Figure 1

The DNA-affinity-purified-chip (DAP-chip) method. (a) DAP-chip strategy. The D. vulgaris RR gene is cloned into E. coli with a carboxy-terminal His-tag. Purified His-tagged protein is phosphorylated with acetyl phosphate, and mixed with sheared D. vulgaris genomic DNA. An aliquot of the binding reaction is saved as input DNA, while the rest is subjected to affinity purification using Ni-NTA resin. The input and the RR-bound DNA are whole genome amplified, and labeled with Cy3 and Cy5, respectively. The labeled DNA is pooled together and hybridized to a tiling array, which is then analyzed to determine the gene targets. (b) Summary of DAP-chip workflow. The flowchart shows a summary of results at the following steps: protein purification, positive target determination, quantitative PCR (qPCR) test for target enrichment, DAP-chip hybridization, target list determination, and binding site motif validation. AAA, σ54 interaction domain; DBD, DNA binding domain; EMSA, electrophoretic mobility shift assay; NTA, nitrilotriacetic acid; REC, receiver domain; RR: response regulator.