Periplasmic export

As reflectins appear to spontaneously associate with membranes, we hoped to promote correct assembly by directing our recombinant reflectins to the [http://en.wikipedia.org/wiki/Periplasmic_space periplasmic space of E. coli].

Design of constructs

Choice of signal sequences

The signal sequence from TorA [http://www.ncbi.nlm.nih.gov/pubmed/11123687 has been used to export GFP to the periplasm] via the TAT pathway. We focused our export plan on this sequence as the TAT pathway exports proteins in their fully folded state, allowing GFP to remain fluorescent (it cannot form a fluorophore in a reducing environment). This allows us to use our reflectin-GFP fusions to provide an easy reporter of export using a confocal microscope.

The amino acid sequence given by Thomas et al was 4 residues shorter than [http://partsregistry.org/Part:BBa_K233307 part BBa_K233307], allowing us to directly synthesise it more cheaply from two primers. Our revised sequence has been submitted as an improved part.

We also designed reflectin fusions with two other signal sequences, from the PelB and PhoD proteins. We are very grateful to Nick Thompson (Physics of Medicine) and Dr David Summers (Dept of Genetics) for their help and advice with choosing signal sequences, and regret that we did not have time to follow through on their suggestions.

GFP imaging

Conclusion