An improved GFP cloning cassette designed for prokaryotic transcriptional fusions

Gene. 1997 Jun 3;191(2):149-53. doi: 10.1016/s0378-1119(97)00051-6.


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.

MeSH terms

  • Cloning, Molecular*
  • Escherichia coli / genetics*
  • Fluorescence
  • Gene Expression
  • Genes, Bacterial
  • Genes, Reporter
  • Genetic Vectors*
  • Green Fluorescent Proteins
  • Iron / pharmacology
  • Luminescent Proteins / genetics*
  • Luminescent Proteins / metabolism
  • Mutation
  • Promoter Regions, Genetic
  • Protein Biosynthesis
  • Pseudomonas / genetics*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Solubility
  • Transcription, Genetic


  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Iron