Expressing reflectin in E. coli

To help pave the way for the manipulation of living structural colour, we investigated the properties of recombinant reflectins in vivo. We designed multiple expression constructs to allow us to study reflectin under different conditions. An outline of our plan is given below.

Isolating the reflectin gene

We initially attempted to clone reflectin genes from Loligo pealeii genomic DNA, but this proved a dead end - extracting squid DNA is notoriously difficult and we were unable to obtain samples fresh enough to create a cDNA library. We are very grateful to Wendy Crookes-Goodson for her kind donation of plasmids containing reflectins codon-optimised for E. coli.

Designing expression constructs

Promoter choice, copy number - justification

In order to work with Reflectin in vivo and in vitro we needed to create DNA constructs that would allow us to control expression level and to tag the protein for purification. The first two constructs we made, GA1, GA2, put reflectin expression under the control of pBAD an arabinose controlled promoter (one on high copy pSB1A2 plasmid, one on the low copy pSB3K3 plasmid), whilst GA3 and GA4 also did this, we incorporated a His-tag to enable purification at a later stage. - pasted from the experiment section

Periplasmic Export

Reflectins associate with membranes in squid to form regular assemblies. In the hope of replicating this behaviour in other cells we attempted to export reflectins into the [http://en.wikipedia.org/wiki/Periplasmic_space periplasmic space] between the two membranes that coat E. coli.