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A simple and highly efficient method for gene silencing in Escherichia coli

Giuseppe Magistro, Christiane Magistro, Christian G. Stief, Sören Schubert
Journal of microbiological methods 2018 v.154 pp. 25-32
Escherichia coli, antisense RNA, bacteria, gene expression, gene silencing, genes, open reading frames, plasmids, protocols, translation (genetics)
Here we present a simple and rapidly achievable protocol for gene silencing in Escherichia coli (E. coli). In this procedure, antisense RNA (asRNA) of 400-nucleotides (nt) length and absolute complementarity to the target is produced by an expression plasmid. The designed asRNA should ideally cover at least the −10 site of the promoter and the Shine-Dalgarno sequence, and additional 300-bp of the following open reading frame of the target gene. We show that the transcription process of the target is not affected at all, whereas the translation process is impaired. Based on high constitutive expression of asRNA we were able to extend the silencing effect to knock-out levels. By inducible expression, we show that also the modulation is possible. This technique should be widely useful to study gene function in E. coli and other bacteria.