Construction of a photo-switchable gene for turning on and off gene expression with light irradiation.
A photoresponsive GFP gene was constructed by attaching a T7 promoter that involves two azobenzene moieties as the photoswitch. The azobenzene moieties tethered on D-threoninol were inserted precisely into the sequence of T7 promoter at two positions in the nontemplate strand. By using azobenzene-tethered DNA as one primer, azobenzene was attached to GFP gene after PCR amplification. However, a single-stranded overhang involving azobenzene was formed because primer extension stopped at the position of azobenzene moiety. Interestingly we found that oligonucleotide complementary to the overhang could be ligated by T4 DNA ligase at the stopped position, and the intact photoresponsive T7 promoter was attached onto GFP gene. Furthermore, the in vitro expression of the constructed photoresponsive GFP gene was successfully switched on and off with light irradiation.
Liang
Xingguo
X
Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Chikusa, Nagoya 464-8603, Japan.
Fujioka
Kenta
K
Tsuda
Yuichiro
Y
Wakuda
Ryuji
R
Asanuma
Hiroyuki
H
D001391:Azo Compounds / Q000737:chemistry; D015870:Gene Expression / Q000528:radiation effects*; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D014466:Ultraviolet Rays
D001391:Azo Compounds; D049452:Green Fluorescent Proteins; C009850:azobenzene