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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|You can write a background of your team here.  Give us a background of your team, the members, etc.  Or tell us more about something of your choosing.
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|[[Image:Cambridge_logo.png|200px|right|frame]]
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''Tell us more about your project.  Give us background.  Use this is the abstract of your project.  Be descriptive but concise (1-2 paragraphs)''
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|[[Image:Cambridge_team.png|right|frame|Your team picture]]
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|align="center"|[[Team:Cambridge | Team Example]]
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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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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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==Bactiridescence - Reflectin' the Future==
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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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Reflectins are a recently identified protein family rich in aromatic and sulphur-containing amino acids, responsible for the 'reflective' camouflage exhibited by certain cephalopods. To date, researchers have isolated the protein, over-expressed it in ''Escherichia coli'' and shown it to exhibit self-assembling behaviour and a changeable morphology which leads to dynamic manipulation of incident light.
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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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===[[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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Under in-vitro conditions ''Kramer et. al'' produced thin films, photonic gratings and fibres which exhibited  structural colour extending across the entire visual spectrum by varying the thickness. In particular the colour change was demonstrated to be reversible. It is hypothesized the colouration is a result of thin film interference.
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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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<html><div style='clear:both'></div></html>
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Within the Atlantic squid ''Loligo pealeii'', ''Morse et.al'' found a multi-layer alternating structure of iridophore platelets of reflectin and an unidentified material, each possessing different refractive indices. By studying tissue samples in-vitro the researchers observed conformational changes in the multi-layer structure due to phosphorylation. 
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==[[Team:Cambridge/Project/Future | Future work]]==
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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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As part of our iGEM project we propose to express reflectin in-vivo within ''Escherichia coli'' to reproduce the same multi-layer structure. Further we wish to demonstrate the ability to dynamically tune structural colour in-vivo through phosphorylation. Our work will directly impact upon the design of next-generation novel biosensors.
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== Project Details==
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== Project Details==
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=== Reflectin and its Properties - A summary of our literature review ===
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In choosing our project we read all the papers we could find on reflectin and several on the squid species that have been worked on.
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What species of squid have reflectin proteins?
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Reflectin was initially identified in the Hawaiian bobtail ''Euprymna scolopes'' as the protein responsible for this species' iridescence and Reflectin 1a from ''E. scolopes'' has been successfully expressed in E.coli and purified (1) (the interesting properties of this purified protein are discussed below). Related proteins in ''Lolliguncula brevis'' and squid of the genus ''Loligo'' have caused much research interest because these species appear to control their iridescence, a property called dynamic iridescence.
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What purpose do reflectins have in these species?
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Reflectins are contained within stacks of repeating membranous structures with in cells in squid skin known as iridophores and together these structures and the reflectin proteins form natural Bragg reflectors (see below). Iridophores and other cell types with optical properties contribute to the squid's ability to camouflage itself and communicate with other animals via manipulating light.
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What is Bragg reflector?
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Bragg reflectors are structures of alternating layers of materials with a high and low refractive indexes. These structures reflect a precise wavelength of light according to the width of the layers and multiple layers allows the amplification of the reflected beam. This principle behind this is that of thin film interference which is responsible for the range of bright colours reflected by oil droplets on the surface of water. In fact the peak reflected wavelength is four times width of the layers in the reflector, as this path difference causes the correct phase difference to allow constructive interference between light waves of this particular wavelength of light.
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What is dynamic iridescence and does work in squid? (membrane association)
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Iridescence describes the property of a material that changes colour as the viewing angle, or the angle of incident light varies. However, the dynamic iridescence seen in certain squid is believed to be neurally controlled. Specifically, the application of the neurotransmitter Acetyl Choline to fresh skin samples resulted in detectable post-translational modifications of the protein, namely phosphorylation. It is believed that phosphorylation of reflectin proteins causes changes in interactions between and within the nanoparticles reflectin forms in vivo. These changes induce a larger scale change, an alteration in the volume of protein platelets of reflectin. Crucially these changes in volume represent a change in the path difference between light reflected from the outer and inner surface of the layers of reflectin. As described above, the path difference determines which wavelength of light constructively interferes and therefore the peak reflected wavelength.
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What is known about the nucleotide sequence of reflectins?
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No introns were found in the reflectin genes when reflectin genes amplified from  ''Euprymna scolopes'' genomic DNA were sequenced. (2)
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What is known about the unusual amino acid content of reflectins?
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What is known about the structure of the reflectin proteins?
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What are the differences and conserved sequences between different reflectins and between reflectins in different species?
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What work has been done on expressing reflectins in E.coli?
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What in vitro experiments have been performed on reflectins?
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What are the properties of in vitro films of reflectin?
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'''References'''
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1. Kramer ''et al.'' nature materials 533-538 VOL 6 JULY 2007
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2. Crookes ''et al.'' p235-238 SCIENCE VOL 303 9 JANUARY 2004
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=== The Experiments ===
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=== Part 3 ===
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== Results ==
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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 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 youtube.

Software

The Gibthon logo

We contributed to 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 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.


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