Team:Cambridge/Project

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Bact<b>iridescence</b> was based around the properties of [[Team:Cambridge/Project/Background | reflectin]], a squid protein with the highest refractive index of any known proteinaceous substance. In squid this protein forms complex platelets which act as [http://en.wikipedia.org/wiki/Bragg_reflector Bragg reflectors] to provide camouflage.
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===Project Goals===
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|The Cambridge 2011 iGEM team is made up of nine undergraduates from diverse disciplines.  We aim to achieve something remarkable using synthetic biology within the short timeslot alloted for our work. If you would like to sponsor Team Bactiridescence please browse our [https://static.igem.org/mediawiki/2011/c/cb/IGEM_brochure_Cambridge_Team_2011_small_version.pdf brochure ].
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'''Bact''iridescence''''' is a project based around the unique properties of reflectin, a squid protein with the highest refractive index of any known proteinaceous substance.  In squid this protein forms complex platelets which act as [http://en.wikipedia.org/wiki/Distributed_Bragg_reflector Bragg reflectors] to provide camouflage. We aim to express reflectin in E. coli and optimise the optical properties, building the groundwork for the manipulation of living structural colour.
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|[[Image:Cambridge_team.png|right|frame|Your team picture]]
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We aimed to [[Team:Cambridge/Project/In_Vivo | 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 [[Team:Cambridge/Project/In_Vitro | over-expression of reflectin in ''E. coli'']], in order to obtain relatively pure samples of the protein for making thin films.
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|align="center"|[[Team:Cambridge | Bactiridescence]]
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=Background to the project=
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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.
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==How to disappear completely (or stand out in a crowd) - Reflectins in cephalopods==
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==Achievements==
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In one short summer the [[Team:Cambridge/Team | 2011 Cambridge team]] has produced a set of [[Team:Cambridge/Parts | BioBrick parts]] to allow future researchers to explore synthetic biology applications for structural colour.
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The beautiful optical properties which reflectin makes possible are used for different purposes in nature. Manipulation of reflectance may allow squid to communicate through polarised light. Altering refractive index to match that of the water column allows cephalopods to hide from their predators - a living invisibility cloak.  
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===[[Team:Cambridge/Project/In_Vivo | In Vivo]]===
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Working with living cells we have;
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*[[Team:Cambridge/Project/Microscopy | Imaged squid tissue using novel techniques]] to explore the in vivo properties of reflectins.
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*Succesfully produced reflectins in ''E. coli''.
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*Characterised best practices for in vivo reflectin production.
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Reflectin was first identified as such in the Hawaiian bobtail squid [http://en.wikipedia.org/wiki/Euprymna_scolopes|''Euprymna scolopes''] as the protein responsible for a reflective layer in the "light organ". This allows light emitted by symbiotic bacteria to be reflected downwards away from the squid, like a [http://en.wikipedia.org/wiki/Headlamp#Reflector_lamps| car headlamp].
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===[[Team:Cambridge/Project/In_Vitro | In Vitro]]===
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[[File:Cam_Multilayer_drop_1.jpg | right | thumb | 150px | A multilayer thin film]]
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By engineering ''E. coli'' to overexpress reflectins we have;
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*[[Team:Cambridge/Experiments/Protein_Purification | Purified reflectin]] and documented best practice for high purity yields.
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*Made [[Team:Cambridge/Project/Microscopy#Reflectin_Thin_Films | thin films]] which show structural colours.
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*Demonstrated the rapid colour changes possible with reflectin.
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**Videos of our thin films are available on [http://www.youtube.com/user/cambridgeigem2011 youtube].
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The Longfin inshore squid [http://en.wikipedia.org/wiki/Longfin_Inshore_Squid|''Loligo pealei''] shows dynamic iridescence controlled through signals from the nervous system which turn on a [http://en.wikipedia.org/wiki/Kinase| protein kinase], an enzyme which adds negatively charged phosphate groups to the positively charged protein.  This alters the attraction/repulsion between the platelet arrangement of reflectins, changing the spacing of the iridophore layers and therefore the colour of light reflected. (Read more about structural colour)
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===[[Team:Cambridge/Project/Gibthon | Software]]===
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[[File:Gibthon2.0beta.png | left | thumb | 100px | The Gibthon logo]]
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We contributed to [http://www.gibthon.org/ Gibthon], an open-source collection of web-based tools for construct design, fully compatible with both BioBrick standards and newer assembly techniques.
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*Greatly improved import and display of fragments (including support for [http://partsregistry.org/Main_Page partsregistry.org]).
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*Added tools to allow management of uploaded parts.
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Squid skin contains multiple specialised cell types designed to allow highly controlled changes of colour and reflectance to optimise camouflage. The beautiful optical properties in light reflecting tissues occur due to a hierarchy of structural arrangements - the structure of reflectin protein itself, the complex platelets which the protein forms, and the shape and layering of reflective cells. A thin tissue layer made up of iridocytes close to the surface of squid skin is made up of ~40% reflectin (out of the total protein content). <sup>[[#Crookes|2]]</sup>  Within iridocytes, reflectins self assemble to form membrane associated platelets.  The changes in refractive index as light moves through the layers of reflectin and cytoplasm forms a natural '[[#Bragg|Bragg reflector]]'<sup>[[#Morse|[3]]]</sup>.
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<html><div style='clear:both'></div></html>
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Iridophores which appear similar in structure to those found in cephalopods are also seen in other members of mollusc phylum - giant clams<sup>[[#Clams|[4]]]</sup>.
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==[[Team:Cambridge/Project/Future | Future work]]==
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They are responsible for the stunning iridescent colours of the mantle, and may play a role in protecting against harmful UV and maximising capture of sunlight for the photosynthetic symbionts which live alongside the clam.
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==Reflectins as novel polymers==
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By creating the first BioBrick parts for production of structural colour, we hope to facilitate further research.  Although time did not allow us to explore the full potential of our project, we have some ideas for what could be done next.
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Reflectins have unique properties in part due to their unique amino acid composition - residues which are normally common in proteins (alanine, isoleucine, leucine and lysine) are nowhere to be seen in any reflectins identified so far, whilst typically rare residues (arginine, methionine, tryptophan and tyrosine) make up ~57% of the protein. The family of reflectin proteins share a repeated domain which may also possess unique optical properties.
 
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The 3D protein structure of reflectin has not yet been characterised.  It may be natively unstructured - Weiss ''et al'' hypothesised that it may form a [http://en.wikipedia.org/wiki/Beta_barrel| beta barrel]-like structure when interacting with membranes, as recombinant reflectin-like proteins associated strongly with artificial membrane structures after cell-free expression.
 
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Kramer ''et al'' and Tao and DeMartini ''et al'' have demonstrated the remarkable capability of reflectin proteins to self assemble in vitro to create complex structures.  Recombinant reflectin, refolded ''in vitro'', can be carefully spread along a silicon slide to make thin films with intense structural colours from thin film interference. Measuring the refractive index of this in vitro arrangement of the protein reveals that it possesses the highest refractive index of any known protein. Kramer ''et al'' also demonstrated the ability of reflectin to form a diffraction grating when the ionic solvents used to dissolve it were diffused away in a water bath.  This ultrastructural arrangement showed iridescence. 
 
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==Light and interference - The physics behind structural colour==
 
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Structural colour is a common occurrence in nature - butterfly wings, fish scales and even [http://en.wikipedia.org/wiki/Tapetum_lucidum|the layer which makes cats's eyes shine at night] contain light reflecting components rather than pigments. It occurs due to the phenomenon of [http://en.wikipedia.org/wiki/Thin-film_interference| '''thin film interference'''].
 
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The difference in [http://en.wikipedia.org/wiki/Refractive_index| refractive index] between a thin film and the substance above and below it leads to some light being reflected (bouncing off) and some passing through the top surface of the film.  When the light which passes through hits the bottom boundary of the film, again some will be reflected.  When the two light waves meet, they will no longer be 'in sync' and interference will occur.  Some wavelengths of light will have destructive interference and be removed from the white light, so not all colours in white light will be reflected from the surface, giving effects like the rainbow colours reflected by oil droplets on the surface of water.
 
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As the viewing angle or the angle of incidence of light is varied the colour seen will change, giving an iridescent effect. Structural colour can also be altered by changing the spacing of the layers with different refractive indices as this will change the peak wavelengths where constructive and destructive interference occur.  This is believed to be the principle by which colour is altered in the skin of squid - the thickness of and spacing between reflectin layers has been observed to change ''in vivo'' due to post translational modification.
 
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=='''References'''==
 
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<div id="Kramer"></div>
 
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[http://www.nature.com/nmat/journal/v6/n7/abs/nmat1930.html] Kramer ''et al.'' '''The self-organizing properties of squid reflectin protein''' Nature Materials 533-538 VOL6 JULY 2007
 
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<div id="Crookes"></div>
 
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[http://www.sciencemag.org/content/303/5655/235.short] Crookes ''et al.'' '''Reflectins: The Unusual Proteins of Squid Reflective Tissues''' SCIENCE 235-238 VOL303 9 JANUARY 2004
 
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<div id="Morse"></div>
 
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[http://www.sciencedirect.com/science/article/pii/S0142961209011442] Morse ''et al.'' '''The role of protein assembly in dynamically tunable bio-optical tissues''' Biomaterials 793-801 VOL31 FEBRUARY 2010
 
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<div id="Clams"></div>
 
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[http://www.publish.csiro.au/paper/ZO9920319.html]Iridophores in the mantle of giant clams
 
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Latest revision as of 02:52, 22 September 2011

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OVERVIEW
home
Bactiridescence was based around the properties of reflectin, a squid protein with the highest refractive index of any known proteinaceous substance. In squid this protein forms complex platelets which act as [http://en.wikipedia.org/wiki/Bragg_reflector Bragg reflectors] to provide camouflage.

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;

In Vitro

A multilayer thin film

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.
    • Videos of our thin films are available on [http://www.youtube.com/user/cambridgeigem2011 youtube].

Software

The Gibthon logo

We contributed to [http://www.gibthon.org/ Gibthon], an open-source collection of web-based tools for construct design, fully compatible with both BioBrick standards and newer assembly techniques.

  • Greatly improved import and display of fragments (including support for [http://partsregistry.org/Main_Page partsregistry.org]).
  • Added tools to allow management of uploaded parts.

Future work

By creating the first BioBrick parts for production of structural colour, we hope to facilitate further research. Although time did not allow us to explore the full potential of our project, we have some ideas for what could be done next.


Retrieved from "http://2011.igem.org/Team:Cambridge/Project"