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DRS001334 |
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"Protocols: To construct a K-12 (W3110) derivative expressing H-NS C-terminally tagged with 12 histidines (12His), we used modified one-step gene inactivation method (Datsenko and Wanner, 2000). The pSTV28-C-12His plasmid was constructed by inserting chemically synthesized 12His coding sequence and kanamycine-resistant gene derived from the pKD4 plasmid (Datsenko and Wanner, 2000), into the multiple cloning site of the pSTV28 plasmid (Takara Bio, Japan). We amplified a DNA fragment containing the 12His coding sequence flanked with Arg-Gly-Ser linker sequence and kanamycine-resistant gene by PCR using the pSTV28-C-12His plasmid and the TOP705-TOP706 primer set. We added ~70 bp sequences of hns coding region and its downstream region in TOP705 and TOP706 sequences, respectively, to facilitate the insertion of the PCR product into chromosome described below. The BW25113 cells harboring the pKD46 plasmid encoding Red recombinase (Datsenko and Wanner, 2000) were transformed by the amplified DNA fragment, and transformants in which linker and 12His sequences were inserted at the 3’ end of chromosomal hns gene through double-crossover at sequences in coding and downstream region of hns were selected by kanamycine-resistance, to obtain the BW25113 H-NS-His12 strain. The hns gene fused with 12His coding sequence was transferred into the W3110 (K-12) chromosome, together with the kanamycine-resistant gene, by P1 transduction.Since strains SE11 and SE15 are resistant to P1 phage, to construct SE11 and SE15 derivatives expressing 12His tagged H-NS, we adopted the gene doctoring method (Lee et al., 2009), using the pDEX plasmid harboring I-Sce-1 recognition site and sucA gene and the pACBSR plasmid harboring I-sce-1 and kanamycine-resistant genes (Herring et al., 2003). The 12His coding sequence and kanamycine-resistant gene on pSTV28-C-12His were obtained by PCR using hns-His12-H1 and hns-His12-H2-1 primers (for SE11) or hns-His12-H1 and hns-His12-H2-2 primers (for SE15), and amplified fragments were inserted into the EcoRV site of pDEX. SE11 and SE15 were co-transformed by two plasmids, pACBSR and appropriate pDEX-H-NS-His12 plasmid, with the selection of Kanamycine- and sucrose-resistance. Then transformants were cultured in LB liquid medium containing 25μg/ml chloramphenicol and 0.2% arabinose, inducing I-Sce-1 to inactivate pDEX-H-NS-His12, for few hours. Cells were harvested by centrifugation and re-grown in LB containing 5% sucrose at 30 ˚C for two hours, to cure pACBSR. Finally, kanamycine- and sucrose-resistant colonies were selected on LB plate containing 50μg/ml kanamycine and 5% sucrose, to isolate transformants in which 12His coding sequence and kanamycine-resistant genes were transferred into the chromosome through homologous recombination at hns coding sequence and sequence downstream of hns introduced at 5’- and 3’-end of PCR products, respectively.Expression of H-NS-12His in thus created strains was confirmed by Western blotting using anti-His tag antibody (MBL, JAPAN). Sequencing of introduced hns gene tagged with 12His revealed a point mutation within hns coding region in the K12 derivative, probably because of an error during the synthesis of the primer used to create the strain. Since identified point mutation (from AAG [136K] to AAA [136K]) did not cause amino acid substitution of H-NS, we used the strain in this analysis.ChAP (chromatin affinity precipitation) was performed according to the original procedure (Ishikawa et al., 2007), using 50 ml cultures of E. coli strains in LB medium under aerobic condition at 37 ˚C. Sequencings of DNA fragments co-purified with H-NS-12His (ChAP) and those in supernatant fraction before ChAP (WCE) were performed using the Illumina GA sequencer, following the instruction for genome sequencing from the supplier (Illumina, USA). We performed ChAP-seq experiments twice for each strains." |
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