Constructs

As part of our project we designed a library of reflectin expression plasmids, as well as constructs with reflectin transcriptional fusions with GFP, and finally plasmids with reflectin targetted for export to the periplasm. Diagrams of all of them are given below.

Plasmids containing synthetic versions of reflectins A1 and A2 (from Loligo) and 1B (from Euprymna) were kindly sent to us by Dr Wendy Crookes-Goodson.

Expression Constructs

GA1

Reflectin A1 is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies).

GA2

Reflectin A1 is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies).

GA3

Reflectin A1 with a HIS tag at the N-terminus is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies).

GA4

Reflectin A1 with a HIS tag at the N-terminus is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies).

GA5

Reflectin A2 is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies).

GA6

Reflectin 1B is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies).

GFP fusions

GA13

Reflectin A1 fused to GFP at the C-terminus is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies).

GA14

Reflectin A1 fused to GFP at the C-terminus is expressed under the control of the arabinose-inducible pBAD promoter on a low copy number plasmid pSB3K3 (10-12 copies).

Export Constructs

GA17

Reflectin A1 is fused to the TorA signal peptide at the N-terminus, and his-tagged at the C-terminus. It is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies). TorA signal targets the protein to the Tat export pathway in E.coli.

GA18

Reflectin A1 is fused to the TorA signal peptide at the N-terminus, and fused to GFP at the C-terminus. It is expressed under the control of the arabinose-inducible pBAD promoter on a high copy number plasmid pSB1A3 (100-300 copies). TorA signal targets the protein to the Tat export pathway in E.coli.