Figure 4.
Deletion of an E. coli genomic region that contains two essential genes. We performed deletion of an E. coli targeted region (b3130–b3163) that contained two nonadjacent essential genes, yraL (b3146) and yhbV (b3159). (A) The targeted genomic region b3130–b3163 was replaced with a linear DNA cassette, b3129-yraL-yhbV-b3164-KmR-sacB-I-SceI-b3163 (E2). Markerless deletion of the introduced selection markers was carried out as described in Figure 2. (B) Correct replacement of the target genomic region and complete removal of E2 were confirmed by PCR using a pair of primers (IE2-f and IE2-r) specific to the ends of E2 and three pairs of primers (b3138F/R, b3152F/R and b3162F/R) specific to the internal genes located in the targeted region. All PCR primers are indicated with arrows in (A). For the sequences of the PCR primers, see Material and methods section. Lanes 1 and 2 indicate PCR products obtained with the E. coli MG1655 genome and the E. coli Δb3130–b3163::E2 genome, respectively, and primers IE2-f and IE2-r. Lane 3 shows a PCR product obtained with the E. coli Δb3130–b3163::yraL yhbV genome and a primers IE2-f and MD4. Lanes 4, 6 and 8 indicate PCR products obtained with the E. coli MG1655 wild-type genome and three pairs of primers (b3138F/R, b3152F/R and b3162F/R) that were specific to internal genes in the targeted region (b3138, b3152 and b3162, respectively). Lanes 5, 7 and 9 also indicate PCR products obtained with the E. coli Δb3130–b3163::yraL yhbV genome and the same primers as in lanes 4, 6 and 8. M indicates the lane that contains the DNA size markers.