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).
Kojima
Takaaki
T
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Japan.
Yamane
Tsuneo
T
Nakano
Hideo
H
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D002474:Cell-Free System; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D008863:Microspheres; D010231:Paracoccus denitrificans / Q000235:genetics* / Q000378:metabolism*; D019151:Peptide Library; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D019151:Peptide Library; D012097:Repressor Proteins; D014157:Transcription Factors