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Figure 4 | BMC Microbiology

Figure 4

From: Use of nfsB, encoding nitroreductase, as a reporter gene to determine the mutational spectrum of spontaneous mutations in Neisseria gonorrhoeae

Figure 4

Schematic illustrating the strategy used to modify the nfsB coding region. Each numbered arrow corresponds to the procedures summarized below: 1: PCR using primers NfsBsmI-3F and NfsBsmI-2R to introduce a BsmI recognition sequence and to alter a poly-A tract. 2: Treatment with S1 nuclease to create blunt ends, polynucleotide kinase to phosphorylate 5' ends, and T4 DNA ligase. E. coli were transformed using this construct (pEC2). Plasmid DNA was isolated by alkaline lysis. 3: Treatment with BsmI to generate pEC1. Digestion product was ligated with T4 DNA ligase. The construct was transformed into E. coli. 4: pEC1 was amplified with primers dwnstrm-F and dwnstrm-R. The product was ligated to the omega fragment, a PCR product of pHP45Σ with the OmegaABC primer, to generate pEC3. The omega fragment is symmetric, so one primer amplifies in both directions

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