Chromosomal Insertion of Gfp Into Bacteria For Quality Control

Abstract

An isolated mutated green fluorescent protein (gfp) gene for chromosomal insertion into a bacterium, wherein the gene is capable of being expressed in bacteria and produce sufficient fluorescence under illumination from a UV lamp in a bacterial colony to be seen by the naked eye. A gene cassette for inserting a gene into a chromosome.

1 - 24 . (canceled) 
     
     
         25 . An isolated mutated green fluorescent protein (gfp) gene having mutations at nucleotides positions 1492, 1493 (Ser72Ala), 1737 (Met153Thr) and 1766 (Val163Ala) in the wild-type gfp gene as set out in SEQ ID NO 1, or mutations for synonymous codons which change the same amino acid positions (Ser72Ala, Met153Thr and Val163Ala). 
     
     
         26 . The isolated gene of  claim 25  having a nucleic acid sequence from bases 1524 to 2240 as set out in SEQ ID NO 4. 
     
     
         27 . An isolated green fluorescent protein (GFP) expressed by the mutated gfp gene according to  claim 25 . 
     
     
         28 . The isolated mutant GFP according to  claim 25  comprising one or more mutations of mutant GFP01, mutant GFP02, mutant GFP03, mutant GFP07, mutant GFP10, mutant GFP15, mutant GFP16, mutant GFP20, mutant GFP21, mutant GFP22, mutant GFP26, mutant GFP27, mutant GFP37, mutant GFP43, mutant GFP44, mutant GFP53, mutant GFP54, or mutant GFP55. 
     
     
         29 . The isolated mutant GFP according to  claim 27  comprising mutant GFP01, mutant GFP02, mutant GFP03, mutant GFP07, mutant GFP10, mutant GFP15, mutant GFP16, mutant GFP20, mutant GFP21, mutant GFP22, mutant GFP26, mutant GFP27, mutant GFP37, mutant GFP43, mutant GFP44, mutant GFP53, mutant GFP54, or mutant GFP55. 
     
     
         30 . The isolated mutant GFP according to  claim 29  having an amino acid sequence as set out in SEQ ID NO 6. 
     
     
         31 . A bacterium or cell containing the mutated gfp gene according to  claim 25 . 
     
     
         32 . The bacterium according to  claim 31  selected from the group consisting of  Acinetobacter lwoffii, Aeromonas hydrophila, Aspergillus niger, Bacillus cereus, Bacillus subtilis, Campylobacter coli, Campylobacter jejuni, Candida albicans, Citrobacter freundii, Clostridium perfringens, Clostridium sporogenes, Edwardsiella tarda, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Escherichia coli  0157,  Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella aerogenes, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentus, Legionella pneumophila, Listeria innocua, Listeria ivanovii, Listeria monocytogenes,  Meth. Resist.  Staph. Aureus, Neisseria gonorrhoeae, Proteus rettgeri, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas fluorescens, Rhodococcus equi, Salmonella abaetetuba, Saccharomyces cerevisiae, Salmonella salford, Salmonella menston, Salmonella sofia, Salmonella Poona, Salmonella typhimurium, Salmonella poona, Serratia marcescens, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Vibrio parahaemolyticus, Yersinia enterocolitica,  and  Zygosaccharomyces rouxii.    
     
     
         33 . The bacterium according to  claim 32  being  Escherichia coli, Salmonella  sp or  Listeria  sp. 
     
     
         34 . The bacterium according to  claim 33  wherein the  Salmonella  sp is  Salmonella typhimurium  or  Salmonella abaetetuba  and the  Listeria  sp is  L. monocytogenes.    
     
     
         35 . The cell according to  claim 31  comprising  Candida albicans, Zygosaccharomyces rouxii  or  Aspergillus niger.