The Tn7 system inserts at the attTn7 site and is oriented specifically such that the right end of Tn7 is adjacent to the 3′ end of the glmS gene , and it has been used for transgene insertion into the chromosome of Escherichia coli, Salmonella, and Shigella . Plasmid pGRG25 is also a temperature-sensitive delivery plasmid that can be cured after transgene insertion at the attTn7 site by culturing at 42°C. Plasmid pBEN276 contains the luxCDABE operon between the Tn7 transposon arms
on plasmid pGRG25, and its expression is driven by the E. coli frr promoter (Figure 1), which controls expression of a house-keeping gene encoding ribosome recycling factor. Thus the lux operon will be expressed constitutively. The chromosomal insertion point is specific and insertion of lux operon does not disrupt the LGX818 in vivo function of glmS gene, therefore it is highly unlikely that bacterial
physiology will be affected adversely . Figure 1 Plasmid pBEN276 vector. tnsABCD are the genes required for transposition. luxCDABE encodes for luciferase and is flanked by Tn7 transposon arms (vertical bars at restriction sites XhoI and NotI). The expression of lux genes is driven by E. coli frr gene promoter between the XhoI sites. Characterizing the bioluminescent properties Tucidinostat order of Salmonella enterica Plasmid pBEN276 was utilized to insert the bacterial lux operon into chromosomes of eleven Salmonella enterica serotypes. Bioluminescence correlated well to bacterial population density in all serotypes used, as exemplified in S. Montevideo (p = < 0.0001, r20.94) (Figure 2). The minimum detectable concentration of all eleven serotypes was, in Tangeritin decreasing order (CFU/mL):
S. Kentucky – 8.00 × 104; S. Mbandaka – 4.99 × 104; S. Enteritidis – 3.10 × 104; S. Schwarzengrund – 2.78 × 104; S. Montevideo – 1.74 × 104; S. Alachua – 1.07 × 104; S. Typhimurium – 6.72 × 103; S. Seftenberg – 6.40 × 103; S. Heidelberg – 5.28 × 103; S. Newport – 4.64 × 103; S. Braenderup – 4.16 × 103. Minimum detectable numbers of Salmonella isolates expressing bioluminescence from the chromosome were higher compared to minimum detectable numbers of Salmonella isolates expressing plasmid-based bioluminescence . One possible explanation for this difference is a copy number effect; a single copy of the lux operon is inserted into the chromosome with the Tn7 system, while multiple copies of the gene are expressed in plasmid systems. Plasmid pAKlux1 is a pBBR1 derived plasmid which characteristically has a medium copy number (~30 copies/cell) . Another possible explanation is due to promoter effect; the frr promoter drives expression of luxCDABE in the Tn7 system, and the lacZ promoter drives expression in the pAKlux1 plasmid system . Our previous work CA4P clinical trial showed bioluminescent Salmonella isolates carrying plasmid pAKlux1 emit, on average, 6.