Regulatory RNA molecules are common tools used in bacterial gene regulation. This paper focuses on the steps in designing an antisense RNA component for insertion into a plasmid in order to silence a gene in the plasmid host cell. By hybridizing to the ribosomal binding site of the target gene mRNA transcript, the antisense RNA transcript from the plasmid is able to inhibit gene translation and lead to eventual mRNA degradation. By regulating or silencing the gene of interest, further experiments could be used to confirm or reject proposed roles for undefined genes. In this paper, wecD silencing using pHN678 plasmid in Escherichia Coli will be used as an example. The design of the antisense RNA component involves three main steps: identification of the gene of interest, selection of the antisense target sequence of the gene, and modification of the target sequence to generate the antisense sequence. In addition, a sense RNA insert could be used as an orientation control in the experiment. The vector plasmid without any sequence insertion should also be included as a negative control. As a result, a successfully designed antisense RNA insert should allow knockdown of the target gene through hybridization to the mRNA transcript and therefore inhibiting gene translation.
Antisense RNA Insert Design for Plasmid Construction to Knockdown Target Gene Expression
Fall 2016 / Winter 2017