Construction of customized small regulatory RNAs

Abstract

For the last decade short noncoding RNAs are discovered to play an important role in genetic regulations. Among them, regulatory small RNAs (sRNA) in Escherichia coli repress translation initiation by hybridizing with a ribosome binding site in mRNA to prevent ribosome binding and by facilitating the degradation of mRNA. Such regulatory small RNAs could provide another way of genetic regulation in synthetic biology so that genetic circuits could be controlled precisely in concert with protein-based regulations. We could construct efficient customized sRNAs for DsRed2 repression and also constructed three different sRNAs for mRNAs of LuxR, AraC, and KanR without cross-reactivity. As a proof of concept, we constructed a temperaturesensitive genetic circuit controlled by artificial sRNA and the circuit operated successfully. The results represent that gene expression can be fine-tuned by designed small RNAs. The possibility of designing small regulatory RNAs opens a new way of regulation in synthetic biology, and which is expected to facilitate the development of precisely regulated synthetic circuits.