Team:Cambridge/Experiments

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OVERVIEW
home
Details of the experiments carried out throughout the project are linked from this page. These experiments should also be linked to from the appropriate blog entry.

Contents

Training Exercise

Initial exercise during our 2 weeks crash course in synthetic biology with the aim of familiarising us with common laboratory methods of preparing and assembling DNA. Find out what we got up to on the blog .

Main Project - 'Bactiridescence'

Obtaining the Reflectin Sequence

Genomic DNA Extraction Attempt

We designed primers to amplify reflectin genes directly from DNA extracted from Loligo tissue. Various combinations of Loligo, primers and DNA extraction protocol were used, ultimately with no success.

Synthesised reflectin sequences were generously donated by Wendy Crookes-Goodson, author of many of the papers on reflectin.

Synthetic Gene Amplification & Plasmid Construction

In anticipation that our genomic DNA extraction might fail, we contacted several researchers who had previously worked on reflectin for advice. Dr. Wendy Crookes-Goodson very kindly offered to donate a sample of synthesised reflectin genes that she used in her research. These arrived on cloning (non-expressing) plasmids that had been spotted onto filter paper.

We extracted and grew up these plasmids, the used their reflectin sequences to assemble constructs with reflectin A1 with and without a his tag, each on high and low copy plasmids. In addition, we put reflectins A2 and 1B on low-copy plasmids.

In Vitro Experiments

Over-Expression & Protein Purification

His-Trap Protein Purification - First attempt

Bacteria expressing his-tagged reflectin were lysed, and the protein was purified using a his-trap column and a denaturing protocol in order to solubilise reflectin. The isolated protein was then precipitated by dialysis and vacuum centrifugation.

His-Trap Protein Purification - Second attempt

As before bacteria expressing his-tagged reflectin were lysed and the protein was purified using a his-trap column but this time the isolated protein was then precipitated by ethanol or acetone precipitation.

Making Thin Films

The culmination of the in vitro work was the production of thin films of reflectin which demonstrate iridescence. We tried a number of different combinations of protein purification protocol and thin films coating method, and produced numerous thin films.

All thin films were made in Nanophotonics Centre, at the West Cambridge site.

We found protein purity to be a major limitation, with crystal structures forming on the film for impure samples.

Reflectin Thin Films I - First films with unfiltered protein

A sub team visited the Nanophotonics Centre Thin Films and Interfaces lab to work with Dr Matthew Hawkeye. We spincoated and flowcoated our purified reflectin samples in addition to several control solutions. We recorded our results using reflected light bright field microscopy and an ocean optics spectrometer.

Reflectin Thin Films II - Improved films with centrifuged reflectin-solvent solution

We refined our methods in order to produce better thin films. We recorded the reflectance spectra of some of the thin films, ran a control using the protein bovine serum albuimin and heat cured the films.

Reflectin Thin Films III - Reflectin Controls

In addition to making some more thin films various controls were implemented in order to verify that the origin of iridescence we were observing were due to the reflectin protein and not due to impurities which may have originated in the elution buffer or at other stages of the purification . The results suggest iridescence is due to the presence of reflectin.

In Vivo Experiments

Part Characterization

Characterization of Parts -- Inclusion Body Formation

Details of the process for characterizing components of our reflectin-producing system.