Experiments

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.

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'

Genomic DNA Extraction Attempt

Amplification of Reflectin Genes from the Squid Genomic DNA - Part 1

We designed primers to amplify reflectin genes directly from [http://en.wikipedia.org/wiki/Loligo Loligo] tissue. Tissue from the Loligo genus was commercially available from fishing bait suppliers and culinary wholesalers. Primers were designed from the nucleotide sequences of three reflectin proteins identified in L. pealei, and used in a PCR reaction upon L. vulgaris genomic DNA.

See week four of our blog for more on this!

Amplification of Reflectin Genes from the Squid Genomic DNA - Part 2

Two new protocols for genomic DNA extraction were used in order to improve yield and purity of DNA. In addition to three sets of primers allowing for amplification of reflectin, an extra 'positive control' pair of primers was used in the PCR reaction.

Assembly of Constructs and Protein Purification

Amplification of Synthesised Reflectin Genes

After failing to isolate reflectin genes from squid genomic DNA, 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. Our first step was to elute the plasmids from the paper, and then to transform them into E. coli for storage and amplification. A standard miniprep then allowed us to recover the DNA from the bacteria.

Assembly of Reflectin Constructs

Assembly and amplification of constructs for reflectin A1 with and without a his tag, each on two different plasmids, and for reflectins A2 and 1B on low-copy plasmids [placeholder].

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

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 ethanol and acetone precipitation.

Making Thin Films

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

On the 24th of August 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

On the 31st of August in order to verify that the origin of iridescence we were observing were due to the reflectin protein and not due to impurities e.g. elution buffer, salts etc. various controls were implemented. The results suggest iridescence is due to the presence of reflectin.

Part Characterization

Characterization of Parts -- Inclusion Body Formation

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