Technological watch

Bacterial small RNAs (sRNAs) that regulate gene expression have been engineered for uses in synthetic biology and metabolic engineering. Here, we designed a novel non?Hfq?dependent sRNA scaffold that makes use of a 20 nt antisense binding region to selectively target mRNAs and influence expression. The system was developed for regulation of a fluorescent reporter in vivo using E. coli, but the system was found to be more responsive and produce statistically significant results when applied to protein synthesis in in vitro cell?free systems (CFS). Antisense binding sequences were designed to target not only translation initiation regions but various secondary structures in the reporter mRNA. Targeting a high energy stem loop structure and the 3’ end of the mRNA produced expression knock?downs that approached 70%. Notably, targeting a low energy stem structure near a potential RNase E binding site led to a statistically significant 65% increase in gene expression. These results were not obtainable in vivo, and the underlying mechanism was translated from the reporter system to achieve better than 75% increase in recombinant diaphorase expression in an in vitro CFS. It is possible the designs developed here can be applied to improve expression of other proteins in a CFS.This article is protected by copyright. All rights reserved.