Engineering Artificial Small RNAs for Conditional Gene Silencing in <i>Escherichia coli</i>

Abstract

It has become increasingly evident that noncoding small RNAs (sRNAs) play a significant and global role in bacterial gene regulation. A majority of the <i>trans</i>-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 <i>Escherichia coli</i>. 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 <i>ompF</i> and <i>fliC</i> 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