In Vitro selection of DNA binding sites for transcription factor, PhaR, from Paracoccus denitrificans using genetic library on microbeads and flow cytometry

We attempted the selection of a site for DNA binding to a transcription factor, PhaR, from Paracoccus denitrificans expressed by cell-free protein synthesis, from a random oligonucleotide library on microbeads that was constructed by the emulsion PCR technique. PhaR is a repressor protein in P. denitrificans that binds to the phaP promoter region. We acquired three types of PhaR-binding DNA fragment using this system. The selected fragments contained a perfect PhaR binding consensus site (TGC I), a sequence similar to that of TGC I, and another PhaR binding site (TGC II).