title: Fluorescent reference strains of bacteria by chromosomal integration of a modified green fluorescent protein gene.
abstract: Fluorescent reference strains of bacteria carrying a stable chromosomally integrated single copy of the gfp gene have been developed. A modified version of the gfp gene has been generated by mutagenesis and expressed under the control of the bacteriophage lambda promoter P(L). A cassette comprising bacteriophage Mu transposon arms flanking the modified gfp gene and regulatory regions was irreversibly integrated as an in-vitro-assembled transposition complex into the genomes of Escherichia coli and Salmonella spp. The modified green fluorescent protein (GFP) protein retained the fluorescence excitation and emission wavelengths of wild-type GFP. However, it fluoresced more brightly in E. coli and Salmonella compared to wild-type GFP, presumably due to improved protein maturation. Fluorescent E. coli and Salmonella strains carrying the gfp gene cassette were easily differentiated from their respective non-fluorescent parental strains on various growth media by visualization under UV light. The bacterial strains produced by this method remained viable and stably fluorescent when incorporated into a matrix for delivery of exact numbers of viable bacterial cells for use as quality control agents in microbiological procedures.
authors.affiliation: Biotechnology Research Institute, Macquarie University, North Ryde, Sydney, New South Wales, 2109, Australia.
authors.forename: L B
authors.initials: LB
authors.lastname: Pinheiro
authors.forename: M D
authors.initials: MD
authors.lastname: Gibbs
authors.forename: G
authors.initials: G
authors.lastname: Vesey
authors.forename: J J
authors.initials: JJ
authors.lastname: Smith
authors.forename: P L
authors.initials: PL
authors.lastname: Bergquist
mesh_terms: D000596:Amino Acids / Q000235:genetics; D002876:Chromosomes, Bacterial / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005453:Fluorescence; D018390:Gene Targeting; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012475:Salmonella / Q000235:genetics*; D013696:Temperature
chemical_list: D000596:Amino Acids; D004251:DNA Transposable Elements; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins