Overexpression of Target sRNAs in Tightly Controlled Multicopy Plasmids

Abstract

Imperfect base-pairing sRNAs might positively or negatively affect the stability of target mRNAs and can directly impact transcription of a target gene. The use of sRNA overexpression plasmids in conjunction with high-throughput RNA sequencing is expected to identify genes that are regulated by sRNA at the mRNA level. Here, we describe the construction of an inducible transcriptional fusion vector as an sRNA overexpression plasmid using the QuikChange^® Lightning Site-Directed Mutagenesis Kit. To investigate the effects of sRNA overexpression in Yersinia pestis, plasmid pBAD/HisA was modified into an inducible transcriptional fusion vector by using the QuikChange^® Lightning Site-Directed Mutagenesis Kit. The resulting plasmid removes the ribosomal binding site region and the start codon and introduces several restriction enzyme sites. The entire sequence of the target sRNA was cloned into the modified plasmid and transformed into Y. pestis cells. The inserted sequence was then transcribed into the sRNA transcript upon addition of arabinose. Finally, the impact of sRNA overexpression was observed in Y. pestis by measuring the mRNA transcript or protein level.