Liang Xingguo, Fujioka Kenta, Tsuda Yuichiro, Wakuda Ryuji, Asanuma Hiroyuki
Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Chikusa, Nagoya 464-8603, Japan.
Nucleic Acids Symp Ser (Oxf). 2008(52):19-20. doi: 10.1093/nass/nrn010.
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.
通过连接一个包含两个偶氮苯部分作为光开关的T7启动子构建了一个光响应性绿色荧光蛋白（GFP）基因。连接在D-苏氨醇上的偶氮苯部分精确地插入到非模板链上T7启动子序列的两个位置。以偶氮苯连接的DNA作为一种引物，在PCR扩增后将偶氮苯连接到GFP基因上。然而，由于引物延伸在偶氮苯部分的位置停止，形成了一个包含偶氮苯的单链突出端。有趣的是，我们发现与突出端互补的寡核苷酸可以被T4 DNA连接酶在停止位置连接，并且完整的光响应性T7启动子被连接到GFP基因上。此外，构建的光响应性GFP基因的体外表达通过光照射成功地实现了开启和关闭。
