Multiple-Gene Silencing Using Antisense RNAs in Escherichia coli

We have developed four expression vectors to express antisense RNAs (asRNAs) by which genes of interest are silenced in Escherichia coli . The vectors are all IPTG-inducible and co-transformable in any combination and target genes are silenced conditionally and concurrently. Furthermore, in order to improve silencing efficacy, the vectors are designed to express uniquely shaped antisense RNAs, named paired termini antisense RNAs (PTasRNAs). The vectors are useful for comprehensive investigation of gene function and are applicable even if the target genes are essential for cell growth. Here, we describe methods to construct PTasRNA-expressing vectors and to evaluate silencing efficacy.