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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==Ideas which deserve more time==
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==Reflectins as novel reporters==
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==Structural colour with recombinant reflectins==
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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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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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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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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.
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-
===Export to the periplasm===
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==Reflectins as optical materials==
-
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.
+
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.
-
===Expressing multiple reflectins===
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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 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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=Groundwork needed=
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===Protein engineering===
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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.
-
Rational engineering of reflectins to add membrane-binding domains may give an approximation of native reflectin membrane association.
+
==Flexible thin films of reflectins==
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===Eukaryotic cells===
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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.
-
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.  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.
+
==Living structural colour with recombinant reflectins==
 +
As of the time of writing we were unable to produce convincing evidence of a [[Team:Cambridge/Project/Microscopy | 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.
-
Reflectins as optical materials
+
===Export to the periplasm===
-
-Flexible substrates - did we have ideas for this?
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-Different methods for controlling colour change
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-Pixel
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==Reflectins as novel biosensors==
+
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.
-
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.]
+
===Expressing multiple reflectins===
-
Why this is cool - colour change is rapid (compare to imperial project and other reporters)
+
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. 
-
What needs to be done - identifying a kinase/engineering kinase recognition sites
+
===Protein engineering===
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engineering a signal transduction pathway
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Reflectin screens
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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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Link paper - reflectin inspired
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No need for backlight
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==Further Work==  
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===Eukaryotic cells===
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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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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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+
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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.
+
 +
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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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 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 - 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, 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 yeast and plant cells - these may hold the key for living structural colour.