An improved GFP cloning cassette designed for prokaryotic transcriptional fusions.
A new gfp cloning cassette designed for prokaryotic transcriptional fusions has been constructed. This cassette consists of gfp (containing the S65T 'red-shift' [Heim et al. (1995) Nature 373, 663-664] and F64L 'protein solubility' [Cormack et al. (1996) Gene 173, 33-38] mutations) 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 80-fold greater fluorescence intensity than wild-type gfp fusion strains. Additionally, this cassette confers sufficient fluorescence to recipient cells to be used in low copy-number plasmids, with promoters conferring low levels of transcription, and in bacterial taxa other than Escherichia, such as Pseudomonas.
Miller
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WG
Department of Plant and Microbial Biology, University of California, Berkeley 94720, USA.
Lindow
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SE
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005453:Fluorescence; D015870:Gene Expression; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007501:Iron / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011549:Pseudomonas / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014158:Transcription, Genetic
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D007501:Iron