An Effective Method for Specific Gene Silencing in Escherichia coli Using Artificial Small RNA

Abstract

Knockdown or silencing of a specific gene presents a powerful strategy for elucidating gene function in a variety of organisms. To date, efficient silencing methods have been established in eukaryotes, but not bacteria. In this chapter, an efficient and versatile gene silencing method using artificial small RNA (afsRNA) is described. For this purpose, target-recognizing sequences were introduced in specially designed RNA scaffolds to exist as single-stranded stretches in afsRNA. The translation initiation region of target genes was used as the sequence for afsRNA recognition, based on the theory that this site is usually highly accessible to ribosomes, and therefore, possibly, afsRNA. Two genes transcribed as monocistrons were tested with our protocol. Both genes were effectively silenced by their cognate afsRNAs.

The small RNA, SdsR, acts as a novel type of toxin inEscherichia coli

“…Plasmids pHM4T and pAKA, which were derived from pACYC184 and pBR322, respectively, were used as cloning vectors to generate RNA expression plasmids, as described previously [33]. RNA expression plasmids for afsRNAs, which can bind to the translation initiation region of each target mRNA, were constructed as described previously [10,71]. Primers and oligonucleotides used for plasmid and strain construction are shown in Table S7.…”