AN IMPROVED GFP CLONING CASSETTE DESIGNED FOR PROKARYOTIC TRANSCRIPTIONAL FUSIONS

A new gfp cloning cassette designed for prokaryotic transcriptional fu sions has been constructed. This cassette consists of gfp (containing the S65T 'red-shift' [Helm et al. (1995) Nature 373, 663-664] and F64L 'protein solubility' [Cormack et al. (1996) Gene 173, 33-38] mutation s) flanked by convenient restriction sites, a translational enhancer, and a consensus ribosome binding site with an optimized spacer region. gfp fusion strains containing this cassette demonstrate from 40- to 8 0-fold greater fluorescence intensity than wild-type gfp fusion strain s. Additionally, this cassette confers sufficient fluorescence to reci pient cells to be used in low copy-number plasmids, with promoters con ferring low levels of transcription, and in bacterial taxa other than Escherichia, such as Pseudomonas. (C) 1997 Elsevier Science B.V.