. Within the
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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.
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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
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that continually reports on changes in signal.
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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.
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=Groundwork needed=
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iGEM is one short summer and [[Team:Cambridge/Team | 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.
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==Flexible thin films of reflectins==
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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.
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==Living structural colour with recombinant reflectins==
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As of the time of writing we were unable to produce convincing evidence of a [[Team:Cambridge/Project/Microscopy | change in the optical
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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.
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===Export to the periplasm===
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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.
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===Expressing multiple reflectins===
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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. 
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===Protein engineering===
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Rational engineering of reflectins to add membrane-binding domains may give an approximation of native reflectin membrane association.
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===Eukaryotic cells===
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We made the decision to focus on expression in ''E. coli'' due to their short replication time and because our [[Team:Cambridge/Team/Advisors | 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 [[Team:UEA-JIC_Norwich | plant cells]] - these may hold the key for living structural colour.
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{{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}

Latest revision as of 22:05, 20 September 2011

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OVERVIEW
home
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

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.