Figure 4
Mini-Tn 5/7 - lux aided promoter identification and capture in E. coli . A) The promoter-less mini-Tn5/7-lux element (indicated by the red arrowhead and expanded inset above it) from pTn5/7LuxK3 was transposed into the DH5α chromosome. Chromosomal DNA from a Kmr and luminescent exconjugant (KVT9; panel B) was isolated, digested with EcoRI, religated and transformed into CC118(λpir +). The lux operon-chromosomal DNA junction on the plasmid was sequenced with the luxC-specific primer P2385. The mini-Tn5/7-lux transposon was inserted in rbsC, the third gene of the rbsDACBK operon required for the transport and initial metabolic steps of ribose. The kup gene located upstream of the rbsDACBK operon encodes a potassium transporter. B) The recovered plasmid was used to transpose the mini-Tn7-P rbs -lux element residing on it to the attTn7 site on the DH5α chromosome resulting in strain KVT11. In a parallel effort, The rbs operon promoter (P rbs ) located in the 167 bp kup-rbsD intergenic region was PCR-amplified and cloned on a 173 bp StuI-DraIII fragment into pTn5/7LuxK4 where it replaced the tnpA gene and flanking MEs to drive transcription of the lux operon. The resulting mini-Tn7-P rbs -lux element was transposed into the attTn7 site on the DH5α chromosome resulting in strain KVT12. Ten μl samples of an overnight culture of the indicated strains were spotted on an LB plate, grown overnight at 37°C and light emission was measured using a Xenogen IVIS imager.