Steps Toward Developing an Antisense RNA Construct for surA in Escherichia coli

Abstract

SurA is a periplasmic chaperone associated with the transport of Bordetella resistance-to-killing (BrkA) protein, which mediates the virulence of Bordetella pertussis. We found that Escherichia coli containing a SurA deletion grow faster than wild-type E. coli — a previously unrecognized phenotype. We also observed that transfection with a BrkA-containing plasmid (pPALMC1) reduced growth rate in SurA mutant E. coli, but not in wild-type. Considering the advantages of inducible gene expression for gene characterization, we sought to knock down surA in E. coli through the expression of a custom surA antisense RNA molecule by the novel pPET plasmid. Our progress toward the creation of the novel pPET plasmid involved the successful creation of the surA asRNA insert fragment and confirmation of the fragment sequence through Sanger sequencing. Future studies can utilize our surA asRNA insert fragment by ligation into pHN678 to create pPET.