Insertion of a heterologous gene construct into a non-functional ORF of the Streptococcus thermophilus chromosome.
An integrative vector was constructed for inserting heterologous genes within a non-functional open reading frame (ORF) on the chromosome of Streptococcus thermophilus. The vector, pINTRS, contained a temperature sensitive origin of replication and an erythromycin resistance gene for initial selection in S. thermophilus. The region of the vector containing unique cloning sites, for insertion of recombinant genes, was flanked by homologous DNA sequences corresponding to a pseudogene in S. thermophilus to facilitate chromosomal integration. The gene encoding green fluorescent protein, regulated by a plasmid borne hsp promoter of S. thermophilus, was cloned into pINTRS to demonstrate proper functioning of the vector.
Renye
John A
JA
Department of Agriculture, Eastern Regional Research Center, Agricultural Research Service, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA. john.renye@ars.usda.gov
Somkuti
George A
GA
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002876:Chromosomes, Bacterial; D004269:DNA, Bacterial / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D004917:Erythromycin / Q000494:pharmacology; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006358:Hot Temperature; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D018741:Replication Origin; D012641:Selection, Genetic; D048249:Streptococcus thermophilus / Q000235:genetics*
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D004917:Erythromycin