Figure 7

Promoter strength and insertion site location determine bioluminescence signal strength in a bacterium with multiple Tn 7 insertion sites. Mini-Tn7 elements in which lux operon expression is directed from the indicated promoters were transposed into the genome of B. pseudomallei strain Bp82.27 and insertion at either glmS1-, glmS2- or glmS3-associated attTn7 sites was determined by PCR. Colonies were patched on LB agar plates with 35 μg/ml kanamycin and incubated at 37°C. Patches were imaged using a Bio-Rad Universal Hood II ChemiDocXRS using high sensitivity chemiluminescence settings. A) and B) Bp82.27 with mini-Tn7-lux insertions derived from transposition from pTn7oLuxK3, pTn7tLuxK3 and pTn7xLuxK3. The promoters directing lux operon expression in these constructs are B. pseudomallei P _ompA , B. pseudomallei P _tolC and P. aeruginosa P _PA4974 , respectively. Patches were grown overnight at 37°C and either imaged using white light and a 0.007 s exposure time (A) or imaged for luminescence using a 30 s exposure time (B). C) Insertion site dependence of bioluminescence intensity. The mini-Tn7-P _ompA -lux element from pTn7oLuxK4 was transposed into the Bp82.27 genome. Insertion sites were determined by PCR. Patches were grown overnight at 37°C and imaged for luminescence using a 30 s exposure time.