Engineering Artificial Small RNAs for Conditional Gene Silencing in Escherichia coli

It has become increasingly evident that noncoding small RNAs (sRNAs) play a significant and global role in bacterial gene regulation. A majority of the trans-acting sRNAs in bacteria interact with the 5' untranslated region (UTR) and/or the translation initiation region of the targeted mRNAs via imperfect base pairing, resulting in reduced translation efficiency and/or mRNA stability. Additionally, bacterial sRNAs often contain distinct scaffolds that recruit RNA chaperones such as Hfq to facilitate gene regulation. In this study, we describe a strategy to engineer artificial sRNAs that can regulate desired endogenous genes in Escherichia colt. Using a fluorescent reporter gene that was translationally fused to a native 5' mRNA leader sequence, active artificial sRNAs were screened from libraries in which natural sRNA scaffolds were fused to a randomized antisense domain. Artificial sRNAs that posttranscriptionally repress two endogenous genes ompF and fliC were isolated and characterized. We anticipate that the artificial sRNAs will be useful for dynamic control and fine-tuning of endogenous gene expression in bacteria for applications in synthetic biology.