Reliable methods for conditional gene silencing in bacteria have been elusive. To improve silencing by expressed antisense RNAs (asRNAs), we systematically altered several design parameters and targeted multiple reporter and essential genes in Escherichia coli. A paired termini (PT) design, where flanking inverted repeats create paired dsRNA termini, proved effective. PTasRNAs targeted against the ackA gene within the acetate kinase-phosphotransacetylase operon (ackA-pta) triggered target mRNA decay and a 78% reduction in AckA activity with high genetic penetrance. PTasRNAs are abundant and stable and function through an RNase III independent mechanism that requires a large stoichiometric excess of asRNA. Conditional ackA silencing reduced carbon flux to acetate and increased heterologous gene expression. The PT design also improved silencing of the essential fabI gene. Full anti-fabI PTasRNA induction prevented growth and partial induction sensitized cells to a FabI inhibitor. PTasRNAs have potential for functional genomics, antimicrobial discovery and metabolic flux control.

译文

细菌中条件性基因沉默的可靠方法一直难以捉摸。为了改善表达的反义rna (asRNAs) 的沉默,我们系统地改变了几个设计参数,并针对大肠杆菌中的多个报告基因和必需基因。事实证明,成对的末端 (PT) 设计是有效的,其中侧翼反向重复创建成对的dsRNA末端。针对乙酸盐激酶-磷酸转乙酰酶操纵子 (ackA-pta) 内的ackA基因的ptasrna触发了靶mRNA衰减,并且具有高遗传外显率的AckA活性78% 降低。Ptasrna丰富且稳定,并通过独立于RNase III的机制起作用,该机制需要大量化学计量过量的asRNA。有条件的ackA沉默减少了碳向乙酸盐的通量,并增加了异源基因的表达。PT设计还改善了必需fabI基因的沉默。完全的抗fabI PTasRNA诱导可防止生长并部分诱导对FabI抑制剂敏感的细胞。Ptasrna具有功能基因组学,抗菌发现和代谢通量控制的潜力。

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