Kato Yusuke
Division of Biotechnology, Institute of Agrobiological Sciences , National Agriculture and Food Research Organization (NARO) , Oowashi 1-2 , Tsukuba , Ibaraki 305-8634 , Japan.
ACS Synth Biol. 2018 Aug 17;7(8):1956-1963. doi: 10.1021/acssynbio.8b00204. Epub 2018 Jul 20.
Tight regulatory system for gene expression, which is ideally controlled by unnatural and bio-orthogonal substances, is a keystone for successful construction of synthetic gene circuits. Here, we present a widely applicable approach to construct tight protein translational switches using site-specific unnatural amino acid (Uaa) incorporation systems. As a key mechanism to obtain excellent tightness, we installed gene circuits for positive feedback derepression. This mechanism dramatically suppressed leakage translation in the absence of the Uaa. In a translational switch with the feedback circuit in Escherichia coli, a 1.4 × 10 ON/OFF ratio was achieved which was 3 × 10-fold greater than that of the parent system and was comparable to that of the well-known tight expression system using the araBAD promoter and the araC regulator. This method offers an avenue for generation of novel tight genetic switches from over a hundred site-specific unnatural amino acid incorporation systems which have already been established. These tight translational switches will facilitate the development of fine gene control systems in synthetic biology, especially for Uaa-auxotrophy-based biological containments and live attenuated vaccines.
由非天然且具有生物正交性的物质理想地控制的基因表达严格调控系统，是成功构建合成基因回路的关键。在此，我们提出一种广泛适用的方法，利用位点特异性非天然氨基酸（Uaa）掺入系统构建严格的蛋白质翻译开关。作为获得优异严格性的关键机制，我们安装了用于正反馈去阻遏的基因回路。该机制在没有Uaa的情况下显著抑制了渗漏翻译。在大肠杆菌中具有反馈回路的翻译开关中，实现了1.4×10的开/关比，比亲本系统高3×10倍，与使用araBAD启动子和araC调节子的著名严格表达系统相当。这种方法为从已建立的一百多种位点特异性非天然氨基酸掺入系统中产生新型严格遗传开关提供了一条途径。这些严格的翻译开关将促进合成生物学中精细基因控制系统的发展，特别是对于基于Uaa营养缺陷型的生物遏制和减毒活疫苗。
