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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''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 in order to build the groundwork for the manipulation of living structural colour. [[Team:Cambridge/Project/Background| Read more about reflectin and structural colour here.]]
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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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=Preliminary observations=
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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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In order to get a real sense of what we were looking to achieve in our project, we felt that it was important to make some observations of native squid reflectin ''in vivo''. We therefore obtained several specimens of ''loligo opalescens'' and ''loligo vulgaris'' squid from a local seafood restaurant and an online fishing bait store for dissection. We chose these species because the whole family of loliginid squid has been identified to contain reflectin, and these particular species were the only members of the family available to us.
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We used a confocal microscope to observe iridescent behaviour in eye and mantle tissue, by the following [https://2011.igem.org/Team:Cambridge/Protocols/Confocal_Microscopy_of_Loligo_Eye_and_Mantle_Dermis_Samples protocol]. The stunning images produced provided a very useful reference to help us to identify what recombinant (well folded) reflectin could look like in E. coli, and definitely enthused the team to obtain bactiridescence!
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=Objective One - Express reflectin in E. coli=
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==Achievements==
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Our first objective was to try to express reflectin, in any shape or form, in E. coli. Ideally, we'd be able to express reflectin in the bacteria at a range of levels using a single construct, so we can both overexpress (for ''in vitro'' studies) or underexpress (to promote ''in vivo'' folding) straightforwardly. In order to do this, we had to create an expression plasmid for reflectin with which we could transform the bacterial cells. Hence, we had to isolate a reflectin gene sequence, choose a suitable promotor and join them on an appropriate backbone in order to engineer what we wanted. These steps are outlined below.
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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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[[File:camflow_1.jpg | 600px | Objective One]]
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==Isolating the reflectin gene==
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===[[Team:Cambridge/Project/In_Vivo | In Vivo]]===
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This was more complicated than we initially expected. Our first idea was to attempt to clone reflectin genes from genomic DNA, extracted from the same squid specimens that we dissected for our [preliminary observations]. We started with a very crude genomic extraction protocol, but unfortunately we did not manage to successfully clone reflectin using this method (details of the experiment can be found [here]). We then attempted two cleaner genomic extraction protocols (described [here]), but unfortunately we remained unsuccessful. After some discussion with Dr. Wendy Goodson, a researcher who was one of the first to study reflectin, we found out that cloning from cephalopod genomic DNA is notoriously difficult, although the cause for this is not understood. She then very kindly offered to send us a quantity of cloning plasmids containing reflectin genes that she has worked with in the past; in this way we obtained genes for reflectin A1, A2 and 1B that had already been codon optimised for expression in E. coli. The amplification of these plasmids is described [here].
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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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==Selecting the promotor==
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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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==Selecting the backbone==
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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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==Creating the expression plasmid==
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<html><div style='clear:both'></div></html>
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Hopefully coming soon!
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==Expression in E. coli==
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==[[Team:Cambridge/Project/Future | Future work]]==
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Hopefully coming soon!
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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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=='''References'''==
 
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<div id="Kramer"></div>[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>[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>[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>[http://www.publish.csiro.au/paper/ZO9920319.html] Griffiths ''et al''. '''Iridophores in the mantle of giant clams.''' Australian Journal of Zoology (1992) Volume: 40, Issue: 3 Pages: 319-326
 
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<div id="Izumi"></div>[http://www.ncbi.nlm.nih.gov/pubmed/19776150] Izumi ''et al''. '''Changes in reflectin protein phosphorylation are associated with dynamic iridescence in squid.''' J. R. Soc. Interface 6 March 2010 vol. 7 no. 44 549-560
 
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<div id="Octopus"></div>[http://www.springerlink.com/content/bba14b73ad35f495/]Brocco ''et al''. '''Reflector cells in the skin of ''Octopus dofleini''''' Cell and Tissue Research, Volume 205, Number 2, 167-186, 1980
 
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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.


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