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Team:Cambridge/Experiments
From 2011.igem.org
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 [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 and created Reflectin A1 : GFP translational fusions.
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 hindrance in progress, with either formation of crystal structure formation or wetting and solvent evaporation problems.
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 on a low copy number plasmid 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.
