Team:Cambridge/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.

Contents 1 Training Exercise 2 Main Project - 'Bactiridescence' 2.1 Obtaining the Reflectin Sequence 2.1.1 Genomic DNA Extraction Attempt 2.1.2 Synthetic Gene Amplification & Plasmid Construction 2.2 In Vitro Experiments 2.2.1 Over-Expression & Protein Purification 2.2.2 Making Thin Films 2.2.3 Reflectin Thin Films I 2.2.4 Reflectin Thin Films II 2.2.5 Reflectin Thin Films III 2.2.6 Colour Intense Films - First Breakthrough 2.2.7 Uniform Films 2.2.8 Multilayers 2.2.9 Attempts to increase film stability lead to first multilayer 2.3 In Vivo Experiments 2.3.1 Low Level Expression 2.3.2 Periplasmic Export 2.3.3 Microscopy 2.4 Part Characterization 2.4.1 Characterization of Parts -- Inclusion Body Formation

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 [http://en.wikipedia.org/wiki/Loligo 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 the DNA, transfected cells and grew up these plasmids, then 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

Using our reflectin constructs, we over expressed reflectin and then tried a number of techniques to purify the protein, including HIS trap purification and an inclusion body prep. We verified our protein by running an SDS PAGE protein gel.

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.

We conducted a number of experiments with the thin films, outlined below.

Reflectin Thin Films I

• Description of the basic method
• Colourful thin films created with reflectin & HFIP
  • Thin film colour not uniform
• HFIP thin film control did not show structural colour

Reflectin Thin Films II

• Method refined in an attempt to reduce impurities
• Bovine Serum Album (BVA) control to test whether generic proteinacious thin films produce colour
  • Did not show structural colour
• Reflectin thin films appear to crystalize on drying

Reflectin Thin Films III

• Control indicates that Urea may be to blame for thin film crystalization
• More controls show that neither GFP nor BSA exhibit structural colour when spun into thin films

Colour Intense Films - First Breakthrough

• Less is more - using the same concentration, spinning a small volume leads to better wetting and more colour intense films
• Introducing the 'Piranha solution' for surface cleaning

Uniform Films

• Quality over quantity - purer reflectin and greater control over spin lead to colour uniformity

Multilayers

• Attempts to create multilayers with alternating PDMS and reflectin.
• Problems with stability of reflectin due to impurities hinder progress

Attempts to increase film stability lead to first multilayer

• Dilution and centrifuging of samples and using only the very top layer seem to provide better stability for urea containing reflectin
• Dialysed protein seemed to be more stable however suffers from dewetting

In Vivo Experiments

We wanted to investigate reflectin's effect on E.Coli when expressed at a low level. Up until this point, researchers had focussed on using E.Coli in order to manufacture large amounts of reflctin for in vitro investigations.

Low Level Expression

Various tests were done on E.Coli with reflectin expressed on a low copy plasmid under an arabinose induced promoter (pBAD). We tested both normal E.Coli cells, and ones with a titratable arabinose response.

While we found that reflectin is surprisingly non-toxic to E.Coli, we did not find any evidence that the reflectin had folded correcty or that it had self assembled into stacks as it does in squid. However, by using our reflectin-GFP fusion as a control, we found that reflectin did not form inclusion bodies as it does under a high copy plasmid, but was distributed throughout the cell.

Periplasmic Export

We attempted to see what would become of reflectin once exported to the periplasm. Our GFP control suggested that our initial attempt at export had failed. There were several possible problems - one being that we were expressing reflectin too strongly and the export machinery was becoming saturated - however due to the unprecedented time constraints of the competition, we simply didn't have the time to try again.

Microscopy

We performed microscopy on both squid cells taken from tissues known to contain reflectin and bacterial cells which were expressing the protein using our construct.

Part Characterization

Characterization of Parts -- Inclusion Body Formation

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