Tight Translational Control Using Site-Specific Unnatural Amino Acid Incorporation with Positive Feedback Gene Circuits,ACS Synthetic Biology

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Tight Translational Control Using Site-Specific Unnatural Amino Acid Incorporation with Positive Feedback Gene Circuits
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-07-06 00:00:00 , DOI: 10.1021/acssynbio.8b00204
Yusuke Kato ₁

Affiliation

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 × 10³ ON/OFF ratio was achieved which was 3 × 10²-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.

中文翻译：

使用位点特定的非天然氨基酸与正反馈基因电路的紧密翻译控制。

理想情况下，由非天然和生物正交物质控制的严格的基因表达调控系统是成功构建合成基因电路的基石。在这里，我们提出了使用位点特异性非天然氨基酸（Uaa）掺入系统构建紧密蛋白翻译开关的广泛适用方法。作为获得极佳密封性的关键机制，我们安装了用于正反馈抑制的基因电路。在没有Uaa的情况下，该机制极大地抑制了泄漏转换。在带有反馈电路的大肠杆菌平移开关中，实现了1.4×10 ^3的开/关比，即3×10 ²-比亲本系统大两倍，并且与使用araBAD启动子和araC调节子的众所周知的紧密表达系统相当。该方法提供了从已经建立的一百多个位点特异性非天然氨基酸掺入系统中产生新的紧密遗传开关的途径。这些紧密的翻译开关将促进合成生物学中精细基因控制系统的开发，尤其是对于基于Uaa-营养缺陷型生物收容所和减毒活疫苗的开发。

更新日期：2018-07-06

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