Tight Translational Control Using Site-Specific Unnatural Amino Acid Incorporation with Positive Feedback Gene...

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.